Research ArticleAlteration of ROS Homeostasis and Decreased Lifespanin S cerevisiae Elicited by Deletion of the MitochondrialTranslocator FLX1
Teresa Anna Giancaspero1 Emilia Dipalo2 Angelica Miccolis2 Eckhard Boles3
Michele Caselle4 and Maria Barile12
1 Istituto di Biomembrane e Bioenergetica CNR Via Amendola 165A 70126 Bari Italy2 Dipartimento di Bioscienze Biotecnologie e Biofarmaceutica Universita degli Studi di Bari ldquoA MorordquoVia Orabona 4 70126 Bari Italy
3 Institute of Molecular Biosciences Goethe University Frankfurt Max-von-Laue Straszlige 9 60438 Frankfurt am Main Germany4Dipartimento di Fisica Via P Giuria 1 10125 Torino Italy
Correspondence should be addressed to Maria Barile mariabarileunibait
Received 31 January 2014 Revised 20 March 2014 Accepted 1 April 2014 Published 8 May 2014
Academic Editor Dina Bellizzi
Copyright copy 2014 Teresa Anna Giancaspero et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
This paper deals with the control exerted by the mitochondrial translocator FLX1 which catalyzes the movement of the redoxcofactor FAD across the mitochondrial membrane on the efficiency of ATP production ROS homeostasis and lifespan of ScerevisiaeThe deletion of the FLX1 gene resulted in respiration-deficient and small-colony phenotype accompanied by a significantATP shortage and ROS unbalance in glycerol-grown cells Moreover the1198911198971199091Δ strain showedH
2O2hypersensitivity and decreased
lifespan The impaired biochemical phenotype found in the 1198911198971199091Δ strain might be justified by an altered expression of theflavoprotein subunit of succinate dehydrogenase a key enzyme in bioenergetics and cell regulation A search for possible cis-acting consensus motifs in the regulatory region upstream SDH1-ORF revealed a dozen of upstream motifs that might respondto induced metabolic changes by altering the expression of Flx1p Among these motifs two are present in the regulatory region ofgenes encoding proteins involved in flavin homeostasis This is the first evidence that the mitochondrial flavin cofactor status isinvolved in controlling the lifespan of yeasts maybe by changing the cellular succinate level This is not the only case in which thehomeostasis of redox cofactors underlies complex phenotypical behaviours as lifespan in yeasts
1 Introduction
Riboflavin (Rf or vitamin B2) is the precursor of flavin
mononucleotide (FMN) and flavin adenine dinucleotide(FAD) the redox cofactors of a large number of dehydroge-nases reductases and oxidases Most of these flavoenzymesare compartmented in the cellular organelles where theyare involved in energy production and redox homeostasisas well as in different cellular regulatory events includ-ing apoptosis chromatin remodelling and interestingly asrecently proposed in epigenetic signalling [1ndash4] Consistentwith the crucial role of flavoenzymes in cell life flavin-dependent enzyme deficiency andor impairment in flavin
homeostasis in humans and experimental animals has beenlinked to several diseases such as cancer cardiovasculardiseases anaemia abnormal fetal development and differentneuromuscular and neurological disorders [5ndash9] The rele-vance of these pathologies merits further research aimed tobetter describe FAD homeostasis and flavoenzyme biogen-esis especially in those organisms that can be a simple andsuitable model for human diseases The conserved biologicalprocesses shared with all eukaryotic cells together with thepossibility of simple and quick genetic manipulation allowedproposing the budding yeast Saccharomyces cerevisiae asthe premier model to understand the biochemistry and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 101286 12 pageshttpdxdoiorg1011552014101286
2 BioMed Research International
molecular biology of mammalian cells and to deciphermolecular mechanisms underlying human diseases [10ndash12]
Formany years S cerevisiae has been used also as amodelto study the complexity of the molecular events involvedin the undesired process of aging in which mitochondriaplay a major role [13 14] The role of mitochondria hasbeen pointed out either because aged respiratory chain is amajor source of cellular ROS [14] or because mitochondriaactively participate in regulating the homeostasis of the redoxcofactor NAD which regulates yeast lifespan by acting as asubstrate of specific deacetylases (EC 351-) named sirtuins[15ndash17] This might not be the only case in which the home-ostasis of redox cofactors underlies complex phenotypicalbehaviours as lifespan in yeasts Here we investigate whetherthe mitochondrial flavin cofactor status may also be involvedin controlling the lifespan of yeasts presumably by changingthe level ofmitochondrial flavoenzymes which are crucial forcell regulation [18 19]
It should be noted that even though mitochondria areplenty of flavin and flavoproteins [20 21] the origin offlavin cofactors starting from Rf in this organelle is still amatter of debate Yeasts have the ability to either synthesiseRf de novo or to take it from outside The first eukaryoticgene coding for a cellular Rf transporter was identifiedin S cerevisiae as the MCH5 gene [22] Intracellular Rfconversion to FAD is a ubiquitous pathway and occurs via thesequential actions of ATP riboflavin 51015840-phosphotransferaseor riboflavin kinase (RFK EC 27126) which phosphorylatesthe vitamin into FMN and of ATP FMN adenylyl transferaseor FAD synthase (FADS EC 2772) which adenylates FMNto FAD The first eukaryotic genes encoding for RFK andFADS were identified in S cerevisiae and named FMN1[23] and FAD1 [24] respectively While there is no doubtabout a mitochondrial localization for Fmn1p [23 25] theexistence of a mitochondrial FADS isoform in yeast is stillcontroversial First a cytosolic localization for Fad1p wasreported [24] thus newly synthesised FAD was expectedto be imported into mitochondria via the FAD translocatorFlx1p [25] However results from our laboratory showed thatbesides in the cytosol FAD-forming activities can be revealedinmitochondria thus requiring uptake of the FADprecursorsinto mitochondria [26 27] FAD synthesised inside theorganelle can be either delivered to a number of nascentclient apo-flavoenzymes or be exported via Flx1p into cytosolto take part of an extramitochondrial posttranscriptionalcontrol of apo-flavoprotein biogenesis [19 26]
Besides synthesis and transport mitochondrial flavinhomeostasis strictly depends also on flavin degradationRecently we have demonstrated that S cerevisiae mito-chondria (SCM) are able to catalyze FAD hydrolysis viaan enzymatic activity which is different from the alreadycharacterized NUDIX hydrolases (ie enzymes that catalyzethe hydrolysis of nucleoside diphosphates linked to othermoieties X) and it is regulated by the mitochondrial NADredox status [17]
To prove the relationship between mitochondrial FADhomeostasis and lifespan in yeast we use as a model aS cerevisiae strain lacking the FLX1 gene which showeda respiratory-deficient phenotype and a derangement in
a number of mitochondrial flavoproteins that is dihy-drolipoamide dehydrogenase (LPD1) succinate dehydro-genase (SDH) and flavoproteins involved in ubiquinonebiosynthesis (COQ6) [18 25 26 28]
We demonstrated here that this deleted strain performedATP shortage and ROS unbalance together with H
2O2
hypersensitivity and altered chronological lifespanThis flx1Δphenotype is correlated to a reduced ability to maintain anappropriate level of the flavoenzyme succinate dehydrogenase(SDH) amember of a complex ldquoflavin networkrdquo participatingin a nucleus-mitochondrion cross-talk
2 Materials and Methods
21 Materials All reagents and enzymes were from Sigma-Aldrich (St Louis MO USA) Zymolyase was from ICN(Abingdon UK) and Bacto Yeast Extract and Bacto Peptonewere from Difco (Franklin Lakes NJ USA) Mitochondrialsubstrates were used as TRIS salts at pH 70 Solvents andsalts used for HPLC were from J T Baker (Center Valley PAUSA) Rat anti-HA monoclonal antibody and peroxidase-conjugated anti-rat IgG secondary antibody were obtainedfrom Roche (Basel Switzerland) and Jackson Immunore-search (West Grove PA USA) respectively
22 Yeast Strains The wild-type S cerevisiae strain(EBY157A or WT genotype MAT120572 ura 3ndash52 MAL2-8cSUC2 p426MET25) used in this work derived from theCENPK series of yeast strains and was obtained fromP Kotter (Institut fur Mikrobiologie Goethe-UniversitatFrankfurt Frankfurt Germany) as already described in [26]The flx1Δmutant strain (EBY167A flx1Δ) was constructed asdescribed in [26] and the WT-HA (EBY157-SDH1-HA) andflx1Δ-HA (EBY167-G418S-SDH1-HA) were constructed asdescribed in [19]
23 Media and Growth Conditions Cells were grown aero-bically at 30∘C with constant shaking in rich liquid medium(YEP 10 gL Yeast Extract 20 gL Bacto Peptone) or inminimal synthetic liquid medium (SM 17 gL yeast nitrogenbase 5 gL ammonium sulphate and 20mgL uracil) supple-mented with glucose or glycerol (2 each) as carbon sourcesThe YEP or SM solid media contained 18 gL agar
24 Chronological Lifespan Determination WT and flx1Δstrains were grown overnight at 30∘C in 5mL YEP liquidmedium supplemented with glucose 05 up to the earlystationary phase Each strain was then cultured in SM liquidmedium at 30∘C for 1 4 and 7 days Five serial dilutions fromeach culture containing 200 cells calculated from A
600 nmwere plated onto SM solid medium and grown at 30∘C fortwo-three days
25 H2O2Sensitivity WT and flx1Δ strains were grown
overnight at 30∘C in 5mL YEP liquid medium supplementedwith glucose 05 up to the early stationary phaseThen eachstrain was inoculated in SM liquid medium (initial A
600 nmequal to 01) containing glucose 2 andH
2O2(005 or 2mM)
BioMed Research International 3
After 5 or 24 h of growth at 30∘C the H2O2sensitivity was
estimated by measuring the A600 nm of the growth culture
26 Malate and Succinate Sensitivity WT and flx1Δ strainswere grown overnight at 30∘C in 5mL YEP liquid mediumsupplemented with glucose 05 up to the early stationaryphaseThen each strain was inoculated in SM liquidmedium(initial A
600 nm equal to 01) containing glucose 2 andsuccinate or malate (5mM) After 24 h of growth at 30∘C theH2O2sensitivity was estimated by measuring the A
600 nm ofthe growth culture
27 Preparation of Spheroplasts Mitochondria and CellularLysates Spheroplasts were prepared using Zymolyase Mito-chondria were isolated from spheroplasts as described in[26] Cellular lysates were obtained by early exponential-phase (5 h) or stationary-phase (24 h) cells harvested bycentrifugation (8000timesg for 5min) washed with sterile waterresuspended in 250 120583L of lysis buffer (10mM Tris-HCl pH76 1mM EDTA 1mM dithiothreitol and 02mM phenyl-methanesulfonyl fluoride supplemented with one tablet ofRoche protease inhibitor cocktail every 10mL of lysis buffer)and vortexed with glass beads for 10min at 4∘C The liquidwas removed and centrifuged at 3000timesg for 5min to removecell debris The protein concentrations of the spheroplastsmitochondria and cellular lysates were assayed according toBradford [29]
28 Quantitation of Flavins ATP and Reactive Oxygen Species(ROS) Rf FMN and FAD content in spheroplasts and SCMwas measured in aliquots (5ndash80120583L) of neutralized perchloricextracts by means of HPLC (Gilson HPLC system includinga model 306 pump and a model 307 pump equipped witha Kontron Instruments SFM 25 fluorometer and Unipointsystem software) essentially as previously described [26]ATP content wasmeasured fluorometrically in cellular lysatesby using the ATP Detecting System essentially as in [30]NADPH formation which corresponds to ATP content(with a 1 1 stoichiometry) was followed with excitationwavelength at 340 nm and emission wavelength at 456 nmROS level was fluorometrically measured on cellular lysatesusing as substrate 21015840-71015840-dichlorofluorescin diacetate (DCF-DA) according to [30] with slight modifications Brieflythe probe DCF-DA (50 120583M) was incubated at 37∘C for 1 hwith 003ndash005mg proteins and converted to fluorescentdichlorofluorescein (DCF) upon reaction with ROS DCFfluorescence of each sample was measured by means ofa LS50S Perkin Elmer spectrofluorometer (excitation andemission wavelengths set at 485 nm and 520 nm resp)
