YPI1 and SDS22 Proteins Regulate the Nuclear Localizationand Function of Yeast Type 1 Phosphatase Glc7*□S
Received for publication, July 28, 2006, and in revised form, October 11, 2006 Published, JBC Papers in Press, December 1, 2006, DOI 10.1074/jbc.M607171200
Leda Pedelini‡1, Maribel Marquina§, Joaquin Arino§, Antonio Casamayor§, Libia Sanz‡, Mathieu Bollen¶,Pascual Sanz‡2, and Maria Adelaida Garcia-Gimeno‡
From the ‡Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientıficas (CSIC), Jaime Roig 11, 46010 Valencia,Spain, the §Department Bioquimica i Biologia Molecular, Facultat de Veterinaria, Universitat Autonoma de Barcelona,08193 Bellaterra, Barcelona, Spain, and the ¶Department of Molecular Cell Biology, Faculty of Medicine,Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
We have recently characterized Ypi1 as an inhibitory subunitof yeast Glc7 PP1 protein phosphatase. In this work we demon-strate that Ypi1 forms a complex with Glc7 and Sds22, anotherGlc7 regulatory subunit that targets the phosphatase to sub-strates involved in cell cycle control. Interestingly, the combina-tion of equimolar amounts of Ypi1 and Sds22 leads to an almostfull inhibition ofGlc7 activity. BecauseYPI1 is an essential gene,we have constructed conditionalmutants that demonstrate thatdepletion of Ypi1 leads to alteration of nuclear localization ofGlc7 and cell growth arrest inmid-mitosis with aberrantmitoticspindle. These phenotypes mimic those produced upon inacti-vation of Sds22. The fact that progressive depletion of eitherYpi1 or Sds22 resulted in similar physiological phenotypes andthat both proteins inhibit the phosphatase activity of Glc7strongly suggest a common role of these two proteins in regulat-ing Glc7 nuclear localization and function.
PP1 (protein Ser/Thr phosphatase-1) is a ubiquitous eukary-otic enzyme that regulates a variety of cellular processes, suchas carbohydrate and lipid metabolism, protein synthesis, andcell cycle progression (for review see Refs. 1–4). The PP1 cata-lytic subunit is highly conserved throughout evolution. In theyeast Saccharomyces cerevisiae, there is only one PP1, namedGlc7, which is essential for cell viability (5, 6). Similarly to itsmammalian counterpart, Glc7 participates in the regulation ofmany different physiological processes, such as glycogenmetabolism, glucose repression, ion homeostasis, cell cycle reg-
ulation, sporulation, vacuole fusion, endocytosis, polyadenyl-ation termination, and the maintenance of cell wall integrity(7–13). The functional versatility of PP1 is achieved by the exist-ence of numerous regulatory subunits that target PP1 to differentsubcellular compartments and/or substrates, confer substratespecificity, and/or modulate enzymatic activity (1–3, 14). Thesesubunits are structurally diverse, but almost all of them contain aconsensusbindingmotif (R/K)(V/I)X(F/W)necessary forPP1 reg-ulation, which also accounts for themutually exclusive binding ofthe different subunits to PP1 (1–3, 14–17).PP1 activity is essential, but it must be tightly controlled as
overexpression or hyperactivation of PP1 phosphatase is dele-terious to the cell. Consequently, a large number of physiolog-ical inhibitors of PP1 have been identified in higher eukaryotes(1, 3, 14, 18). We have recently identified the first inhibitorysubunit of Glc7 in budding yeast. It is a small (155 amino acids),hydrophilic, heat-stable protein that we named Ypi1 (YeastPhosphatase Inhibitor 1). This protein contains the typical con-sensus binding motif (R/K)(V/I)X(F/W) necessary to bind PP1.Deletion ofYPI1 is lethal, suggesting a relevant role of the inhib-itor in yeast physiology. On the other hand, overexpression ofYpi1 displays a number of phenotypes consistent with an inhib-itory role of this protein on Glc7 activity. Structural homo-logues of Ypi1 can be found in yeast, plants, and animals, sug-gesting a strongly conserved function of this protein (19).In this work, we provide evidence that Ypi1 interacts with
Sds22, another regulatory subunit of Glc7 that targets the phos-phatase to substrates involved inmitosis and chromosome seg-regation (20–23). Sds22 lacks the consensus (R/K)(V/I)X(F/W)recognition motif found in other Glc7 regulatory subunits (15,17). However, interaction between the human orthologue ofSds22 and PP1 is mediated by the 11 leucine-rich repeats(LRR)3 that Sds22 has in its central domain and occurs at a sitein PP1 different from the one used to bind the (R/K)(V/I)X(F/W)motif (24). In yeast, Sds22 is an essential protein of 40kDa largely found in the nucleus, despite the absence of a clearnuclear localization sequence (NLS). In addition to its role inmitosis, Sds22 plays a role in maintaining the normal nuclearlocalization of Glc7 (25). In this work, we show that Ypi1 andSds22 form a complex with Glc7, and present data suggesting
* This work was supported in part by Grants BMC2002-00208 (to P. S.),BFU2004-01432 (to Juan Jose Calvete), BMC2002-04011-C05-04,BFU2005_06388-C4-04 (to J. A.), and BFU2004-00014 (to A. C.) from theSpanish Ministry of Education and Science and Fondo Europeo de Desar-rollo Regional, an “Ajut de Suport als Grups de Recerca de Catalunya” Grant2001SGR00193 (to J. A.), the Instituto de Salud Carlos III Network GrantsRCMN C03/08 and RGDM G03/212 (to P. S.), Grant PNL2004-8 from Univer-sitat Autonoma de Barcelona, and Grant MIRG-CT-2004-003794 from theEuropean Commission (to A. C.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.
The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) CAB11073.
1 Supported by Predoctoral I3P Fellowship from the CSIC.2 To whom correspondence should be addressed. Tel.: 3496-3391779; Fax:
that, similarly to Sds22, Ypi1 may function within the nucleus,regulating cell growth and also maintaining the nuclear local-ization of Glc7. The fact that Glc7, Ypi1, and Sds22 are proteinsconserved among all eukaryotes suggests that the proposedmodel of regulation might be also conserved.