29 Enzymatic Assays Succinate dehydrogenase (SDH EC1351) and fumarase (FUM EC 4212) activities weremeasured as in [26] Glutathione reductase (GR EC1642) activity was spectrophotometrically assayed by mon-itoring the absorbance at 340 nm due to NADPH oxi-dation after glutathione addition (1mM) essentially asin [30] Superoxide dismutase (SOD EC 11511) activity
was spectrophotometrically measured by the xanthine oxi-dasexanthinecytochrome cmethod essentially as describedin [31]
210 Statistical Analysis All experiments were repeated atleast three times with different cell preparations Resultsare presented as mean plusmn standard deviation (SD) Statisticalsignificance was evaluated by Studentrsquos 119905-test Values of 119875 lt005 were considered statistically significant
3 Results
31 Phenotypical and Biochemical Consequences of FLX1Deletion In order to study the relevance of mitochondrialflavin cofactor homeostasis on cellular bioenergetics weintroduced a yeast strain lacking the FLX1 gene encodingthe mitochondrial FAD transporter [26] This deleted strainshowed a small-colony phenotype on both fermentable andnonfermentable carbon sources due to an impairment inthe aerobic respiratory chain pathway [32] The deletedstrain flx1Δ grew normally on glucose medium but failed togrow on nonfermentable carbon sources (ie glycerol) thusindicating a respiration-deficient phenotype (Figure 1(a))The growth defect on nonfermentable carbon source whichwas restored by complementing the deleted strain with theYEpFLX1 plasmid [26] was not rescued by the additionof tricarboxylic acid (TCA) cycle intermediates such assuccinate or malate (Figure 1(a))
Among the mitochondrial flavoenzymes which weredemonstrated to be altered in flx1Δ strain [25 26 28]we showed before [19 32] and confirmed in Figure 1(b)a significant reduced level of the apo-flavoprotein Sdh1presulting in an altered functionality of SDH or complexII of the respiratory chain This reduction was revealedby creating a strain in which three consecutive copies ofthe human influenza hemagglutinin epitope (HA epitopeYPYDVPDYA) were fused in frame to the 31015840end of the SDH1ORF in the genome of both the WT and flx1Δ strains Thechimera protein namely Sdh1-HAp carrying the HA-tag atthe C-terminal end of Sdh1p lost the ability to covalentlybind the flavin cofactor FAD [19 33] but not its regulatorybehaviour that is its inducible expression in galactose or innonfermentable carbon sources In all the growth conditionstested the FAD-independent fumarase (FUM) activity usedas a control was not affected by FLX1 deletion (see histogramin Figure 1(b))
A significant decrease of Sdh1-HAp level was accom-panied in galactose but not in glycerol by a profoundderangement of flavin cofactors particularly evident in cellgrown at the early exponential phase (Table 1) in agreementwith [25 26] respectively The reason for these carbonsource-dependent flavin level changes which is not easilyexplainable is addressed in Section 4
Consistent with an altered functionality of SDH the flx1Δstrain also showed impaired isolated mitochondria oxygenconsumption activity specifically detectable when succinatewas used as a respiratory substrate [19] Similar phenotypewas also observed in yeast strains carrying either a deletion
4 BioMed Research International
8
6
4
2
0
Glucose GlycerolCTR WTCTR flx1Δ
WT flx1Δ WT flx1Δ
A600
nm
+Mal 5mM+Succ 5mM
(a)FU
M sp
ecifi
c act
ivity
250
200
150
100
50
Glycerol Galactose
Glycerol Galactose
0
Act1p
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
Sdh1-HAp
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
(nm
olmiddotminminus1middotm
gminus1)
(b)
Figure 1 (a) Respiratory-deficient phenotype of flx1Δ strain effect of succinate and malate addition WT and flX1Δ cells were cultured at30∘C in YEP liquidmedium supplemented with either glucose or glycerol (2 each) as carbon sourceWhere indicated either 5mM succinate(Succ) or 5mM malate (Mal) was added Cell growth was estimated at the stationary phase (24 h) by measuring the absorbance at 600 nm(119860600 nm) of a ten-fold dilution of each growth culture consistently corrected for the dilution factor The values reported in the histogram
are the means (plusmnSD) of three experiments (b) Changes in the recombinant Sdh1-HAp level in flx1Δ strain Cellular lysates were preparedfromWT-HA and flX1Δ-HA cells grown at 30∘C up to the exponential growth phase (5 h) in YEP liquid medium supplemented with eitherglycerol or galactose (2 each) as carbon source Proteins from cellular lysates (005mg) were separated by SDSPAGE and transferred ontoa PVDF membrane In each extract Sdh1-HA protein was detected by using an 120572-HA and its amount was densitometrically evaluated Thevalues reported in the histogram are the means (plusmnSD) of three experiments performed with different cellular lysates preparations Statisticalevaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005) As a control the specific activity of the enzyme fumarase (FUM) wasdetermined in each cellular lysate preparation
BioMed Research International 5
Table 1 Endogenous flavin content in spheroplasts and mitochondria
Spheroplasts and mitochondria (SCM) were prepared fromWT and 1198911198971199091Δ cells grown in glycerol or galactose (2) up to the exponential growth phase (5 h)FAD and FMN content was determined in neutralized perchloric acid extracts as described inMaterials andMethods Riboflavin amount was not relevant andthus its value has not been reportedThemeans (plusmnSD) of the flavin endogenous content determined in three experiments performedwith different preparationsare reported ∘Data published in (Bafunno et al 2004) [26] statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
120
100
80
60
40
20
05 24
Glucose Glycerol
120
100
80
60
40
20
05 24 5 24 5 24
( o
f eac
h co
ntro
l)
( o
f eac
h co
ntro
l)
flx1Δ flx1ΔWTWT
A600
nm
A600
nm
+H2O2 005mM+H2O2 2mM
Figure 2 Sensitivity to H2O2 WT and flX1Δ cells were cultured
at 30∘C in YEP liquid medium supplemented with either glucoseor glycerol (2 each) as carbon source Where indicated H
2O2at
the indicated concentration was added Cell growth was estimatedat the exponential (5 h) and stationary phase (24 h) by measuringthe absorbance at 600 nm (119860
600 nm) In the histogram the 119860600 nm
of the cell cultures grown in the presence of H2O2is reported as
a percentage of the control (ie the 119860600 nm of cell cultures grown
in the absence of H2O2 set arbitrary equal to 100) The values
reported in the histogram are themeans (plusmnSD) of three experiments
of SDH1 [34] or a deletion of SDH5 which encodes amitochondrial protein involved in Sdh1p flavinylation [35]Another respiration-related phenotype of flx1Δ strain wasinvestigated in Figure 2 by testing H
2O2hypersensitivity
of cells grown on both fermentable and nonfermentablecarbon sources In glucose the WT cells grew up to thestationary phase (24 h) in the presence of H
2O2(005 or
2mM) essentially as the control cells grown in the absence ofH2O2 In glycerol their ability to grow up to 24 hwas reduced
of about 20 at 005mM H2O2and of 60 at 2mM with
respect to the control cells in which no H2O2was added
In glucose flx1Δ cells did not showH2O2hypersensitivity
at 005mM At 2mM H2O2 their ability to grow was
significantly reduced (of about 85) with respect to flx1Δcells grown in the absence of H
2O2 The ability of the flx1Δ
cells to grow in glycerol which was per se drastically reducedby deletion was reduced at 24 h by the addition of 005mMH2O2(about 50 with respect to the control cells grown in
24h
96h
168h
flx1ΔWT
Figure 3 Chronological lifespan determination WT and flX1Δstrains were cultured in SM liquid medium at 30∘C Dilutions fromeach culture containing about 200 cells (as calculated from 119860
600 nmby taking into account that one 119860
600 nm is equivalent to 3 times 107cellmL) were harvested after 24 96 and 168 h and plated onto SMsolid medium and grown at 30∘C for two-three days
the absence of H2O2) An even higher sensitivity toH
2O2was
observed in the presence of 2mMH2O2 having their growth
ability reduced of about 85 with respect to control cells inwhich no addition was made The impairment in the abilityto grow under H
2O2stress conditions clearly demonstrates
an impairment in defence capability of the flx1Δ strainInterestingly the same phenotype was observed also in theyeast sdh5Δ [35] sdh1Δ and sdh2Δ [36] strains
To understand whether mitochondrial flavoproteinimpairment due to FLX1 deletion influenced aging in yeastwe carried out measurements of chronological lifespanon both WT and flx1Δ cells cultured at 30∘C in SM liquidmedium supplemented with glucose 2 as carbon source(Figure 3) Following 24 h (1 day) 96 h (4 days) and 168 h(7 days) of growth the number of colonies was determinedby spotting five serial dilutions of the liquid culture andincubating the plates for two-three days at 30∘C The resultsof a typical experiment are reported in Figure 3 A reducednumber of small colonies were counted for the flx1Δ strainwith respect to the number of colonies counted for theWT strain This phenotype particularly evident after 96 hand 168 h of growth time clearly indicated a decrease inchronological lifespan of the flx1Δ strain Essentially thesame phenotype was observed in sdh1Δ and sdh5Δ strains[35] Thus it seems quite clear that a correct biogenesis ofmitochondrial flavoproteome and in particular assembly ofSDH ensures a correct aging rate in yeast When flx1Δ cellswere grown on glycerol they lost the ability to form coloniesfollowing 24 h growth time (data not shown)
6 BioMed Research International
80
6
4
2
05 24
ATP
leve
l
ATP
leve
l
20
16
12
08
04
0
(a998400)
5 5 524
lowast
lowast
(a)WTWT flx1Δ flx1Δ
(nm
olmiddotm
gminus1)
(nm
olmiddotm
gminus1)
20
16
08
12
04
05 24
20
16
12
08
04
0
(b998400)
5 5 524
lowast
(b)flx1WTflx1WT
ROS
leve
l (ΔFmiddot120583
gminus1 )
ROS
leve
l (ΔFmiddot120583
gminus1 )
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
molecular biology of mammalian cells and to deciphermolecular mechanisms underlying human diseases [10ndash12]
Formany years S cerevisiae has been used also as amodelto study the complexity of the molecular events involvedin the undesired process of aging in which mitochondriaplay a major role [13 14] The role of mitochondria hasbeen pointed out either because aged respiratory chain is amajor source of cellular ROS [14] or because mitochondriaactively participate in regulating the homeostasis of the redoxcofactor NAD which regulates yeast lifespan by acting as asubstrate of specific deacetylases (EC 351-) named sirtuins[15ndash17] This might not be the only case in which the home-ostasis of redox cofactors underlies complex phenotypicalbehaviours as lifespan in yeasts Here we investigate whetherthe mitochondrial flavin cofactor status may also be involvedin controlling the lifespan of yeasts presumably by changingthe level ofmitochondrial flavoenzymes which are crucial forcell regulation [18 19]
It should be noted that even though mitochondria areplenty of flavin and flavoproteins [20 21] the origin offlavin cofactors starting from Rf in this organelle is still amatter of debate Yeasts have the ability to either synthesiseRf de novo or to take it from outside The first eukaryoticgene coding for a cellular Rf transporter was identifiedin S cerevisiae as the MCH5 gene [22] Intracellular Rfconversion to FAD is a ubiquitous pathway and occurs via thesequential actions of ATP riboflavin 51015840-phosphotransferaseor riboflavin kinase (RFK EC 27126) which phosphorylatesthe vitamin into FMN and of ATP FMN adenylyl transferaseor FAD synthase (FADS EC 2772) which adenylates FMNto FAD The first eukaryotic genes encoding for RFK andFADS were identified in S cerevisiae and named FMN1[23] and FAD1 [24] respectively While there is no doubtabout a mitochondrial localization for Fmn1p [23 25] theexistence of a mitochondrial FADS isoform in yeast is stillcontroversial First a cytosolic localization for Fad1p wasreported [24] thus newly synthesised FAD was expectedto be imported into mitochondria via the FAD translocatorFlx1p [25] However results from our laboratory showed thatbesides in the cytosol FAD-forming activities can be revealedinmitochondria thus requiring uptake of the FADprecursorsinto mitochondria [26 27] FAD synthesised inside theorganelle can be either delivered to a number of nascentclient apo-flavoenzymes or be exported via Flx1p into cytosolto take part of an extramitochondrial posttranscriptionalcontrol of apo-flavoprotein biogenesis [19 26]
Besides synthesis and transport mitochondrial flavinhomeostasis strictly depends also on flavin degradationRecently we have demonstrated that S cerevisiae mito-chondria (SCM) are able to catalyze FAD hydrolysis viaan enzymatic activity which is different from the alreadycharacterized NUDIX hydrolases (ie enzymes that catalyzethe hydrolysis of nucleoside diphosphates linked to othermoieties X) and it is regulated by the mitochondrial NADredox status [17]
To prove the relationship between mitochondrial FADhomeostasis and lifespan in yeast we use as a model aS cerevisiae strain lacking the FLX1 gene which showeda respiratory-deficient phenotype and a derangement in
a number of mitochondrial flavoproteins that is dihy-drolipoamide dehydrogenase (LPD1) succinate dehydro-genase (SDH) and flavoproteins involved in ubiquinonebiosynthesis (COQ6) [18 25 26 28]
We demonstrated here that this deleted strain performedATP shortage and ROS unbalance together with H
2O2
hypersensitivity and altered chronological lifespanThis flx1Δphenotype is correlated to a reduced ability to maintain anappropriate level of the flavoenzyme succinate dehydrogenase(SDH) amember of a complex ldquoflavin networkrdquo participatingin a nucleus-mitochondrion cross-talk
2 Materials and Methods
21 Materials All reagents and enzymes were from Sigma-Aldrich (St Louis MO USA) Zymolyase was from ICN(Abingdon UK) and Bacto Yeast Extract and Bacto Peptonewere from Difco (Franklin Lakes NJ USA) Mitochondrialsubstrates were used as TRIS salts at pH 70 Solvents andsalts used for HPLC were from J T Baker (Center Valley PAUSA) Rat anti-HA monoclonal antibody and peroxidase-conjugated anti-rat IgG secondary antibody were obtainedfrom Roche (Basel Switzerland) and Jackson Immunore-search (West Grove PA USA) respectively
22 Yeast Strains The wild-type S cerevisiae strain(EBY157A or WT genotype MAT120572 ura 3ndash52 MAL2-8cSUC2 p426MET25) used in this work derived from theCENPK series of yeast strains and was obtained fromP Kotter (Institut fur Mikrobiologie Goethe-UniversitatFrankfurt Frankfurt Germany) as already described in [26]The flx1Δmutant strain (EBY167A flx1Δ) was constructed asdescribed in [26] and the WT-HA (EBY157-SDH1-HA) andflx1Δ-HA (EBY167-G418S-SDH1-HA) were constructed asdescribed in [19]
23 Media and Growth Conditions Cells were grown aero-bically at 30∘C with constant shaking in rich liquid medium(YEP 10 gL Yeast Extract 20 gL Bacto Peptone) or inminimal synthetic liquid medium (SM 17 gL yeast nitrogenbase 5 gL ammonium sulphate and 20mgL uracil) supple-mented with glucose or glycerol (2 each) as carbon sourcesThe YEP or SM solid media contained 18 gL agar
24 Chronological Lifespan Determination WT and flx1Δstrains were grown overnight at 30∘C in 5mL YEP liquidmedium supplemented with glucose 05 up to the earlystationary phase Each strain was then cultured in SM liquidmedium at 30∘C for 1 4 and 7 days Five serial dilutions fromeach culture containing 200 cells calculated from A
600 nmwere plated onto SM solid medium and grown at 30∘C fortwo-three days
25 H2O2Sensitivity WT and flx1Δ strains were grown
overnight at 30∘C in 5mL YEP liquid medium supplementedwith glucose 05 up to the early stationary phaseThen eachstrain was inoculated in SM liquid medium (initial A
600 nmequal to 01) containing glucose 2 andH
2O2(005 or 2mM)
BioMed Research International 3
After 5 or 24 h of growth at 30∘C the H2O2sensitivity was
estimated by measuring the A600 nm of the growth culture
26 Malate and Succinate Sensitivity WT and flx1Δ strainswere grown overnight at 30∘C in 5mL YEP liquid mediumsupplemented with glucose 05 up to the early stationaryphaseThen each strain was inoculated in SM liquidmedium(initial A
600 nm equal to 01) containing glucose 2 andsuccinate or malate (5mM) After 24 h of growth at 30∘C theH2O2sensitivity was estimated by measuring the A
600 nm ofthe growth culture
27 Preparation of Spheroplasts Mitochondria and CellularLysates Spheroplasts were prepared using Zymolyase Mito-chondria were isolated from spheroplasts as described in[26] Cellular lysates were obtained by early exponential-phase (5 h) or stationary-phase (24 h) cells harvested bycentrifugation (8000timesg for 5min) washed with sterile waterresuspended in 250 120583L of lysis buffer (10mM Tris-HCl pH76 1mM EDTA 1mM dithiothreitol and 02mM phenyl-methanesulfonyl fluoride supplemented with one tablet ofRoche protease inhibitor cocktail every 10mL of lysis buffer)and vortexed with glass beads for 10min at 4∘C The liquidwas removed and centrifuged at 3000timesg for 5min to removecell debris The protein concentrations of the spheroplastsmitochondria and cellular lysates were assayed according toBradford [29]
28 Quantitation of Flavins ATP and Reactive Oxygen Species(ROS) Rf FMN and FAD content in spheroplasts and SCMwas measured in aliquots (5ndash80120583L) of neutralized perchloricextracts by means of HPLC (Gilson HPLC system includinga model 306 pump and a model 307 pump equipped witha Kontron Instruments SFM 25 fluorometer and Unipointsystem software) essentially as previously described [26]ATP content wasmeasured fluorometrically in cellular lysatesby using the ATP Detecting System essentially as in [30]NADPH formation which corresponds to ATP content(with a 1 1 stoichiometry) was followed with excitationwavelength at 340 nm and emission wavelength at 456 nmROS level was fluorometrically measured on cellular lysatesusing as substrate 21015840-71015840-dichlorofluorescin diacetate (DCF-DA) according to [30] with slight modifications Brieflythe probe DCF-DA (50 120583M) was incubated at 37∘C for 1 hwith 003ndash005mg proteins and converted to fluorescentdichlorofluorescein (DCF) upon reaction with ROS DCFfluorescence of each sample was measured by means ofa LS50S Perkin Elmer spectrofluorometer (excitation andemission wavelengths set at 485 nm and 520 nm resp)
29 Enzymatic Assays Succinate dehydrogenase (SDH EC1351) and fumarase (FUM EC 4212) activities weremeasured as in [26] Glutathione reductase (GR EC1642) activity was spectrophotometrically assayed by mon-itoring the absorbance at 340 nm due to NADPH oxi-dation after glutathione addition (1mM) essentially asin [30] Superoxide dismutase (SOD EC 11511) activity
was spectrophotometrically measured by the xanthine oxi-dasexanthinecytochrome cmethod essentially as describedin [31]
210 Statistical Analysis All experiments were repeated atleast three times with different cell preparations Resultsare presented as mean plusmn standard deviation (SD) Statisticalsignificance was evaluated by Studentrsquos 119905-test Values of 119875 lt005 were considered statistically significant
3 Results
31 Phenotypical and Biochemical Consequences of FLX1Deletion In order to study the relevance of mitochondrialflavin cofactor homeostasis on cellular bioenergetics weintroduced a yeast strain lacking the FLX1 gene encodingthe mitochondrial FAD transporter [26] This deleted strainshowed a small-colony phenotype on both fermentable andnonfermentable carbon sources due to an impairment inthe aerobic respiratory chain pathway [32] The deletedstrain flx1Δ grew normally on glucose medium but failed togrow on nonfermentable carbon sources (ie glycerol) thusindicating a respiration-deficient phenotype (Figure 1(a))The growth defect on nonfermentable carbon source whichwas restored by complementing the deleted strain with theYEpFLX1 plasmid [26] was not rescued by the additionof tricarboxylic acid (TCA) cycle intermediates such assuccinate or malate (Figure 1(a))
Among the mitochondrial flavoenzymes which weredemonstrated to be altered in flx1Δ strain [25 26 28]we showed before [19 32] and confirmed in Figure 1(b)a significant reduced level of the apo-flavoprotein Sdh1presulting in an altered functionality of SDH or complexII of the respiratory chain This reduction was revealedby creating a strain in which three consecutive copies ofthe human influenza hemagglutinin epitope (HA epitopeYPYDVPDYA) were fused in frame to the 31015840end of the SDH1ORF in the genome of both the WT and flx1Δ strains Thechimera protein namely Sdh1-HAp carrying the HA-tag atthe C-terminal end of Sdh1p lost the ability to covalentlybind the flavin cofactor FAD [19 33] but not its regulatorybehaviour that is its inducible expression in galactose or innonfermentable carbon sources In all the growth conditionstested the FAD-independent fumarase (FUM) activity usedas a control was not affected by FLX1 deletion (see histogramin Figure 1(b))
A significant decrease of Sdh1-HAp level was accom-panied in galactose but not in glycerol by a profoundderangement of flavin cofactors particularly evident in cellgrown at the early exponential phase (Table 1) in agreementwith [25 26] respectively The reason for these carbonsource-dependent flavin level changes which is not easilyexplainable is addressed in Section 4
Consistent with an altered functionality of SDH the flx1Δstrain also showed impaired isolated mitochondria oxygenconsumption activity specifically detectable when succinatewas used as a respiratory substrate [19] Similar phenotypewas also observed in yeast strains carrying either a deletion
4 BioMed Research International
8
6
4
2
0
Glucose GlycerolCTR WTCTR flx1Δ
WT flx1Δ WT flx1Δ
A600
nm
+Mal 5mM+Succ 5mM
(a)FU
M sp
ecifi
c act
ivity
250
200
150
100
50
Glycerol Galactose
Glycerol Galactose
0
Act1p
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
Sdh1-HAp
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
(nm
olmiddotminminus1middotm
gminus1)
(b)
Figure 1 (a) Respiratory-deficient phenotype of flx1Δ strain effect of succinate and malate addition WT and flX1Δ cells were cultured at30∘C in YEP liquidmedium supplemented with either glucose or glycerol (2 each) as carbon sourceWhere indicated either 5mM succinate(Succ) or 5mM malate (Mal) was added Cell growth was estimated at the stationary phase (24 h) by measuring the absorbance at 600 nm(119860600 nm) of a ten-fold dilution of each growth culture consistently corrected for the dilution factor The values reported in the histogram