EXPERIMENTAL PROCEDURES
Strains and Culture Conditions
Escherichia coli DH5� was used as the recipient cell for allplasmids and constructs, whereas E. coli BL21 (DE3) codonplus-RIL (Stratagene) was used to produce recombinant pro-teins. S. cerevisiae strains used in this work are described inTable 1. Strain MMR09-4, in which the expression of YPI1 isunder the control of the tetO7 promoter, was constructed asfollows. A PCR-amplified KanMX4-tetO7 cassette was madewith oligonucleotides 5�TETO-YPI1 and 3�TETO-YPI1 (Table2); this cassette was inserted immediately upstream from theinitiating ATG codon of the chromosomal YPI1 coding regionby homologous recombination in the CML476 strain.Strain MMR11-1 was made as follows. A 2.7-kbp DNA frag-
ment containing the entire GLC7-yEmCitrine::SpHis5MX cas-sette, flanked by 206 nucleotides upstream from theGLC7 startcodon and 209 nucleotides immediately downstream from theGLC7 stop codon, was PCR-amplified from genomic DNAfrom the KT2422 strain using Glc7-1256 and Glc7-1673 oligo-nucleotides (Table 2) as primers. This fragment was integratedin the genome of the MMR09-4 strain by homologous recom-bination to generate the MMR11-1 strain. The indicatedGLC7-yEmCitrine cassette was also introduced in the strainSAY302 (sds22-5ts) to yield strain MMR13-4.
Standard methods for genetic analysis and transformationwere used. Yeast cultures were grown in richmedium (YPD) orsynthetic complete (SC) medium lacking appropriate supple-ments to maintain selection for plasmids (26), containing theindicated carbon sources.
Oligonucleotides
Oligonucleotides used in the present study are described inTable 2.
Reverse Transcription-PCR
Cells from 20 ml of yeast culture (MMR09-4 strain) wereused for each time point after doxycycline or mock treatments.Yeast cells were collected at 4 °C and washed in cold water, andthe dried cell pellets kept at �80 °C. Total RNA was extractedusing the RiboPure-Yeast kit (Ambion) following the manufac-turer’s instructions. RNA quality was assessed by denaturingagarose gel electrophoresis, and the RNA quantification wascarried out in a BioPhotometer (Eppendorf). 1 �g of RNA wasused to generate cDNAwith the OneStep RT-PCR kit (Invitro-gen) according to the manufacturer’s instructions using theRT_YPI1_UP and RT_YPI1_DO oligonucleotides (Table 2). 24cycles of PCR were performed.
Plasmids
Yeast expression plasmids pWS-GST-Ypi1 (GST-Ypi1),pWS-Ypi1 (HA-Ypi1), pBTM-Ypi1 (LexA-Ypi1), pBTM-Ypi1W53A (LexA-Ypi1W53A), and pACT2-Ypi1 (GAD-Ypi1)and bacterial plasmids pGEX-Ypi1 (GST-Ypi1) and pUC-Ypi1were described previously (19). Plasmids pBTM-Ypi1-Nterm(LexA-Ypi1-(1–93)) and pBTM-Ypi1-Cterm (LexA-Ypi1-(97–155)) were constructed in the following way: plasmid pBTM-Ypi1 was digested with EcoRI/BamHI and then the fragmentsubcloned into pBTM116 to obtain plasmid pBTM-Ypi1-Nterm, or digested with BamHI/SalI and the fragment sub-cloned into pUC18, resulting in plasmid pUC-Ypi1-Cterm, andthen an EcoRI/SalI fragment from the latter was subcloned intopBTM116 to obtain plasmid pBTM-Ypi1-Cterm.Plasmid pADH1-Ypi1-GFPwas obtained in several steps. First
we amplified by PCR the coding region ofYPI1 using oligonucleo-tidesYFR-1 andYpi1-GFP, andFY250 genomicDNAas template.The amplified fragment was digested with EcoRI/NotI and sub-cloned in vector pRS426 (27) to obtain plasmid pRS426-Ypi1. ANotI fragment obtained from plasmid pSF-GFP (28) was sub-cloned in-frame into the NotI site of pRS426-Ypi1 to obtain plas-mid pRS426-Ypi1-GFP. Finally, a SalI/EcoRI fragment containing
TABLE 1Yeast strains used in this study
Strain Genotype SourceFY250 MAT� his3�200 leu2�1 trp1�63 ura3-52 F. Winston (35)TAT7 MATa ade2 his3 leu2 trp1 gal4 gal80 LYS2::lexAop-HIS3 URA3::lexAop-lacZ R. Sternglanz (35)KT2422 MATa ura3-52 leu2 his3 GLC7-yEmCitrine::SpHis5Mx K. TatchellMMR18 MATa/MAT� M5 his4/� leu2/leu2 trp1/trp1 ura3/ura3 YPI1/ypi1�::KanMX 19CML476 MATa ura3-52 leu�1 his3�200 GAL2 CMVp(tetR�-SSN6)::LEU2 trp1::tTA 57MMR09-4 MATa CML476 KanMX4-(tetO7):YPI1 This workSAY302 MATaW303 sds22::TRP1 leu2-3,112::YIp22-5 25MMR11-1 MATaMMR09-4 GLC7::yEMCitrine::SpHis5Mx This workMMR13-4 MATa SAY302 GLC7::yEMCitrine::SpHis5Mx This workSBY214 MATa ura3-1 leu2,3-112, his3-11::pCUP1-GFP12-lacI12::HIS3 trp1--1:lacO:TRP1 lys2� ade2-1 can1-100 bar1� 48SBY322 MATa ura3-1 leu2,3-112, his3-11::pCUP1-GFP12-lacI12::HIS3 trp1-1:lacO:TRP1 lys2� ade2-1 can1-100 bar1� ipl1-321 48
TABLE 2Oligonucleotides used in the present study
Name SequenceYFR-1 5�-GTCTGAATTCATGAGTGGAAATCAAATGG-3�Ypi1-GFP 5�-TTATAGCGGCCGCCGTCCTTCTTTTCCTGCTGT
the ADH1 promoter was subcloned into the latter plasmid toobtain pADH1-Ypi1-GFP. All Ypi1 fusion proteins were fullyfunctional as deduced by rescue of the lethal phenotype of anypi1::KanMXmutant (strainMMR18; data not shown).