are the means (plusmnSD) of three experiments (b) Changes in the recombinant Sdh1-HAp level in flx1Δ strain Cellular lysates were preparedfromWT-HA and flX1Δ-HA cells grown at 30∘C up to the exponential growth phase (5 h) in YEP liquid medium supplemented with eitherglycerol or galactose (2 each) as carbon source Proteins from cellular lysates (005mg) were separated by SDSPAGE and transferred ontoa PVDF membrane In each extract Sdh1-HA protein was detected by using an 120572-HA and its amount was densitometrically evaluated Thevalues reported in the histogram are the means (plusmnSD) of three experiments performed with different cellular lysates preparations Statisticalevaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005) As a control the specific activity of the enzyme fumarase (FUM) wasdetermined in each cellular lysate preparation
BioMed Research International 5
Table 1 Endogenous flavin content in spheroplasts and mitochondria
Spheroplasts and mitochondria (SCM) were prepared fromWT and 1198911198971199091Δ cells grown in glycerol or galactose (2) up to the exponential growth phase (5 h)FAD and FMN content was determined in neutralized perchloric acid extracts as described inMaterials andMethods Riboflavin amount was not relevant andthus its value has not been reportedThemeans (plusmnSD) of the flavin endogenous content determined in three experiments performedwith different preparationsare reported ∘Data published in (Bafunno et al 2004) [26] statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
120
100
80
60
40
20
05 24
Glucose Glycerol
120
100
80
60
40
20
05 24 5 24 5 24
( o
f eac
h co
ntro
l)
( o
f eac
h co
ntro
l)
flx1Δ flx1ΔWTWT
A600
nm
A600
nm
+H2O2 005mM+H2O2 2mM
Figure 2 Sensitivity to H2O2 WT and flX1Δ cells were cultured
at 30∘C in YEP liquid medium supplemented with either glucoseor glycerol (2 each) as carbon source Where indicated H
2O2at
the indicated concentration was added Cell growth was estimatedat the exponential (5 h) and stationary phase (24 h) by measuringthe absorbance at 600 nm (119860
600 nm) In the histogram the 119860600 nm
of the cell cultures grown in the presence of H2O2is reported as
a percentage of the control (ie the 119860600 nm of cell cultures grown
in the absence of H2O2 set arbitrary equal to 100) The values
reported in the histogram are themeans (plusmnSD) of three experiments
of SDH1 [34] or a deletion of SDH5 which encodes amitochondrial protein involved in Sdh1p flavinylation [35]Another respiration-related phenotype of flx1Δ strain wasinvestigated in Figure 2 by testing H
2O2hypersensitivity
of cells grown on both fermentable and nonfermentablecarbon sources In glucose the WT cells grew up to thestationary phase (24 h) in the presence of H
2O2(005 or
2mM) essentially as the control cells grown in the absence ofH2O2 In glycerol their ability to grow up to 24 hwas reduced
of about 20 at 005mM H2O2and of 60 at 2mM with
respect to the control cells in which no H2O2was added
In glucose flx1Δ cells did not showH2O2hypersensitivity
at 005mM At 2mM H2O2 their ability to grow was
significantly reduced (of about 85) with respect to flx1Δcells grown in the absence of H
2O2 The ability of the flx1Δ
cells to grow in glycerol which was per se drastically reducedby deletion was reduced at 24 h by the addition of 005mMH2O2(about 50 with respect to the control cells grown in
24h
96h
168h
flx1ΔWT
Figure 3 Chronological lifespan determination WT and flX1Δstrains were cultured in SM liquid medium at 30∘C Dilutions fromeach culture containing about 200 cells (as calculated from 119860
600 nmby taking into account that one 119860
600 nm is equivalent to 3 times 107cellmL) were harvested after 24 96 and 168 h and plated onto SMsolid medium and grown at 30∘C for two-three days
the absence of H2O2) An even higher sensitivity toH
2O2was
observed in the presence of 2mMH2O2 having their growth
ability reduced of about 85 with respect to control cells inwhich no addition was made The impairment in the abilityto grow under H
2O2stress conditions clearly demonstrates
an impairment in defence capability of the flx1Δ strainInterestingly the same phenotype was observed also in theyeast sdh5Δ [35] sdh1Δ and sdh2Δ [36] strains
To understand whether mitochondrial flavoproteinimpairment due to FLX1 deletion influenced aging in yeastwe carried out measurements of chronological lifespanon both WT and flx1Δ cells cultured at 30∘C in SM liquidmedium supplemented with glucose 2 as carbon source(Figure 3) Following 24 h (1 day) 96 h (4 days) and 168 h(7 days) of growth the number of colonies was determinedby spotting five serial dilutions of the liquid culture andincubating the plates for two-three days at 30∘C The resultsof a typical experiment are reported in Figure 3 A reducednumber of small colonies were counted for the flx1Δ strainwith respect to the number of colonies counted for theWT strain This phenotype particularly evident after 96 hand 168 h of growth time clearly indicated a decrease inchronological lifespan of the flx1Δ strain Essentially thesame phenotype was observed in sdh1Δ and sdh5Δ strains[35] Thus it seems quite clear that a correct biogenesis ofmitochondrial flavoproteome and in particular assembly ofSDH ensures a correct aging rate in yeast When flx1Δ cellswere grown on glycerol they lost the ability to form coloniesfollowing 24 h growth time (data not shown)
6 BioMed Research International
80
6
4
2
05 24
ATP
leve
l
ATP
leve
l
20
16
12
08
04
0
(a998400)
5 5 524
lowast
lowast
(a)WTWT flx1Δ flx1Δ
(nm
olmiddotm
gminus1)
(nm
olmiddotm
gminus1)
20
16
08
12
04
05 24
20
16
12
08
04
0
(b998400)
5 5 524
lowast
(b)flx1WTflx1WT
ROS
leve
l (ΔFmiddot120583
gminus1 )
ROS
leve
l (ΔFmiddot120583
gminus1 )
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
After 5 or 24 h of growth at 30∘C the H2O2sensitivity was
estimated by measuring the A600 nm of the growth culture
26 Malate and Succinate Sensitivity WT and flx1Δ strainswere grown overnight at 30∘C in 5mL YEP liquid mediumsupplemented with glucose 05 up to the early stationaryphaseThen each strain was inoculated in SM liquidmedium(initial A
600 nm equal to 01) containing glucose 2 andsuccinate or malate (5mM) After 24 h of growth at 30∘C theH2O2sensitivity was estimated by measuring the A
600 nm ofthe growth culture
27 Preparation of Spheroplasts Mitochondria and CellularLysates Spheroplasts were prepared using Zymolyase Mito-chondria were isolated from spheroplasts as described in[26] Cellular lysates were obtained by early exponential-phase (5 h) or stationary-phase (24 h) cells harvested bycentrifugation (8000timesg for 5min) washed with sterile waterresuspended in 250 120583L of lysis buffer (10mM Tris-HCl pH76 1mM EDTA 1mM dithiothreitol and 02mM phenyl-methanesulfonyl fluoride supplemented with one tablet ofRoche protease inhibitor cocktail every 10mL of lysis buffer)and vortexed with glass beads for 10min at 4∘C The liquidwas removed and centrifuged at 3000timesg for 5min to removecell debris The protein concentrations of the spheroplastsmitochondria and cellular lysates were assayed according toBradford [29]
28 Quantitation of Flavins ATP and Reactive Oxygen Species(ROS) Rf FMN and FAD content in spheroplasts and SCMwas measured in aliquots (5ndash80120583L) of neutralized perchloricextracts by means of HPLC (Gilson HPLC system includinga model 306 pump and a model 307 pump equipped witha Kontron Instruments SFM 25 fluorometer and Unipointsystem software) essentially as previously described [26]ATP content wasmeasured fluorometrically in cellular lysatesby using the ATP Detecting System essentially as in [30]NADPH formation which corresponds to ATP content(with a 1 1 stoichiometry) was followed with excitationwavelength at 340 nm and emission wavelength at 456 nmROS level was fluorometrically measured on cellular lysatesusing as substrate 21015840-71015840-dichlorofluorescin diacetate (DCF-DA) according to [30] with slight modifications Brieflythe probe DCF-DA (50 120583M) was incubated at 37∘C for 1 hwith 003ndash005mg proteins and converted to fluorescentdichlorofluorescein (DCF) upon reaction with ROS DCFfluorescence of each sample was measured by means ofa LS50S Perkin Elmer spectrofluorometer (excitation andemission wavelengths set at 485 nm and 520 nm resp)
29 Enzymatic Assays Succinate dehydrogenase (SDH EC1351) and fumarase (FUM EC 4212) activities weremeasured as in [26] Glutathione reductase (GR EC1642) activity was spectrophotometrically assayed by mon-itoring the absorbance at 340 nm due to NADPH oxi-dation after glutathione addition (1mM) essentially asin [30] Superoxide dismutase (SOD EC 11511) activity
was spectrophotometrically measured by the xanthine oxi-dasexanthinecytochrome cmethod essentially as describedin [31]
210 Statistical Analysis All experiments were repeated atleast three times with different cell preparations Resultsare presented as mean plusmn standard deviation (SD) Statisticalsignificance was evaluated by Studentrsquos 119905-test Values of 119875 lt005 were considered statistically significant
3 Results
31 Phenotypical and Biochemical Consequences of FLX1Deletion In order to study the relevance of mitochondrialflavin cofactor homeostasis on cellular bioenergetics weintroduced a yeast strain lacking the FLX1 gene encodingthe mitochondrial FAD transporter [26] This deleted strainshowed a small-colony phenotype on both fermentable andnonfermentable carbon sources due to an impairment inthe aerobic respiratory chain pathway [32] The deletedstrain flx1Δ grew normally on glucose medium but failed togrow on nonfermentable carbon sources (ie glycerol) thusindicating a respiration-deficient phenotype (Figure 1(a))The growth defect on nonfermentable carbon source whichwas restored by complementing the deleted strain with theYEpFLX1 plasmid [26] was not rescued by the additionof tricarboxylic acid (TCA) cycle intermediates such assuccinate or malate (Figure 1(a))
Among the mitochondrial flavoenzymes which weredemonstrated to be altered in flx1Δ strain [25 26 28]we showed before [19 32] and confirmed in Figure 1(b)a significant reduced level of the apo-flavoprotein Sdh1presulting in an altered functionality of SDH or complexII of the respiratory chain This reduction was revealedby creating a strain in which three consecutive copies ofthe human influenza hemagglutinin epitope (HA epitopeYPYDVPDYA) were fused in frame to the 31015840end of the SDH1ORF in the genome of both the WT and flx1Δ strains Thechimera protein namely Sdh1-HAp carrying the HA-tag atthe C-terminal end of Sdh1p lost the ability to covalentlybind the flavin cofactor FAD [19 33] but not its regulatorybehaviour that is its inducible expression in galactose or innonfermentable carbon sources In all the growth conditionstested the FAD-independent fumarase (FUM) activity usedas a control was not affected by FLX1 deletion (see histogramin Figure 1(b))
A significant decrease of Sdh1-HAp level was accom-panied in galactose but not in glycerol by a profoundderangement of flavin cofactors particularly evident in cellgrown at the early exponential phase (Table 1) in agreementwith [25 26] respectively The reason for these carbonsource-dependent flavin level changes which is not easilyexplainable is addressed in Section 4
Consistent with an altered functionality of SDH the flx1Δstrain also showed impaired isolated mitochondria oxygenconsumption activity specifically detectable when succinatewas used as a respiratory substrate [19] Similar phenotypewas also observed in yeast strains carrying either a deletion
4 BioMed Research International
8
6
4
2
0
Glucose GlycerolCTR WTCTR flx1Δ
WT flx1Δ WT flx1Δ
A600
nm
+Mal 5mM+Succ 5mM
(a)FU
M sp
ecifi
c act
ivity
250
200
150
100
50
Glycerol Galactose
Glycerol Galactose
0
Act1p
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
Sdh1-HAp
WT-HA flx1Δ-HA WT-HA flx1Δ-HA
(nm
olmiddotminminus1middotm
gminus1)
(b)