SDS22 coding region was amplified by PCR with oligonu-cleotides SDS22-1 and SDS22-2 (see Table 2) using FY250genomic DNA as template. The amplified fragment wasdigested with BamHI/SalI and subcloned into vectorspBTM116 (29) pWS93 (30), and pWS-GST (31) to obtain theyeast expression plasmids pBTM-Sds22 (LexA-Sds22), pWS-Sds22 (HA-Sds22), and pWS-GST-Sds22 (GST-Sds22), respec-tively. Using the following combination of oligonucleotides,SDS22�41/SDS22-2, SDS22-1/SDS22�316, and SDS22�41/SDS22�316 (Table 2), we amplified truncated forms of Sds22lacking either an N-terminal fragment (from amino acids1–40), a C-terminal fragment (from amino acids 316–338), orboth. Using oligonucleotides SDS22LRRcap/SDS22-2, we alsoamplified the LRR-cap domain (from amino acids 316–338) ofSds22. These fragments were digested with BamHI/SalI andsubcloned into vector pBTM116 to obtain plasmids pBTM-Sds22�41, pBTM-Sds22�316, pBTM-Sds22�41�316, andpBTM-Sds22LRRcap respectively, which expressed LexA-Sds22(41–338), LexA-Sds22(1–315), LexA-Sds22(41–315),and LexA-Sds22(316–338) fusion proteins (in parentheses arethe amino acid sequences being expressed). Plasmid pGEX-Glc7 (GST-Glc7) was described in Ref. 32, plasmid YEpACT-Glc7 (HA-Glc7) in Ref. 33, and plasmid pACT-Glc7 in Ref. 34.
Gel Filtration
Twomgof yeast crude extract fromcells expressingHA-Ypi1(plasmid pWS-Ypi1), HA-Glc7 (plasmid YEpACT-Glc7), andLexA-Sds22 (plasmid pBTM-Sds22) was loaded to a calibratedFPLC Superdex 200 HR 10/30 gel filtration column previouslyequilibrated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1mM dithiothreitol. Aliquots of 300 �l were collected and ana-lyzed by SDS-PAGE andWestern blotting using anti-LexA andanti-HA antibodies (see below).
Co-immunoprecipitation Assays and Immunoblot Analysis
Preparation of yeast protein extracts for co-immunoprecipita-tion assays was essentially as described previously (35). Extractionbuffer was 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% TritonX-100, 1mM dithiothreitol, and 10% glycerol and contained 2mMphenylmethylsulfonyl fluoride and a complete protease inhibitormixture (Roche Applied Science). Yeast extracts (500 �g) wereincubated with either 1 �l of anti-LexA polyclonal antibody(Invitrogen), 1�l of anti-HAmonoclonal antibody (Sigma), or 1�lof anti-GST polyclonal antibody (AmershamBiosciences), and 50�l of protein A-Sepharose beads for 1 h at 4 °C and then washedfour times with extraction buffer. Proteins retained by the affinitysystem were detected by SDS-PAGE followed by immunoblotusing anti-HA monoclonal, anti-LexA polyclonal, and anti-GSTpolyclonal antibodies and chemiluminescence reagents (ECL,Amersham Biosciences).
Purification of Recombinant Proteins in E. coli and Yeast
Purification of the fusion protein GST-Ypi1 expressed inE. coli was carried out as described previously (36). Transfor-
mants were grown at 37 °C until the absorbance at 600 nmreached a value of around 0.3. Isopropyl 1-thio-�-D-galactopy-ranoside was then added to a concentration of 0.1 mM, and thecultures were grown overnight at 25 °C. Cells were harvestedand resuspended in 20ml of sonication buffer (50mMTris-HCl,pH 7.6, 0.2 mM EGTA, 150mMNaCl, 10% glycerol, 0.1% TritonX-100, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluo-ride, and complete protease inhibitor mixture (Roche AppliedScience)). Cells were disrupted by sonication, and the fusionproteins were purified by passing the extracts through a 1-mlbed volume of glutathione-Sepharose columns (AmershamBiosciences). GST fusion proteins were eluted from the columnwith 10 mM glutathione. Samples were stored at �80 °C. Bac-terial expression and purification of GST-Glc7 fusion proteinwere described previously (19). GST-Sds22 fusion protein wasexpressed in yeast and purified as above.
Effect of Depletion of Ypi1 and Sds22 on Cell Growth
To evaluate the effect of depletion of Ypi1 in cell growth, wildtype CML476 and the conditional tetO:YPI1 mutant(MMR09-4) were grown overnight in YPD, diluted at A600 of0.01, and treated with 100 �g/ml doxycycline (or vehicle), andgrowth was resumed for 12 h at 30 °C. The culture was dilutedagain until an A600 of 0.01 and again received 100 �g/ml doxy-cycline before growth was resumed. Samples were taken at theindicated intervals, and the A600 of the culture measured. Thedoxycycline treatment was renewed every 12 h to account forpossible degradation of the drug.To check which step of the cell cycle the Ypi1-depleted cells
were arrested, cultures (15 ml) of the MMR09-4 strain in YPDat A660 of 0.01 were treated with doxycycline (100 �g/ml), andgrowth was resumed at 30 °C for 12 h. The �-factor (10 �g/ml)and fresh doxycycline (100 �g/ml) were then added, and after2 h, cells were washed and resuspended in 15 ml of fresh YPDcontaining doxycycline (100 �g/ml) and treated with 0.2 Mhydroxyurea for 30 min. Cells were washed again, resuspendedin 15ml of freshYPDwith doxycycline, and growth resumed for4 h. The same protocol was used for cells untreated with doxy-cycline and for wild type cells (CML476). Strain SAY306 (wildtype) and SAY302 (sds22-5ts) were synchronized as above,except that growth temperature was 24 °C. After blockage withhydroxyurea, cells were shifted to the nonpermissive tempera-ture (37 °C) and growth resumed for 4 h. In all cases, at differenttimes after the release from the blockage, aliquots of cells werefixed with 3.7% formaldehyde for 60 min at room temperatureand stained with DAPI to visualize the nuclei. 120min after therelease, cellswere also collected for tubulin staining (see below).
Tubulin Staining
Cells were prepared for indirect immunofluorescence as in Ref.37 and incubated with 1:250 diluted rat monoclonal antibody YL1/2 raised against yeast�-tubulin (Serotech) (a generous gift ofDr.Jesus Avila, CBM, Madrid, Spain) and subsequently with 1:100diluted fluorescein isothiocyanate-conjugated goat anti-rat IgG(Alexa Fluor 488; Molecular Probes), to label microtubules. AColor View 12 CCD camera coupled to a Nikon Eclipse E800microscope was used in combination with the Analyze 3.0 soft-ware (Soft Imaging System) to capture the images.
Ypi1 and Sds22 Regulate Glc7 Nuclear Function
3284 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 5 • FEBRUARY 2, 2007
Subcellular Localization of Ypi1-GFP and Glc7-yEmCitrine
Exponentially growing yeast cells containing plasmidpADH1-Ypi1-GFPwere used to visualize Ypi1-GFP fusion. Ali-quots (2 �l) of the cultures were placed on microscope slidesand covered with 18 � 18-mm coverslips. The images of Ypi1-GFP and DAPI-stained nuclei were directly captured by fluo-rescence microscopy, using a Leica DMRXA2 microscope andthe Leica FW4000 software.MMR11-1 transformants (expressing Glc7-yEmCitrine)
were used to localize Glc7 in the absence of a functional Ypi1protein in the following way: yeast cells were inoculated intoYPD, grown to saturation, diluted at anA600 of 0.01 in YPD, andgrowth resumed for 1 h. Doxycycline was then added from a 5mg/ml stock solution (made in 50% ethanol) to achieve a finalconcentration of 100 �g/ml. Control cells received the samevolume of vehicle. Growth was resumed, and after 12 h, thesame amount of doxycycline or vehicle was added to accountfor degradation of the antibiotic. Cells were collected after 24 hand fixed by resuspension in phosphate-buffered saline/form-aldehyde (2%) for 5 min at room temperature. Cells werewashed several times with phosphate-buffered saline and keptat 4 °C until further use. Similarly, strainMMR13-4 was used tomonitor localization of Glc7 in the absence of a functionalSds22 protein. Cells were grown at 26 °C in YPDuntil anA600 of0.5 and then maintained at the same temperature or shifted to37 °C for 1 h. Samples were taken and fixed as described above.Fluorescently labeled Glc7 and DAPI-stained nuclei wereobserved under the microscope (Nikon Eclipse E-800) by mix-ing on a slide 2�l of the samples with 2�l of a DAPI-containingmounting solution as described previously (38).
Other Techniques
Protein Phosphatase Assays—Protein phosphatase activityusing p-nitrophenyl phosphate as substrate was determinedessentially as described previously (39). The reaction buffer was50mMTris-HCl, pH 7.5, 0.1mMEGTA, 2mMMnCl2, and 1mMdithiothreitol. Samples were incubated 10min at 30 °C, and thereaction was then stopped by adding 1% Tris (final concentra-tion). For phosphatase inhibition assays, different amounts ofthe purified inhibitors were incubated with the purified phos-phatases during 5 min at 30 °C, prior to the addition of p-nitro-phenyl phosphate.
�-Galactosidase Assay—�-Galactosidase activity was assayedin permeabilized cells and expressed inMiller units as describedin Ref. 40.
RESULTS
Ypi1 Interacts with Sds22 and Glc7—We have recently char-acterized Ypi1 as an inhibitory subunit of yeast Glc7 proteinphosphatase and shown that it performs an essential function(19). Recent evidence obtained through large scale affinity co-immunoprecipitation approaches suggested that Ypi1 couldinteract physically with Sds22 (41, 42). Because Sds22 is anessential protein of 40 kDa that interacts with Glc7 and targetsthe phosphatase to substrates involved in mitosis and chromo-some segregation (see Introduction), we considered that theYpi1-Sds22 interaction might have functional relevance. How-ever, because it is known that a large number of interactions
defined by high throughput methods turn out to be false posi-tives (43), we decided to validate such interaction by differentmethods. We first confirmed the interaction between Sds22andYpi1 by a direct co-immunoprecipitationmethod using cellextracts from yeast expressing GST-Sds22, LexA-Ypi1, andHA-Glc7. Using anti-GST antibodies (to immunoprecipitateGST-Sds22), immunoblot analysis revealed the presence ofboth Ypi1 (LexA-Ypi1) andGlc7 (HA-Glc7) in the immunopre-cipitates (Fig. 1A). Similarly, when the cell extracts were immu-noprecipitated with anti-LexA antibodies (to immunoprecipi-tate LexA-Ypi1), we were able to recover Sds22 (GST-Sds22)and Glc7 (HA-Glc7) in the immunoprecipitates (Fig. 1B), andfinally, when we immunoprecipitated the cell extracts withanti-HA antibodies (to immunoprecipitate HA-Glc7), werecovered Ypi1 (LexA-Ypi1) and Sds22 (GST-Sds22) in theimmunoprecipitates (Fig. 1C). These results indicated thatSds22, Ypi1, and Glc7 were able to associate physically withinthe yeast cell. To analyze whether these three proteins formed acomplex, we used a triple-hybrid system. This approach hasbeen successfully applied by a number of groups to demon-strate that co-expression of an auxiliary bait is sufficient tostrengthen ternary interactions (44, 45). As shown in Table 3,overexpression of Ypi1 improved the two-hybrid interactionbetween Sds22 and Glc7 by 14-fold. Similarly, overexpressionof Sds22 also improved the magnitude of the two-hybrid inter-action between Ypi1 and Glc7 by 150-fold (Table 3). Theseresults suggested that Sds22, Ypi1, and Glc7 may form a stableternary complex. To confirm these results, we analyzed by gelfiltration a crude extract of cells expressing LexA-Sds22, HA-Ypi1, and HA-Glc7. As shown in Fig. 2, most of the three pro-teins appeared in high molecular weight fractions, with an esti-matedmolecular mass of around 130 kDa (fractions 39 and 41),very close to the expected molecular mass of a putative ternarycomplex (65 kDa (LexA-Sds22) � 40 kDa (HA-Glc7) � 33 kDa
FIGURE 1. Sds22 associates physically with Ypi1 and Glc7. Crude yeastextracts (500 �g) were prepared from FY250 cells growing exponentially inglucose and expressing different combinations of plasmids. A, crude yeastextracts from yeast transformants expressing GST or GST-Sds22, LexA-Ypi1,and HA-Glc7 were immunoprecipitated (IP) with 1 �l of anti-GST polyclonalantibody. B, crude extracts from yeast transformants expressing LexA orLexA-Ypi1, GST-Sds22, and HA-Glc7 were immunoprecipitated with 1 �l ofanti-LexA polyclonal antibody. C, crude extracts from yeast transformantsexpressing HA or HA-Glc7, GST-Sds22, and LexA-Ypi1 were immunoprecipi-tated with 1 �l of anti-HA monoclonal antibody. Co-immunoprecipitated pro-teins were analyzed by SDS-PAGE and immunodetected with the mentionedantibodies. Tagged proteins in the crude extracts (CE; 1 �g) were also immu-nodetected. Migration of size standards is indicated in kDa.