Figure 1 (a) Respiratory-deficient phenotype of flx1Δ strain effect of succinate and malate addition WT and flX1Δ cells were cultured at30∘C in YEP liquidmedium supplemented with either glucose or glycerol (2 each) as carbon sourceWhere indicated either 5mM succinate(Succ) or 5mM malate (Mal) was added Cell growth was estimated at the stationary phase (24 h) by measuring the absorbance at 600 nm(119860600 nm) of a ten-fold dilution of each growth culture consistently corrected for the dilution factor The values reported in the histogram
are the means (plusmnSD) of three experiments (b) Changes in the recombinant Sdh1-HAp level in flx1Δ strain Cellular lysates were preparedfromWT-HA and flX1Δ-HA cells grown at 30∘C up to the exponential growth phase (5 h) in YEP liquid medium supplemented with eitherglycerol or galactose (2 each) as carbon source Proteins from cellular lysates (005mg) were separated by SDSPAGE and transferred ontoa PVDF membrane In each extract Sdh1-HA protein was detected by using an 120572-HA and its amount was densitometrically evaluated Thevalues reported in the histogram are the means (plusmnSD) of three experiments performed with different cellular lysates preparations Statisticalevaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005) As a control the specific activity of the enzyme fumarase (FUM) wasdetermined in each cellular lysate preparation
BioMed Research International 5
Table 1 Endogenous flavin content in spheroplasts and mitochondria
Spheroplasts and mitochondria (SCM) were prepared fromWT and 1198911198971199091Δ cells grown in glycerol or galactose (2) up to the exponential growth phase (5 h)FAD and FMN content was determined in neutralized perchloric acid extracts as described inMaterials andMethods Riboflavin amount was not relevant andthus its value has not been reportedThemeans (plusmnSD) of the flavin endogenous content determined in three experiments performedwith different preparationsare reported ∘Data published in (Bafunno et al 2004) [26] statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
120
100
80
60
40
20
05 24
Glucose Glycerol
120
100
80
60
40
20
05 24 5 24 5 24
( o
f eac
h co
ntro
l)
( o
f eac
h co
ntro
l)
flx1Δ flx1ΔWTWT
A600
nm
A600
nm
+H2O2 005mM+H2O2 2mM
Figure 2 Sensitivity to H2O2 WT and flX1Δ cells were cultured
at 30∘C in YEP liquid medium supplemented with either glucoseor glycerol (2 each) as carbon source Where indicated H
2O2at
the indicated concentration was added Cell growth was estimatedat the exponential (5 h) and stationary phase (24 h) by measuringthe absorbance at 600 nm (119860
600 nm) In the histogram the 119860600 nm
of the cell cultures grown in the presence of H2O2is reported as
a percentage of the control (ie the 119860600 nm of cell cultures grown
in the absence of H2O2 set arbitrary equal to 100) The values
reported in the histogram are themeans (plusmnSD) of three experiments
of SDH1 [34] or a deletion of SDH5 which encodes amitochondrial protein involved in Sdh1p flavinylation [35]Another respiration-related phenotype of flx1Δ strain wasinvestigated in Figure 2 by testing H
2O2hypersensitivity
of cells grown on both fermentable and nonfermentablecarbon sources In glucose the WT cells grew up to thestationary phase (24 h) in the presence of H
2O2(005 or
2mM) essentially as the control cells grown in the absence ofH2O2 In glycerol their ability to grow up to 24 hwas reduced
of about 20 at 005mM H2O2and of 60 at 2mM with
respect to the control cells in which no H2O2was added
In glucose flx1Δ cells did not showH2O2hypersensitivity
at 005mM At 2mM H2O2 their ability to grow was
significantly reduced (of about 85) with respect to flx1Δcells grown in the absence of H
2O2 The ability of the flx1Δ
cells to grow in glycerol which was per se drastically reducedby deletion was reduced at 24 h by the addition of 005mMH2O2(about 50 with respect to the control cells grown in
24h
96h
168h
flx1ΔWT
Figure 3 Chronological lifespan determination WT and flX1Δstrains were cultured in SM liquid medium at 30∘C Dilutions fromeach culture containing about 200 cells (as calculated from 119860
600 nmby taking into account that one 119860
600 nm is equivalent to 3 times 107cellmL) were harvested after 24 96 and 168 h and plated onto SMsolid medium and grown at 30∘C for two-three days
the absence of H2O2) An even higher sensitivity toH
2O2was
observed in the presence of 2mMH2O2 having their growth
ability reduced of about 85 with respect to control cells inwhich no addition was made The impairment in the abilityto grow under H
2O2stress conditions clearly demonstrates
an impairment in defence capability of the flx1Δ strainInterestingly the same phenotype was observed also in theyeast sdh5Δ [35] sdh1Δ and sdh2Δ [36] strains
To understand whether mitochondrial flavoproteinimpairment due to FLX1 deletion influenced aging in yeastwe carried out measurements of chronological lifespanon both WT and flx1Δ cells cultured at 30∘C in SM liquidmedium supplemented with glucose 2 as carbon source(Figure 3) Following 24 h (1 day) 96 h (4 days) and 168 h(7 days) of growth the number of colonies was determinedby spotting five serial dilutions of the liquid culture andincubating the plates for two-three days at 30∘C The resultsof a typical experiment are reported in Figure 3 A reducednumber of small colonies were counted for the flx1Δ strainwith respect to the number of colonies counted for theWT strain This phenotype particularly evident after 96 hand 168 h of growth time clearly indicated a decrease inchronological lifespan of the flx1Δ strain Essentially thesame phenotype was observed in sdh1Δ and sdh5Δ strains[35] Thus it seems quite clear that a correct biogenesis ofmitochondrial flavoproteome and in particular assembly ofSDH ensures a correct aging rate in yeast When flx1Δ cellswere grown on glycerol they lost the ability to form coloniesfollowing 24 h growth time (data not shown)
6 BioMed Research International
80
6
4
2
05 24
ATP
leve
l
ATP
leve
l
20
16
12
08
04
0
(a998400)
5 5 524
lowast
lowast
(a)WTWT flx1Δ flx1Δ
(nm
olmiddotm
gminus1)
(nm
olmiddotm
gminus1)
20
16
08
12
04
05 24
20
16
12
08
04
0
(b998400)
5 5 524
lowast
(b)flx1WTflx1WT
ROS
leve
l (ΔFmiddot120583
gminus1 )
ROS
leve
l (ΔFmiddot120583
gminus1 )
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Figure 1 (a) Respiratory-deficient phenotype of flx1Δ strain effect of succinate and malate addition WT and flX1Δ cells were cultured at30∘C in YEP liquidmedium supplemented with either glucose or glycerol (2 each) as carbon sourceWhere indicated either 5mM succinate(Succ) or 5mM malate (Mal) was added Cell growth was estimated at the stationary phase (24 h) by measuring the absorbance at 600 nm(119860600 nm) of a ten-fold dilution of each growth culture consistently corrected for the dilution factor The values reported in the histogram
are the means (plusmnSD) of three experiments (b) Changes in the recombinant Sdh1-HAp level in flx1Δ strain Cellular lysates were preparedfromWT-HA and flX1Δ-HA cells grown at 30∘C up to the exponential growth phase (5 h) in YEP liquid medium supplemented with eitherglycerol or galactose (2 each) as carbon source Proteins from cellular lysates (005mg) were separated by SDSPAGE and transferred ontoa PVDF membrane In each extract Sdh1-HA protein was detected by using an 120572-HA and its amount was densitometrically evaluated Thevalues reported in the histogram are the means (plusmnSD) of three experiments performed with different cellular lysates preparations Statisticalevaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005) As a control the specific activity of the enzyme fumarase (FUM) wasdetermined in each cellular lysate preparation
BioMed Research International 5
Table 1 Endogenous flavin content in spheroplasts and mitochondria
Spheroplasts and mitochondria (SCM) were prepared fromWT and 1198911198971199091Δ cells grown in glycerol or galactose (2) up to the exponential growth phase (5 h)FAD and FMN content was determined in neutralized perchloric acid extracts as described inMaterials andMethods Riboflavin amount was not relevant andthus its value has not been reportedThemeans (plusmnSD) of the flavin endogenous content determined in three experiments performedwith different preparationsare reported ∘Data published in (Bafunno et al 2004) [26] statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
120
100
80
60
40
20
05 24
Glucose Glycerol
120
100
80
60
40
20
05 24 5 24 5 24
( o
f eac
h co
ntro
l)
( o
f eac
h co
ntro
l)
flx1Δ flx1ΔWTWT
A600
nm
A600
nm
+H2O2 005mM+H2O2 2mM
Figure 2 Sensitivity to H2O2 WT and flX1Δ cells were cultured
at 30∘C in YEP liquid medium supplemented with either glucoseor glycerol (2 each) as carbon source Where indicated H
2O2at
the indicated concentration was added Cell growth was estimatedat the exponential (5 h) and stationary phase (24 h) by measuringthe absorbance at 600 nm (119860
600 nm) In the histogram the 119860600 nm
of the cell cultures grown in the presence of H2O2is reported as
a percentage of the control (ie the 119860600 nm of cell cultures grown
in the absence of H2O2 set arbitrary equal to 100) The values
reported in the histogram are themeans (plusmnSD) of three experiments
of SDH1 [34] or a deletion of SDH5 which encodes amitochondrial protein involved in Sdh1p flavinylation [35]Another respiration-related phenotype of flx1Δ strain wasinvestigated in Figure 2 by testing H
2O2hypersensitivity
of cells grown on both fermentable and nonfermentablecarbon sources In glucose the WT cells grew up to thestationary phase (24 h) in the presence of H
2O2(005 or
2mM) essentially as the control cells grown in the absence ofH2O2 In glycerol their ability to grow up to 24 hwas reduced
of about 20 at 005mM H2O2and of 60 at 2mM with
respect to the control cells in which no H2O2was added
In glucose flx1Δ cells did not showH2O2hypersensitivity
at 005mM At 2mM H2O2 their ability to grow was
significantly reduced (of about 85) with respect to flx1Δcells grown in the absence of H
2O2 The ability of the flx1Δ
cells to grow in glycerol which was per se drastically reducedby deletion was reduced at 24 h by the addition of 005mMH2O2(about 50 with respect to the control cells grown in
24h
96h
168h
flx1ΔWT
Figure 3 Chronological lifespan determination WT and flX1Δstrains were cultured in SM liquid medium at 30∘C Dilutions fromeach culture containing about 200 cells (as calculated from 119860
600 nmby taking into account that one 119860
600 nm is equivalent to 3 times 107cellmL) were harvested after 24 96 and 168 h and plated onto SMsolid medium and grown at 30∘C for two-three days
the absence of H2O2) An even higher sensitivity toH
2O2was
observed in the presence of 2mMH2O2 having their growth
ability reduced of about 85 with respect to control cells inwhich no addition was made The impairment in the abilityto grow under H
2O2stress conditions clearly demonstrates
an impairment in defence capability of the flx1Δ strainInterestingly the same phenotype was observed also in theyeast sdh5Δ [35] sdh1Δ and sdh2Δ [36] strains
To understand whether mitochondrial flavoproteinimpairment due to FLX1 deletion influenced aging in yeastwe carried out measurements of chronological lifespanon both WT and flx1Δ cells cultured at 30∘C in SM liquidmedium supplemented with glucose 2 as carbon source(Figure 3) Following 24 h (1 day) 96 h (4 days) and 168 h(7 days) of growth the number of colonies was determinedby spotting five serial dilutions of the liquid culture andincubating the plates for two-three days at 30∘C The resultsof a typical experiment are reported in Figure 3 A reducednumber of small colonies were counted for the flx1Δ strainwith respect to the number of colonies counted for theWT strain This phenotype particularly evident after 96 hand 168 h of growth time clearly indicated a decrease inchronological lifespan of the flx1Δ strain Essentially thesame phenotype was observed in sdh1Δ and sdh5Δ strains[35] Thus it seems quite clear that a correct biogenesis ofmitochondrial flavoproteome and in particular assembly ofSDH ensures a correct aging rate in yeast When flx1Δ cellswere grown on glycerol they lost the ability to form coloniesfollowing 24 h growth time (data not shown)