Ypi1 and Sds22 Regulate Glc7 Nuclear Function
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(HA-Ypi1)). We were also able to detect the three proteins invery highmolecular weight fractions (fraction 23), very close tothe void volume of the column (fraction 21), suggesting that thethree proteins might form part of a supramolecular complex.The amount of HA-Glc7 in these very high molecular weightfractions was higher than HA-Ypi1, probably suggesting thatHA-Glc7 may participate in different very high molecularweight complexes (46).We next mapped the regions involved in the interaction
between Ypi1 and Sds22. Deletion of the Sds22 N-terminalregion (from residues 1–40; Fig. 3A) only reduced slightly theinteraction between Sds22 and Ypi1 (Fig. 3B), whereas removal
of the last C-terminal 22 amino acids (from residues 316–338)of Sds22, which largely consists of the LRR-cap domain, pre-vented the interaction of Sds22 with Ypi1 (Fig. 3B). Deletion ofboth N-terminal and C-terminal regions of Sds22 gave similarresults to the deletion of only the C-terminal region (data notshown). To determine whether the LRR-cap domain wasresponsible for the interaction, we constructed a fusion proteincontaining only this domain (residues 316–338), but we did notobserve any interaction with Ypi1 (Fig. 3B). These results sug-gested that the LRR-cap domain was necessary but not suffi-cient for the interaction with Ypi1. We also tested the interac-
FIGURE 2. Gel filtration chromatography of yeast extracts. A, crude yeastextracts from FY250 cells growing exponentially in glucose and expressingLexA-Sds22 (plasmid pBTM-Sds22), HA-Ypi1 (plasmid pWS-Ypi1), andHA-Glc7 (plasmid YEpACT-Glc7) were loaded on an FPLC Superdex 200 HR10/30 gel filtration column. Aliquots were analyzed by SDS-PAGE and immu-nodetected with either anti-HA antibodies (upper panel) or anti-LexA anti-bodies (lower panel). One �g of the original crude extract (CE) was also ana-lyzed in the same way. The position of standard molecular mass markers isindicated (alcohol dehydrogenase (ADH) 146 kDa; bovine serine albumin(BSA) 67 kDa; ovalbumin (Ovo) 42 kDa; soybean trypsin inhibitor (STI) 20 kDa;cytochrome c (CytC) 13 kDa). B, calibration of the gel filtration column. TheVe/Vo of the markers used to calibrate the column was plotted against thelogarithm of their corresponding molecular weight. The dashed line corre-sponds to fraction 40, with an estimated molecular mass of 130 kDa.
FIGURE 3. Identification of the domains involved in the interactionamong Sds22, Ypi1, and Glc7. A, diagram of motifs present in Sds22 protein;LRR, leucine rich repeats; LRR-cap, motif downstream of the last and incom-plete LRR. B and C, two-hybrid interaction between GAD-Ypi1 (B) or GAD-Glc7(C) and different truncated forms of Sds22. TAT7 yeast cells were transformedwith pACT2-Ypi1 (B) or pACT-Glc7 (C) and the appropriate pBTM-Sds22 plas-mids (pBTM-Sds22, pBTM-Sds22�41, pBTM-Sds22�316, and pBTM-Sds22LRRcap). Transformants were analyzed for two-hybrid interaction asdescribed in the legend of Table 3. Values were normalized to the activitypresent in the interaction with the full-length forms (LexA-Sds22 � GAD-Ypi1, 12.9 �-galactosidase units; LexA-Sds22 � GAD-Glc7, 49.9 �-galactosid-ase units). Bars indicate standard deviation. Crude extracts were preparedfrom representative transformants expressing GAD-Ypi1 and analyzed byWestern blotting using anti-LexA antibodies, to check the production of thedifferent LexA-Sds22 derivatives. Similar results were obtained with transfor-mants expressing GAD-Glc7 (not shown). D, diagram of motifs present inYpi1; RVXW, consensus site for Glc7 binding; NLS?, putative bipartite nuclearlocalization sequence. E and F, two-hybrid interaction between GAD-Sds22(E) and GAD-Glc7 (F) and different truncated forms of Ypi1. TAT7 yeast cellswere transformed with pACT2-Sds22 (E) or pACT-Glc7 (F) and the appropri-ated pBTM-Ypi1 plasmids (pBTM-Ypi1, pBTM-Ypi1-Nterm, and pBTM-Ypi1-Cterm). Transformants were analyzed as above. Values were normalized tothe activity present in the interaction with the full-length form (LexA-Ypi1 �GAD-Sds22, 347.7 �-galactosidase units; LexA-Ypi1 � GAD-Glc7, 5.8 �-galac-tosidase units). Bars indicate standard deviation. Crude extracts were pre-pared from representative transformants expressing GAD-Sds22 and ana-lyzed by Western blotting using anti-LexA antibodies, to check theproduction of the different LexA-Ypi1 derivatives. Similar results wereobtained with transformants expressing GAD-Glc7 (not shown).
TABLE 3Two-hybrid interaction between Sds22 and Glc7 is enhanced byoverexpression of Ypi1S. cerevisiae TAT7 cells containing either plasmid pBTM-Sds22 (LexA-Sds22) orpBTM-Ypi1 (LexA-Ypi1) were transformedwith the indicated combination of plas-mids as follows: pACT-Glc7 (GAD-Glc7), pACT2-Ypi1 (GAD-Ypi1), and pACT2(GAD) and plasmids pWS93 (HA), pWS-Ypi1 (HA-Ypi1), or pWS-Sds22 (HA-Sds22). Plasmid pSK-Ypi1 (HA-Ypi1) was used in the control experiment. Trans-formants were grown until exponential phase (A600 0.5) in selective SC mediumcontaining 2% glucose. Protein interactionwas estimated using the yeast two-hybridsystem, by measuring the �-galactosidase activity. Values correspond to meansfrom 4 to 6 different transformants � S.D.
Bait Prey Additional protein �-Galactosidase activityunits
tion between Glc7 and the different fragments of Sds22 andobtained similar results, although we observed a better interac-tion of Glc7 with the N-terminal truncated form of Sds22 incomparison with the full-length protein (Fig. 3C).Western blotanalyses indicated that the truncated proteinswere produced atsimilar levels in all the cases (Fig. 3B).Similarly, we constructed N-terminal and C-terminal dele-
tions of Ypi1 (Fig. 3D) and found that Sds22 interacted mainlywith the C-terminal part of Ypi1 (from residue 97–155), whichcontained a putative bipartite NLS (Fig. 3E), whereas Glc7interacted mainly with the N-terminal region of Ypi1 (fromresidues 1–93), which contained the (R/K)(V/I)X(F/W) motif(Fig. 3F). The interaction with this fragment was much betterthan with full-length Ypi1, probably because of the higher levelof expression of the truncated form of the protein (Fig. 3E)and/or to a better accessibility to the (R/K)(V/I)X(F/W)motif inthe truncated form. Both domains of Ypi1 were necessary foractivity because the expression of any of the truncated forms ofYpi1 could not rescue the lethal phenotype of a ypi1::KanMXmutant (strain MMR18; data not shown).
Ypi1 Is Largely Located Inside the Nucleus—Next, we ana-lyzed the subcellular localization of Ypi1. As observed in Fig. 4,a Ypi1-GFP fusion protein was enriched in the nucleus. Theseresults were consistent with the nuclear enrichment of Sds22and Glc7 (25).Inhibitory Capacity of Ypi1 Is Enhanced by Sds22—Because
Ypi1 inhibits the phosphatase activity of Glc7 (Fig. 5) (19), wetested whether Sds22 had the same properties. As shown in Fig.5, a GST-Sds22 fusion protein produced in yeast also inhibited(53% inhibition) the activity of Glc7 used in the assay. Moreinterestingly, the combination of equimolar amounts of Ypi1and Sds22 (0.224 �M each) displayed a higher inhibitory capac-ity onGlc7, leading to an almost full inhibition (Fig. 5). Ypi1 andSds22 displayed an additive inhibitory capacity as indicated bythe dose-response curve of equimolar amounts of both proteins(Fig. 5).Mutations in YPI1 and SDS22 Produce Similar Phenotypes—
The results presented thus far suggested that the function ofYpi1 and Sds22 could be related. To study this possibility, we
FIGURE 4. Ypi1-GFP fusion protein has a nuclear localization. FY250 yeastcells expressing Ypi1-GFP fusion protein were grown to exponential phase inglucose-containing medium. Aliquots were taken and analyzed as describedunder “Experimental Procedures.” Images of the Nomarski optics, the greenfluorescent protein (GFP), and DAPI and the merge of these two fluorescencesof representative samples are shown.
FIGURE 5. Inhibitory capacity of Ypi1 is enhanced by Sds22. Five �g ofpurified GST-Glc7 (0.158 �M) were incubated in the presence of p-nitrophenylphosphate (40 mM) as substrate. Equimolar amounts (0.224 �M) of GST-Ypi1(5 �g) and GST-Sds22 (7.4 �g) produced in bacteria and yeast, respectively,were added alone or in combination to the reaction mixture. Combinations oflower amounts of GST-Ypi1 and GST-Sds22 were also assayed. GST (0.224 �M)was used as a control. Values represent the percentage of activity of Glc7phosphatase with respect to control assay without additions. Values aremeans of at least two different experiments (bars indicate standarddeviation).
FIGURE 6. Effect of conditional depletion of Ypi1 on cell growth. A,CML476 (wt, circles) and MMR09-4 (tetO7, triangles) strains were cultured inYPD at 28 °C in the absence (open symbols) or presence (filled symbols) ofdoxycycline (DOX) (100 �g/ml) as indicated under “Experimental Proce-dures,” and the growth of the cultures (A600) was monitored for 40 h. B, RT-PCRwas performed on total RNA isolated from the MMR09-4 strain grown in YPDin the presence or absence of doxycycline (100 �g/ml) for different timesusing the specific primers RT_YPI1_UP and RT_YPI1_DO (Table 2). Theexpected 400-bp DNA fragment is shown. C, MMR09-4 (tetO7:YPI1) cells weretransformed with a centromeric plasmid containing YPI1 regulated under itsown promoter (pRS316-Ypi1) or with an empty plasmid (pRS316). Transfor-mants were cultured in YPD at 28 °C in the absence (open symbols) or pres-ence (filled symbols) of doxycycline, as above.
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first tested whether the function of Sds22 could be replaced byYpi1 overexpression and vice versa. However, this was not thecase because overexpression of Sds22 could not rescue the
lethal phenotype of a ypi1::KanMXmutant (strain MMR18), and over-expression of Ypi1 was not able torescue the lethal phenotype of ansds22-5tsmutant at the nonpermis-sive temperature (strain SAY302;data not shown). Therefore, thefunctions of Ypi1 and Sds22 werenot interchangeable.If the functions of Ypi1 and Sds22
were related, we reasoned that itshould be possible to identify simi-lar cellular phenotypes as a result ofthe inactivation of any of these twoproteins. Because the deletion ofYPI1 is lethal (19), we constructedconditional mutants. Among thedifferent strategies tested, placingYPI1 under the control of a tetO7promoter at its own chromosomallocation (strain MMR09-4; see“Experimental Procedures”) gavethe best results. In this strain, YPI1expression could be switched off byaddition of doxycycline. As shownin Fig. 6A, addition of doxycycline tothe MMR09-4 strain resulted in adramatic inhibition of cell growth.RT-PCR analysis clearly showed amarked decrease of YPI1 mRNAlevels several hours after addition ofdoxycycline (Fig. 6B). The growthdefect was specifically because ofthe lack of Ypi1 because introduc-tion in strainMMR09-4 of a centro-meric plasmid expressing YPI1withits own promoter (pRS316-Ypi1)completely rescued the growthdefect in doxycycline-containingmedia (Fig. 6C). These data con-firmed a key role of Ypi1 in cellphysiology.It has been described that Sds22 is
essential for the progression frommetaphase to anaphase in the cellcycle and that sds22-deficient cellsare arrested in mid-mitosis withcondensed chromosomes and shortmitotic spindle (23). Therefore, wedecided to test whether the lack ofYpi1 could result in an equivalentphenotype. To this end, cells weresynchronized in S-phase in the pres-ence of doxycycline and the positionand number of nuclei monitored
after release from the blockage. As observed (Fig. 7A), the con-ditional tetO:YPI1 mutant (strain MMR09-4) suffers a severeblockage during anaphase, similarly to what it is observed in
FIGURE 7. DAPI staining of tetO:YPI1 and sds2-5ts conditional mutants. A, wild type strain CML476 and itstetO:YPI1 derivative (MMR09-4) were synchronized with �-factor and hydroxyurea (see “Experimental Procedures”),and finally resuspended in YPD medium containing 100 �g/ml doxycycline to resume growth. B, strains SAY306(wild type) and SAY302 (sds22-5ts) were synchronized as above (except that growth temperature was 24 °C). Cellswere resuspended in fresh YPD and shifted from 24 to 37 °C, and growth was resumed. In all cases culture sampleswere taken at different time intervals, fixed with 3.7% formaldehyde for 60 min, and stained with DAPI to visualizethe nuclei. A total of 300 cells in each time were classified into four groups according to the position and number ofnuclei (see schematic), and the results were plotted as a function of the time after release from the blockage, in thepresence of doxycycline (A) or after the shift to 37 °C (B). The experiment was carried out twice with similar results.Micrographs are examples of cultures after 180 min of release, in all cases.