6 BioMed Research International
80
6
4
2
05 24
ATP
leve
l
ATP
leve
l
20
16
12
08
04
0
(a998400)
5 5 524
lowast
lowast
(a)WTWT flx1Δ flx1Δ
(nm
olmiddotm
gminus1)
(nm
olmiddotm
gminus1)
20
16
08
12
04
05 24
20
16
12
08
04
0
(b998400)
5 5 524
lowast
(b)flx1WTflx1WT
ROS
leve
l (ΔFmiddot120583
gminus1 )
ROS
leve
l (ΔFmiddot120583
gminus1 )
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Spheroplasts and mitochondria (SCM) were prepared fromWT and 1198911198971199091Δ cells grown in glycerol or galactose (2) up to the exponential growth phase (5 h)FAD and FMN content was determined in neutralized perchloric acid extracts as described inMaterials andMethods Riboflavin amount was not relevant andthus its value has not been reportedThemeans (plusmnSD) of the flavin endogenous content determined in three experiments performedwith different preparationsare reported ∘Data published in (Bafunno et al 2004) [26] statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
120
100
80
60
40
20
05 24
Glucose Glycerol
120
100
80
60
40
20
05 24 5 24 5 24
( o
f eac
h co
ntro
l)
( o
f eac
h co
ntro
l)
flx1Δ flx1ΔWTWT
A600
nm
A600
nm
+H2O2 005mM+H2O2 2mM
Figure 2 Sensitivity to H2O2 WT and flX1Δ cells were cultured
at 30∘C in YEP liquid medium supplemented with either glucoseor glycerol (2 each) as carbon source Where indicated H
2O2at
the indicated concentration was added Cell growth was estimatedat the exponential (5 h) and stationary phase (24 h) by measuringthe absorbance at 600 nm (119860
600 nm) In the histogram the 119860600 nm
of the cell cultures grown in the presence of H2O2is reported as
a percentage of the control (ie the 119860600 nm of cell cultures grown
in the absence of H2O2 set arbitrary equal to 100) The values
reported in the histogram are themeans (plusmnSD) of three experiments
of SDH1 [34] or a deletion of SDH5 which encodes amitochondrial protein involved in Sdh1p flavinylation [35]Another respiration-related phenotype of flx1Δ strain wasinvestigated in Figure 2 by testing H
2O2hypersensitivity
of cells grown on both fermentable and nonfermentablecarbon sources In glucose the WT cells grew up to thestationary phase (24 h) in the presence of H
2O2(005 or
2mM) essentially as the control cells grown in the absence ofH2O2 In glycerol their ability to grow up to 24 hwas reduced
of about 20 at 005mM H2O2and of 60 at 2mM with
respect to the control cells in which no H2O2was added
In glucose flx1Δ cells did not showH2O2hypersensitivity
at 005mM At 2mM H2O2 their ability to grow was
significantly reduced (of about 85) with respect to flx1Δcells grown in the absence of H
2O2 The ability of the flx1Δ
cells to grow in glycerol which was per se drastically reducedby deletion was reduced at 24 h by the addition of 005mMH2O2(about 50 with respect to the control cells grown in
24h
96h
168h
flx1ΔWT
Figure 3 Chronological lifespan determination WT and flX1Δstrains were cultured in SM liquid medium at 30∘C Dilutions fromeach culture containing about 200 cells (as calculated from 119860
600 nmby taking into account that one 119860
600 nm is equivalent to 3 times 107cellmL) were harvested after 24 96 and 168 h and plated onto SMsolid medium and grown at 30∘C for two-three days
the absence of H2O2) An even higher sensitivity toH
2O2was
observed in the presence of 2mMH2O2 having their growth
ability reduced of about 85 with respect to control cells inwhich no addition was made The impairment in the abilityto grow under H
2O2stress conditions clearly demonstrates
an impairment in defence capability of the flx1Δ strainInterestingly the same phenotype was observed also in theyeast sdh5Δ [35] sdh1Δ and sdh2Δ [36] strains
To understand whether mitochondrial flavoproteinimpairment due to FLX1 deletion influenced aging in yeastwe carried out measurements of chronological lifespanon both WT and flx1Δ cells cultured at 30∘C in SM liquidmedium supplemented with glucose 2 as carbon source(Figure 3) Following 24 h (1 day) 96 h (4 days) and 168 h(7 days) of growth the number of colonies was determinedby spotting five serial dilutions of the liquid culture andincubating the plates for two-three days at 30∘C The resultsof a typical experiment are reported in Figure 3 A reducednumber of small colonies were counted for the flx1Δ strainwith respect to the number of colonies counted for theWT strain This phenotype particularly evident after 96 hand 168 h of growth time clearly indicated a decrease inchronological lifespan of the flx1Δ strain Essentially thesame phenotype was observed in sdh1Δ and sdh5Δ strains[35] Thus it seems quite clear that a correct biogenesis ofmitochondrial flavoproteome and in particular assembly ofSDH ensures a correct aging rate in yeast When flx1Δ cellswere grown on glycerol they lost the ability to form coloniesfollowing 24 h growth time (data not shown)
6 BioMed Research International
80
6
4
2
05 24
ATP
leve
l
ATP
leve
l
20
16
12
08
04
0
(a998400)
5 5 524
lowast
lowast
(a)WTWT flx1Δ flx1Δ
(nm
olmiddotm
gminus1)
(nm
olmiddotm
gminus1)
20
16
08
12
04
05 24
20
16
12
08
04
0
(b998400)
5 5 524
lowast
(b)flx1WTflx1WT
ROS
leve
l (ΔFmiddot120583
gminus1 )
ROS
leve
l (ΔFmiddot120583
gminus1 )
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Figure 4 Bioenergetic and redox impairment in flx1Δ strain ATP and ROS content Cellular lysates were prepared from WT and flx1Δmutant strains grown in glycerol ((a) (b)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840)) up to theexponential phase (5 h) ATP content ((a) (a1015840)) was enzymatically determined following perchloric acid extraction and neutralization ROScontent ((b) (b1015840)) was fluorometrically measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) ofthree experiments performed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test(lowast119875 lt 005)
In order to correlate the observed phenotype with animpairment of cellular bioenergetics we compared the ATPcontent and the ROS amount of the flx1Δ strain with that ofthe WT In Figure 4 panel (a) the ATP cellular content wasenzymatically measured in neutralized perchloric extractsprepared from WT and flx1Δ cells grown on glycerol Atthe exponential growth phase (5 h) a significant reductionwas detected in the flx1Δ cells in comparison with theWT (021 versus 105 nmolsdotmgminus1 protein) At the stationarygrowth phase (24 h) the ATP content increased significantlyin WT cells (34 nmolsdotmgminus1 protein) and even more in thedeleted strain (52 nmolsdotmgminus1 protein)The temporary severedecrease in ATP content induced by the absence of Flx1p wasnot observed in glucose-grown cells (Figure 4 panel (a1015840)) asexpected when fermentation is themain way to produce ATP
FLX1 deletion induced also a significant increase inthe amount of ROS (135 with respect to the WT cells)as estimated with the fluorescent dye DCFH-DA on thecellular lysates prepared from cells grown in glycerol up tothe exponential growth phase (Figure 4 panel (b)) At thestationary phase the flx1Δ cells presented almost the sameROS amount measured in the WT cells (Figure 4 panel (b))In glucose-grown cells the amount of cellular ROS in theflx1Δ strain was not significantly changed with respect to theWT (Figure 4 Panel (b1015840)) as expected when a mitochondrialdamage is the major cause of ROS unbalance
In line with the unique role of flavin cofactor in oxygenmetabolism and ROS defence systems [20 30 37 38] wefurther investigated whether the impairment of the ROS levelin glycerol-grown flx1Δ strain was due to a derangement inenzymes involved in ROS detoxification such as the flavo-protein glutathione reductase (GR) or the FAD-independent
superoxide dismutase (SOD) their specific enzymatic activ-ities were measured in cellular lysates from WT and flx1Δcells grown on glycerol and glucose while assaying the FAD-independent enzyme FUM as control (Figure 5) Figure 5panel (a) shows a significant increase in GR specific activityin flx1Δ strain (65) at the exponential growth phase withrespect to that measured in WT The GR specific activityin the flx1Δ reached the same value measured in the WTcells (about 35 nmolsdotmgminus1 protein) at the stationary phase Incells grown in glucose up to the exponential growth phase(Figure 5 panel (a1015840)) a slight but not significant reductionin GR specific activity was detected in the flx1Δ strain withrespect to the WT (25 versus 31 nmolsdotmgminus1 protein)
As regards SOD in the glycerol-grown flx1Δ cells after 5 hgrowth time (Figure 5 panel (b)) the SOD specific activitywas significantly higher than the value measured in the WTcells (16 versus 9 standard Usdotmgminus1) At the stationary phasethe SOD specific activity in the flx1Δ significantly decreasedreaching a value of 66 standard Usdotmgminus1 that is about two-fold lower than the SOD specific activity measured in WTcells In glucose-grown cells after 5 h growth time (Figure 5panel (b1015840)) a slight but significant reduction in SOD specificactivity can be detected in the flx1Δ strain with respect tothe WT (92 versus 122 nmolsdotmgminus1 protein) This reductionmight be explained by a defect in FAD dependent proteinfolding as previously observed in [30 39]
In all the growth conditions tested the FUMactivity usedas a control was not affected by FLX1 deletion (Figure 5panels (c) and (c1015840))
32 The Role of Flx1p in a Retrograde Cross-Talk ResponseRegulating Cell Defence and Lifespan Results described in
BioMed Research International 7
GR
spec
ific a
ctiv
ity
GR
spec
ific a
ctiv
ity
60
50
40
30
20
10
0
60
50
40
30
20
10
05 24 5 24 5 24WT WT
(a) (a998400 )
lowast
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
20
16
12
4
8
0
20
16
12
4
5 55 5
8
0
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
SOD
spec
ific a
ctiv
ity(s
tand
ard
unitmiddot
mgminus1)
24 24WTWT
(b) (b998400 )
lowast
lowastlowast
flx1Δ flx1ΔFU
M sp
ecifi
c act
ivity
FUM
spec
ific a
ctiv
ity
250
200
150
100
50
0
50
40
30
20
10
055 5 52424
WT WT(c) (c998400 )
flx1Δ flx1Δ
(nm
olmiddotminminus1middotm
gminus1)
(nm
olmiddotminminus1middotm
gminus1)
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Figure 5 GR and SOD activities in flx1Δ strain Cellular lysates were prepared fromWT and flx1Δmutant strains grown in glycerol ((a) (b)and (c)) up to either the exponential (5 h) or the stationary phase (24 h) or in glucose ((a1015840) (b1015840) and (c1015840)) up to the exponential phase (5 h) GR((a) (a1015840)) and SOD ((b) (b1015840)) specific activities were spectrophotometrically determined as described in Section 2 As control FUM specificactivity ((c) (c1015840)) was measured as described in Section 2 The values reported in the histograms are the means (plusmnSD) of three experimentsperformed with different cellular lysate preparations Statistical evaluation was carried out according to Studentrsquos 119905-test (lowast119875 lt 005)
the previous paragraph strengthen the relevance of Flx1p inensuring cell defence and correct aging by maintaining thehomeostasis of mitochondrial flavoproteome As concernsSDH in [19] we gained some insight into the mechanism bywhich Flx1p could regulate Sdh1p apo-protein expression asdue to a control that involves regulatory sequences locatedupstream of the SDH1 coding sequence (as reviewed in[40])