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SAY302 cells, carrying a thermosensitive sds22 allele (sds22-5ts), shifted to the nonpermissive temperature (Fig. 7B). Fur-thermore, when we checked the length of themitotic spindle inypi1-deficient cells using an anti-tubulin antibody (Fig. 8), wecould observe that, whereas wild type cells presented longmitotic spindles, indicative of normal chromosome segrega-tion, in Ypi1-depleted cells the mitotic spindles were short andsimilar to the ones observed in the sds22-5ts mutant grown atthe nonpermissive temperature (Fig. 8). Wild type cells treatedwith doxycycline or incubated at the nonpermissive tempera-ture presented long mitotic spindles (data not shown).Because it has been suggested that Sds22 plays a role in the
nuclear localization of Glc7 (25), we also evaluated the possiblerole of Ypi1 in regulating this process. With this aim, we intro-duced a GLC7-yEmCitrine cassette (a generous gift fromDr. K.Tatchell) into strainMMR09-4 and examined cells grown in theabsence or presence of doxycycline. As shown in Fig. 9, fluores-cently labeled Glc7 was mostly localized in the nucleus in cellsexpressing Ypi1. However, in Ypi1-depleted cells, Glc7-derivedfluorescencewasmore diffuse, no longer nuclear, and occasion-ally presented a punctate pattern, suggesting that localization ofGlc7 was altered in the absence of Ypi1. This phenotype resem-bled the one observed in strain MMR13-4, which expressedGLC7-yEmCitrine in an sds22-5ts background, grown at the
nonpermissive temperature (Fig. 9), in agreement with a previ-ous report (25). These data suggested a common role of Ypi1and Sds22 in regulating Glc7 nuclear localization.Sds22 has also been found as a dosage suppressor of the tem-
perature-sensitive phenotype of the ipl1-321mutant (47). Ipl1is a protein kinase involved in the regulation of kinetochore-microtubule interactions and microtubule function, whoseactivity is antagonized by Glc7 (9, 47–49). It was suggested thatSds22 could act as a Glc7 chaperone that could titrate Glc7away from essential Ipl1 targets (47). To know whether Ypi1had the same effect, we overexpressed Ypi1 in an ipl1-321mutant and found that it suppressed the lethal phenotype of themutant grown at the nonpermissive temperature, as in the caseof overexpressing Sds22 (Fig. 10). These results suggested thatoverexpression of Ypi1 or Sds22 caused a decrease in the spe-cific activity of Glc7 toward a particular substrate related to theIpl1 protein kinase pathway.
DISCUSSION
It has been described that among other physiological pro-cesses, Glc7 PP1 phosphatase regulates cell cycle progression(2, 9, 10). Some of the functions of Glc7 in cell cycle progressionare achieved by its binding to Sds22, a regulatory subunit thattargets Glc7 to substrates involved in regulation of mitosis andchromosome segregation (20–23). Sds22 is an atypical Glc7regulatory subunit because it lacks the consensus (R/K)(V/I)X(F/W)motif present inmostGlc7 regulatory subunits. How-ever, despite the absence of this motif, Sds22 still binds to Glc7,and it does so through its central domain composed of 11leucine-rich repeats. Binding occurs at a site in Glc7 differentfrom the one used to bind RVXF motif-containing interactors.In fact, Sds22 binds to a domain in PP1, composed of �4, �5,and �6 helices, that is located far away from other well knownregulatory binding sites of PP1, such as the RVXF hydrophobicbinding channel, the �12-�13 loop, and the acidic groove,involved in binding of RVXF motif-containing interactors (24).The observation that the C-terminal half of PP1, including allresidues that contribute to the RVXF-binding channel, is notrequired for the interaction with Sds22 (24, 50) raises the inter-esting possibility that in Sds22-associated PP1 holoenzymesthis channel is free for interaction with a specific additionalRVXF-containing subunit. In thisway, Sds22-PP1holoenzymeswould resemble other trimeric PP1 holoenzymes known tocontain an RVXF-containing and an RVXF-less regulator, suchas the CPI-17-Mypt1-PP1 complex (51).In this study, we provide strong evidence indicating that
Ypi1, a recently identified inhibitory subunit of Glc7 phospha-tase containing a typical consensus (R/K)(V/I)X(F/W)-bindingmotif (19), interacts physically with both Sds22 andGlc7. Inter-estingly, overexpression of Ypi1 enhances the interactionbetween Sds22 and Glc7, and overexpression of Sds22enhances the interaction between Ypi1 and Glc7. A possibleexplanation for these results is that the overexpression of eitherYpi1 or Sds22 could stabilize the interaction between the othertwo components (Glc7-Sds22 or Glc7-Ypi1, respectively)and/or displace other endogenous regulatory subunits fromtheir binding to Glc7, making the phosphatase more availablefor interaction with the other component of a putative ternary
FIGURE 8. Tubulin staining. A, growth of MMR09-4 (tetO:YPI1) and SAY302(sds22-5ts) cells was synchronized as in the legend to Fig. 7. After 120 min ofrelease from the blockage, aliquots were prepared for indirect immunofluo-rescence using anti-tubulin antibody. B, data indicate the mean of the lengthof the mitotic spindle in 300 cells at mid-mitosis for each condition. dox,doxycycline.