To gain further insight into this mechanism we searchedhere for elements that could be relevant in modulating Sdh1pexpression in response to alteration in flavin cofactor home-ostasis Therefore first we searched for cis-acting elements inthe regulatory regions located upstream of the SDH1 ORFfirst of all in the 51015840UTR region as defined by [41] whichcorresponds to the first 71 nucleotides before the start codonof SDH1 ORF No consensus motifs were found in thisregion by using the bioinformatic tool ldquoYeast ComparativeGenomicsmdashBroad Instituterdquo [42] Indeed it should be notedthat no further information is at the moment available on theactual length of the 51015840UTR of SDH1
Thus we extended our analysis along the 1 kbp upstreamregion of SDH1 ORF and we found twelve consensus motifsthat could bind regulatory proteins six of which are ofunknown function Among these motifs summarised inTable 2 the most relevant at least in the scenario described
by our experiments seemed to be a motif which is located atminus80 nucleotides upstream the start codon of SDH1 ORF andnamely motif 29 (consensus sequence shRCCCYTWDt)that perfectly overlaps with motif 38 (consensus sequenceCTCCCCTTAT) This motif is also present in the upstreamregion of the mitochondrial flavoprotein ARH1 involved inubiquinone biosynthesis [28] but not in that of flavoproteinLPD1 and COQ6 [25 26 28] Interestingly this motif 29is also present in the upstream regions of the membersof the machinery that maintained Rf homeostasis that isthe mitochondrial FAD transporter FLX1 [25] the FADforming enzyme FAD1 [25] and the Rf translocator MCH5[22] Moreover this motif is also present in the upstreamregulatory region of the mitochondrial isoenzyme SOD2 butnot in the cytosolic one SOD1 and in one of the five nuclearsuccinate sensitive JmjC-domain-containing demethylasesthat is RPH1 [43] According to [42] this motif is bound bytranscription factor Msn2p and its close homologue Msn4p(referred to as Msn24p) which under nonstress conditionsare located in the cytoplasm Upon different stress condi-tions among which oxidative stress Msn24p are hyper-phosphorylated and shuttled from the cytosol to the nucleus[44] The pivotal role played by Msn24p in chronologicallifespan in yeast was first discovered by [45] and recentlyexhaustively reviewed by [46]
8 BioMed Research International
C
-AA(n)A-3998400
PDH
Posttrancriptionalcontrol
Transcriptional controlEpigenetic control
Rox1p
GTP + RIBULOSE-5P
Rib 1-57p
Rf
Rf
Mch5p
ADP
ADP
AMPATP
ATP ATP
ATP
Fmn1p
Fmn1p FMN
PPi
PPi
Fad1p FAD
Msn24p
JmjC
IM
OM
Rf
mt-FADS
H2OFMN
RfT
FAD
FAD
FAD
FAD
FAD
FAD
Sdh5p
Sdh5pFlavinylation
Sdh1p
Processing
Sdh2p
Sdh2p
TMP62 Sdh6pSdh3p Sdh4p
Sdh3p Sdh4p
AssemblyTCAcycle
Fumarate
CRATPROS
TOMcomplex
TOM20
Dic1p
SDH1 mRNA
I
()
5998400-m7GppN-
TIMcomplex
X
Succinate
Succinate
flx1p flx1p
H2N
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Figure 6 A possible correlation between mitochondrial FAD homeostasis and chronological lifespan The scheme summarizes resultsfrom studies described in this and other papers [17 19 22 26 35 36 40 50 53] Mch5p plasma membrane Rf transporter Rib1-57penzymes involved in Rf de novo biosynthesis Rf
FAD synthase Flx1p mitochondrial FAD exporter I FAD pyrophosphatase Sdh1p succinate dehydrogenase flavoprotein subunit Sdh5pprotein required for Sdh1p flavinylation Sdh234p other subunits of succinate dehydrogenase complex Tmp62pSdh6p factors requiredfor SDH complex assembly TCA cycle tricarboxylic acid cycle TOM complexTIM complex proteins involved in mitochondrial proteinimportDic1pmitochondrial dicarboxylic acid carrier PDH prolyl hydroxylase JmjC JmjC-domain-containing demethylases Rox1p heme-dependent repressor of hypoxic genes Msn24p transcriptional factors activated in stress conditions
A further comparison between the 51015840UTRs of SDH1and of proteins involved in FAD homeostasis revealedanother common motif of unknown function located atndash257 nucleotides upstream the start codon of SDH1 ORF
namely the motif 14 (consensus sequence YCTATTGTT)[42] Besides SDH1 this motif is also present in the upstreamregion of MCH5 and its homologue MCH4 in FAD1 andalso in a number of mitochondrial flavoproteins including
BioMed Research International 9
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
Table 2 List of motifs localized in the 1000 nucleotides upstream region of SDH1 ORF and identified by enriched conservation among allSaccharomyces species genome using the ldquoYeast Comparative GenomicsmdashBroad Instituterdquo database
Number Motif Number of ORFs Binding factor Function2 RTTACCCGRM 865 Reb1 RNA polymerase I enhancer binding protein14 YCTATTGTT 561 Unknown 26 DCGCGGGGH 285 Mig1 Involved in glucose repression29 hRCCCYTWDt 442 Msn24 Involved in stress conditions38 CTCCCCTTAT 218 Msn24 Involved in stress conditions39 GCCCGG 152 Unknown Filamentation41 CTCSGCS 77 Unknown 47 TTTTnnnnnnnnnnnngGGGT 359 Unknown 57 CGGCnnMGnnnnnnnCGC 84 Gal4 Involved in galactose induction61 GKBAGGGT 363 TBF1 Telobox-containing general regulatory factor63 GGCSnnnnnGnnnCGCG 80 mbp1-like Involved in regulation of cell cycle progression from G1 to S70 CGCGnnnnnGGGS 156 Unknown
HEM14 NDI1 and NCP1 The binding factor and thefunctional role of the motif 14 have not yet annotated inldquoYeast Comparative GenomicsmdashBroad Instituterdquo (Table 2)Searching in the biological database ldquoBiobase-Gene-regulation-Transfacrdquo we found that this motif is reported asbound by Rox1p (YPR065W a heme-dependent repressor ofhypoxic genesmdashSGD information) Rox1p is involved in theregulation of the expression of proteins involved in oxygen-dependent pathways such as respiration heme and sterolsbiosynthesis [47]Thus SDH1 expression is downregulated inrox1Δ strain under aerobiosis [47] This finding strengthensthe well-described relationship between oxygenhememetabolism and flavoproteins [18 37] A possible involve-ment of this transcriptional pathway in the scenario depictedby deletion of FLX1 remains at the moment only speculative
4 Discussion
This paper deals with the role exerted by the mitochondrialtranslocator Flx1p in the efficiency of ATP production ROShomeostasis H
2O2sensitivity and chronological lifespan
in S cerevisiae starting from the previous demonstrationsof the derangements in specific mitochondrial flavoproteinswhich are crucial for mitochondrial bioenergetics includingCoq6p [28] Lpd1p and Sdh1p [19 25 26] The alteration inSdh1p expression level in different carbon source is confirmedhere (Figure 1) and it is accompanied by an alteration inflavin cofactor amount in galactose but not in glycerol-growncells (Table 1) in agreement with [19 25] respectively Inthe attempt to rationalize the reason for the carbon sourcedependence of the flavin level changes we hypothesizeddifferent subcellular localization for Fad1p in response tocarbon sources Experiments are going on in our laboratoryto evaluate this possibility
The flx1Δ strain showed impaired succinate-dependentoxygen consumption [19] Since no reduction in the oxygenconsumption rate was found by using alternative substratessuch as NADH or glycerol 3-phosphate possible defectsin the ubiquinone or heme biosynthesis [28] could not be
relevant for mitochondrial respiration at least under thisnonstress condition
To evaluate the consequences of FLX1 deletion on bioen-ergetics and cellular redox balance the ATP content andROS level (Figure 4) were compared inWT and flx1Δ strainsaccompanied by measurements of the enzymatic activitiesof GR and SOD enzymes involved in ROS detoxification(Figure 5) ATP shortage and ROS unbalance were observedin flx1Δ cells grown in glycerol up to the exponential growthphase but not in cells grown in glycerol up to the stationaryphase or in glucose The findings are in agreement with themitochondrial origin of these biochemical parameters Moreimportantly the observation that lifespan was changed inglucose (not accompanied by a detectable ROS unbalance)allows us to propose that the lifespan shortage inducedby the mitochondrial alteration due to absence of FLX1gene (correlated to flavoprotein impairment) may act alsoindependently of ROS level increase
The flx1Δ strain showed also H2O2hypersensitivity
(Figure 2) Since the same respiratory-deficient phenotypewas previously observed in the yeast strain sdh1Δ and sdh5Δstrains [35] these results could be explained by the incapa-bility of the flx1Δ strain to increase the amount of Sdh1p inresponse to oxidative stress
In this paper for the first time a correlation betweendeletion of FLX1 and altered chronological lifespan wasreported (Figure 3) A similar phenotype was also previouslydemonstrated for sdh5Δ strains [35]Thus it seems quite clearthat a correct biogenesis ofmitochondrial flavoproteome andin particular assembly of SDH ensures a correct aging ratein yeast This conclusion is also consistent with the recentobservations made in another model organism that is Celegans in which the FAD forming enzyme FADS coded byflad-1 gene was silenced [30 48]
To understand the molecular mechanism by which FADhomeostasis derangement and flavoproteome level mainte-nance are correlated a bioinformatic analysis was performedwhich revealed at least two cis-acting motifs which arelocated in the upstream region of genes encoding SDH1other mitochondrial flavoproteins and some members of
10 BioMed Research International
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
the machinery that maintain cellular FAD homeostasisTherefore the analysis describes the ability of yeast cells toimplement under H
2O2stress condition and aging a strategy
of gene expression coordinating flavin cofactor homeostasiswith the biogenesis of a number of mitochondrial flavoen-zymes involved in various aspects of metabolism rangingfrom oxidative phosphorylation to heme and ubiquinonebiosynthesis Even though no experimental evidence stillexists to test the direct involvement of these cis-acting motifsin flavin-dependent cell defence and chronological lifespantheir involvement in the scenario depicted by deletion ofFLX1 appeared to be a fascinating purpose to be pursuedExperiments in this direction are at the moment going on inour laboratory
In [19] we demonstrated that the early-onset change inapo-Sdh1p content observed in the flx1Δ strain appearedconsistent with a posttranscriptional control exerted by Flx1pas depicted in Figure 6 Thus an inefficient translation ofSDH1-mRNA is expected in flx1Δ strain due to the posttran-scriptional control [19] evenwhen putativemRNA levelsmaychange in response to cell stress andor aging In this pathwaythe transcription factors Msn24p and Rox1p could play acrucial role
Moreover scheme in Figure 6 outlines how FLX1 dele-tion causing a change in expression level of Sdh1p couldactivate a sort of retrograde cross-talk directed to nucleusIn our hypothesis besides ROS increase a key moleculemediating nucleus-mitochondrion cross-talk should be theTCA cycle intermediate succinate whose amount is expectedto increase when altering the activity of SDH The increasedamount of succinate in turn may alter the activity of the120572-ketoglutarate- and Fe(II)-depending dioxygenases amongwhich there are (i) the JmjC-domain-containing demethy-lases [36] which may be causative of epigenetic events at thebasis of precocious aging (for an exhaustive review on thispoint see [49]) and (ii) the prolyl hydroxylase (PDH) whichmay mimic a hypoxia condition in the cell [50]
5 Conclusions
Here we prove that in S cerevisiae deletion of the mito-chondrial translocator FLX1 results in H
2O2hypersensitivity
and altered chronological lifespan which is associated withATP shortage and ROS unbalance in nonfermentable carbonsourceWe propose that this yeast phenotype is correlated to areduced ability to maintain an appropriate level of succinatedehydrogenase flavoprotein subunit [19] which in turn caneither derange epigenetic regulation or mimic a hypoxic con-dition Thus flx1Δ strain provides a useful model system forstudying human aging and degenerative pathologic conditionassociated with alteration in flavin homeostasis which can berestored by Rf treatment [51 52]
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from PON-Ricerca eCompetitivita 2007ndash2013 (PON Project 01 00937 ldquoModelliSperimentali Biotecnologici Integrati per la Produzione edil Monitoraggio di Biomolecole di Interesse per la SalutedellrsquoUomordquo) to M Barile The authors thank Dr A M SLezza for her critical reading of the paper The excellenttechnical assistance of V Giannoccaro is gratefully acknowl-edged
References
[1] V Joosten and W J van Berkel ldquoFlavoenzymesrdquo CurrentOpinion in Chemical Biology vol 11 no 2 pp 195ndash202 2007
[2] P MacHeroux B Kappes and S E Ealick ldquoFlavogenomicsmdasha genomic and structural view of flavin-dependent proteinsrdquoFEBS Journal vol 278 no 15 pp 2625ndash2634 2011
[3] S Hino A Sakamoto K Nagaoka et al ldquoFAD-dependentlysine-specific demethylase-1 regulates cellular energy expendi-turerdquo Nature Communications vol 3 article 758 2012
[4] B R Selvi D V Mohankrishna Y B Ostwal and T KKundu ldquoSmall molecule modulators of histone acetylation andmethylation a disease perspectiverdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 10-12 pp810ndash828 2010
[5] R H Houtkooper E Pirinen and J Auwerx ldquoSirtuins asregulators of metabolism and healthspanrdquo Nature ReviewsMolecular Cell Biology vol 13 no 4 pp 225ndash238 2012
[6] H J Powers ldquoRiboflavin (vitamin B-2) and healthrdquo The Amer-ican Journal of Clinical Nutrition vol 77 no 6 pp 1352ndash13602003
[7] R Horvath ldquoUpdate on clinical aspects and treatment ofselected vitamin-responsive disorders II (riboflavin andCoQ10)rdquo Journal of Inherited Metabolic Disease vol 35 no 4
pp 679ndash687 2012[8] F Depeint W R Bruce N Shangari R Mehta and P J
OrsquoBrien ldquoMitochondrial function and toxicity role of the Bvitamin family onmitochondrial energymetabolismrdquoChemico-Biological Interactions vol 163 no 1-2 pp 94ndash112 2006
[9] L Guarente ldquoMitochondria-A nexus for aging calorie restric-tion and sirtuinsrdquo Cell vol 132 no 2 pp 171ndash176 2008
[10] C Pimentel L Batista-Nascimento C Rodrigues-Pousada andR A Menezes ldquoOxidative stress in Alzheimerrsquos and Parkinsonrsquosdiseases insights from the yeast Saccharomyces cerevisiaerdquoOxidative Medicine and Cellular Longevity vol 2012 Article ID132146 9 pages 2012
BioMed Research International 11
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
[11] D Botstein and G R Fink ldquoYeast an experimental organismfor 21st century biologyrdquo Genetics vol 189 no 3 pp 695ndash7042011
[12] S Tenreiro and T F Outeiro ldquoSimple is good yeast modelsof neurodegenerationrdquo FEMS Yeast Research vol 10 no 8 pp970ndash979 2010
[13] M H Barros F M da Cunha G A Oliveira E B Tahara andA J Kowaltowski ldquoYeast as a model to study mitochondrialmechanisms in ageingrdquo Mechanisms of Ageing and Develop-ment vol 131 no 7-8 pp 494ndash502 2010
[14] Y Pan ldquoMitochondria reactive oxygen species and chronolog-ical aging amessage from yeastrdquoExperimental Gerontology vol46 no 11 pp 847ndash852 2011
[15] M B Wierman and J S Smith ldquoYeast sirtuins and theregulation of agingrdquo FEMS Yeast Research vol 14 no 1 pp 73ndash88 2014
[16] L Guarente ldquoSirtuins aging and metabolismrdquo Cold SpringHarbor Laboratory of Quantitative Biology vol 76 pp 81ndash902011
[17] T A Giancaspero V Locato andM Barile ldquoA regulatory role ofNAD redox status on flavin cofactor homeostasis in S cerevisiaemitochondriardquo Oxidative Medicine and Cellular Longevity vol2013 Article ID 612784 16 pages 2013
[18] V Gudipati K Koch W D Lienhart and P MacherouxldquoThe flavoproteome of the yeast Saccharomyces cerevisiaerdquoBiochimica et Biophysica ActamdashProteins and Proteomics vol1844 no 3 pp 535ndash544 2013
[19] T A Giancaspero R Wait E Boles and M Barile ldquoSuc-cinate dehydrogenase flavoprotein subunit expression in Sac-charomyces cerevisiaemdashinvolvement of the mitochondrial FADtransporter Flx1prdquo FEBS Journal vol 275 no 6 pp 1103ndash11172008
[20] M Barile T A Giancaspero C Brizio et al ldquoBiosynthesis offlavin cofactors in man implications in health and diseaserdquoCurrent Pharmaceutical Design vol 19 no 14 pp 2649ndash26752013
[21] AAHeikal ldquoIntracellular coenzymes as natural biomarkers formetabolic activities and mitochondrial anomaliesrdquo Biomarkersin Medicine vol 4 no 2 pp 241ndash263 2010
[22] P Reihl and J Stolz ldquoThe monocarboxylate transporterhomolog Mch5p catalyzes riboflavin (vitamin B2) uptake inSaccharomyces cerevisiaerdquo Journal of Biological Chemistry vol280 no 48 pp 39809ndash39817 2005
[23] M A Santos A Jimenez and J L Revuelta ldquoMolecular charac-terization of FMN1 the structural gene for the monofunctionalflavokinase of Saccharomyces cerevisiaerdquo Journal of BiologicalChemistry vol 275 no 37 pp 28618ndash28624 2000
[24] M Wu B Repetto D M Glerum and A Tzagoloff ldquoCloningand characterization of FAD1 the structural gene for flavinadenine dinucleotide synthetase of Saccharomyces cerevisiaerdquoMolecular and Cellular Biology vol 15 no 1 pp 264ndash271 1995
[25] A Tzagoloff J Jang D M Glerum and M Wu ldquoFLX1 codesfor a carrier protein involved inmaintaining a proper balance offlavin nucleotides in yeast mitochondriardquo Journal of BiologicalChemistry vol 271 no 13 pp 7392ndash7397 1996
[26] V Bafunno T A Giancaspero C Brizio et al ldquoRiboflavinuptake and FAD synthesis in saccharomyces cerevisiae mito-chondria Involvement of the flx1p carrier in fad exportrdquo Journalof Biological Chemistry vol 279 no 1 pp 95ndash102 2004
[27] M L Pallotta C Brizio A Fratianni C De Virgilio M Barileand S Passarella ldquoSaccharomyces cerevisiae mitochondria can
synthesise FMN and FAD from externally added riboflavin andexport them to the extramitochondrial phaserdquoFEBS Letters vol428 no 3 pp 245ndash249 1998
[28] M Ozeir U Muhlenhoff H Webert R Lill M Fontecave andF Pierrel ldquoCoenzyme Q biosynthesis Coq6 is required for theC5-hydroxylation reaction and substrate analogs rescue Coq6deficiencyrdquo Chemistry and Biology vol 18 no 9 pp 1134ndash11422011
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] V C Liuzzi T A Giancaspero E Gianazza C Banfi MBarile and C De Giorgi ldquoSilencing of FAD synthase gene inCaenorhabditis elegans upsets protein homeostasis and impactson complex behavioral patternsrdquo Biochimica et BiophysicaActamdashGeneral Subjects vol 1820 no 4 pp 521ndash531 2012
[31] J M McCord ldquoUnit 73 Analysis of superoxide dismutaseactivityrdquo in Current Protocols in Toxicology 2001
[32] T A Giancaspero C Brizio R Wait E Boles and M BarileldquoExpression of succinate dehydrogenase flavoprotein subunitin Saccharomyces cerevisiae studied by lacZ reporter strategyEffect of FLX1 deletionrdquo Italian Journal of Biochemistry vol 56no 4 pp 319ndash322 2007
[33] H J Kim M Y Jeong U Na and D R Winge ldquoFlavinylationand assembly of succinate dehydrogenase are dependent onthe C-terminal tail of the flavoprotein subunitrdquo The Journal ofBiological Chemistry vol 287 no 48 pp 40670ndash40679 2012
[34] K B Chapman S D Solomon and J D Boeke ldquoSDH1 the geneencoding the succinate dehydrogenase flavoprotein subunitfrom Saccharomyces cerevisiaerdquoGene vol 118 no 1 pp 131ndash1361992
[35] H-X Hao O Khalimonchuk M Schraders et al ldquoSDH5 agene required for flavination of succinate dehydrogenase ismutated in paragangliomardquo Science vol 325 no 5944 pp 1139ndash1142 2009
[36] E H Smith R Janknecht and J L Maher III ldquoSuccinateinhibition of 120572-ketoglutarate-dependent enzymes in a yeastmodel of paragangliomardquo Human Molecular Genetics vol 16no 24 pp 3136ndash3148 2007
[37] T A Giancaspero V Locato M C De Pinto L De Garaand M Barile ldquoThe occurrence of riboflavin kinase and FADsynthetase ensures FAD synthesis in tobacco mitochondria andmaintenance of cellular redox statusrdquo FEBS Journal vol 276 no1 pp 219ndash231 2009
[38] P Chaiyen M W Fraaije and A Mattevi ldquoThe enigmaticreaction of flavins with oxygenrdquo Trends in Biochemical Sciencesvol 37 no 9 pp 373ndash380 2012
[39] RWerner K CManthey J B Griffin and J Zempleni ldquoHepG2cells develop signs of riboflavin deficiency within 4 days ofculture in riboflavin-deficient mediumrdquo Journal of NutritionalBiochemistry vol 16 no 10 pp 617ndash624 2005
[40] H J Kim andD RWinge ldquoEmerging concepts in the flavinyla-tion of succinate dehydrogenaserdquoBiochimica et Biophysica Actavol 1827 no 5 pp 627ndash636 2013
[41] B J De La Cruz S Prieto and I E Scheffler ldquoThe role ofthe 51015840 untranslated region (UTR) in glucose-dependent mRNAdecayrdquo Yeast vol 19 no 10 pp 887ndash902 2002
[42] M Kellis N Patterson M Endrizzi B Birren and E S LanderldquoSequencing and comparison of yeast species to identify genesand regulatory elementsrdquoNature vol 423 no 6937 pp 241ndash2542003
12 BioMed Research International
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
[43] D-W Kwon and S H Ahn ldquoRole of yeast JmjC-domain con-taining histone demethylases in actively transcribed regionsrdquoBiochemical and Biophysical Research Communications vol 410no 3 pp 614ndash619 2011
[44] M Jacquet G Renault S Lallet J De Mey and A GoldbeterldquoOscillatory nucleocytoplasmic shuttling of the general stressresponse transcriptional activators Msn2 and Msn4 in Saccha-romyces cerevisiaerdquo Journal of Cell Biology vol 161 no 3 pp497ndash505 2003
[45] P Fabrizio F Pozza S D Pletcher C M Gendron and V DLongo ldquoRegulation of longevity and stress resistance by Sch9 inyeastrdquo Science vol 292 no 5515 pp 288ndash290 2001
[46] K A Morano C M Grant and W S Moye-Rowley ldquoTheresponse to heat shock and oxidative stress in saccharomycescerevisiaerdquo Genetics vol 190 no 4 pp 1157ndash1195 2012
[47] K E Kwast L-C Lai N Menda D T James III S Arefand P V Burke ldquoGenomic analyses of anaerobically inducedgenes in Saccharomyces cerevisiae functional roles of Rox1 andother factors in mediating the anoxic responserdquo Journal ofBacteriology vol 184 no 1 pp 250ndash265 2002
[48] C B Edwards N Copes A G Brito J Canfield and P C Brad-shaw ldquoMalate and fumarate extend lifespan in Caenorhabditiselegansrdquo PLoS ONE vol 8 no 3 Article ID e58345 2013
[49] A R Cyr and F E Domann ldquoThe redox basis of epigeneticmodifications from mechanisms to functional consequencesrdquoAntioxidants and Redox Signaling vol 15 no 2 pp 551ndash5892011
[50] A P Wojtovich C O Smith C M Haynes K W Nehrkeand P S Brookes ldquoPhysiological consequences of complexII inhibition for aging disease and the mKATP channelrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1827 no 5 pp598ndash611 2013
[51] E Gianazza L Vergani R Wait et al ldquoCoordinated andreversible reduction of enzymes involved in terminal oxida-tive metabolism in skeletal muscle mitochondria from ariboflavin-responsive multiple acyl-CoA dehydrogenase defi-ciency patientrdquo Electrophoresis vol 27 no 5-6 pp 1182ndash11982006
[52] N Gregersen B S Andresen C B Pedersen R K J Olsen TJ Corydon and P Bross ldquoMitochondrial fatty acid oxidationdefectsmdashremaining challengesrdquo Journal of Inherited MetabolicDisease vol 31 no 5 pp 643ndash657 2008
[53] J Rutter D R Winge and J D Schiffman ldquoSuccinatedehydrogenasemdashassembly regulation and role in human dis-easerdquoMitochondrion vol 10 no 4 pp 393ndash401 2010
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