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complex. This complex was confirmed by co-immunoprecipi-tation and gel filtration analyses. In the latter we found thatmost of the Sds22, Glc7, and Ypi1 proteins were present infractions corresponding to an estimated molecular mass of 130kDa. The existence of this complex would provide an expla-nation for the isolation in Schizosaccharomyces pombe, bygel filtration, of a trimeric complex of 105 kDa containingSds22, Sds21 (PP1), and an unidentified phosphoprotein of25 kDa (23). In this case, the 25-kDa unidentified protein could
be the S. pombe orthologue of Ypi1(GenBankTM accession numberCAB11073).We have also mapped the
domains involved in the interactionamong these three proteins, and ourresults suggest that the C-terminaldomain of Ypi1 is responsible forthe interaction with Sds22, whereasGlc7 binds to the N-terminaldomain of Ypi1, which indeed har-bors the PP1-binding (R/K)(V/I)X(F/W)motif.We have also notedthat binding of Sds22 toYpi1may beindependent of the presence of Glc7in the complex. This notion cameout from our finding that a Ypi1-W35A mutant, which binds Glc7very poorly because of an alteredRVXW motif (19), interacted withSds22 with the same strength aswild type (data not shown).We havealso observed that the N-terminalpart of Sds22 is dispensable for theinteraction with Glc7 (in agreementwith previous results (24)) and withYpi1, and that the LRR-cap domainis necessary but not sufficient forthe interaction with both Ypi1 and
Glc7. The latter resultsmay suggest that the LRR-cap domain isrequired for the proper folding of Sds22. One could envisagethat the binding of the RVXF-containing interactors to Glc7,like Ypi1, would not compete with the binding of Sds22,because both interactors bind Glc7 at separate locations. How-ever, binding of Sds22 and RVXF-containing interactors toGlc7 can also be mutually exclusive, as in the case of Gac1, anRVXF-containing regulator that targets Glc7 to substratesinvolved in glycogen metabolism (52).We also present strong evidence indicating that the func-
tions of Ypi1 and Sds22 are related, because the inactivation ofany of these two proteins results in similar cellular phenotypes.We demonstrate that upon Ypi1 depletion, Glc7 shows analtered subcellular localization. This phenotype is similar to theone observed upon inactivation of Sds22, indicating that bothYpi1 and Sds22 are necessary for the nuclear localization ofGlc7. Because Glc7 regulates cell cycle progression (2, 9, 10),the mislocalization of the phosphatase would lead to cellgrowth inhibition. In agreement with this suggestion, we haveobserved that depletion of Ypi1 caused cell growth arrest atmid-mitosis, and that this phenotype was similar to the oneobserved by inactivating Sds22 (25). Microscopic examinationof Ypi1-depleted cells revealed that these cells contained shortmitotic spindles, as in the case of sds22-5ts cells grown at thenonpermissive temperature. Therefore, the presence of bothproteins is required for Glc7 to perform its nuclear essentialfunctions. In addition, the function of Ypi1 and Sds22 is notredundant because overexpression of one of these regulatory
FIGURE 9. Depletion of Ypi1 and Sds22 affect Glc7 localization. Strain MMR11-1 (tetO:YPI1), which containsan integrated GLC7-yEmCitrine construct, was grown in the absence or presence of 100 �g/ml doxycycline(dox) as described under “Experimental Procedures.” Cells were taken after 25 h of growth and fixed for micro-scopic observation. Similarly, strain MMR13-4 (sds22-5ts), containing the same GLC7-yEmCitrine construct, wasgrown at 26 °C until an A660 of 0.5 and maintained at 26 °C or switched to 37 °C for 1 additional h. Fluorescenceof GLC7-yEmCitrine expression was detected by using a fluorescein isothiocyanate filter. Samples were alsoprocessed for DAPI staining.
FIGURE 10. Overexpression of Ypi1 or Sds22 suppresses the lethal pheno-type of ipl1-321 mutant grown at the nonpermissive temperature.SBY214 (wild type (WT)) and SBY322 (ipl1-321) cells were transformed withplasmids pWS93 (empty), pWS-Ypi1 and pWS-Sds22. Transformants weregrown at 30 °C in selective SC-2% glucose until they reached the exponentialphase (A660 0.5). 10-Fold dilutions of the cultures were prepared, and 3 �l ofeach were spotted on selective SC-2% glucose plates. Plates were then incu-bated at 30 or 37 °C for 48 h.
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subunits cannot suppress the lethal phenotype derived fromthe absence of the other.In this study we also show that both Ypi1 and Sds22 have, in
vitro, the ability to inhibit Glc7 and that the combination ofequimolar amounts of both proteins leads to an almost com-plete inhibition of Glc7 activity. The inhibitory role of Sds22 onPP1 activity has already been documented in higher eukaryotessuch as rat hepatocytes (53) and Schistosoma mansoni (54). Inaddition, in bovine spermatozoids, it is known that Sds22 inhib-its PP1�2 phosphatase activity. In these cells, the inhibitory roleof Sds22 is controlled by binding to an unknown protein of 17kDa, forming an inactive complex that is unable to bind PP1.Only when Sds22 is released from its binding to p17, is it able tobind and inhibit PP1 (55, 56). We think that the situation inyeast is different, because our triple-hybrid analysis suggeststhat the expression of either Ypi1 or Sds22 improves the inter-action between the other two components of a putative ternarycomplex. In addition, if the function of Ypi1 was to maintainSds22 in an inactive state, in Ypi1-depleted cells Sds22would befree to interact with PP1 and inhibit its function. However, ourphenotype experiments indicate that depletion of Ypi1 givessimilar phenotypes as impairing Sds22 function, which is notcompatible with the proposed model. The inhibitory ability ofYpi1 and Sds22 is consistent with the fact that overexpressionof any of these two proteins can suppress the lethal phenotypeof ipl1-321 mutants grown at the nonpermissive temperature.A possible explanation for these results could be that overex-pression of Ypi1 would inhibit Glc7 activity toward substratesinvolved in the Ipl1 protein kinase pathway, as already sug-gested for Sds22 (47).The fact that all the proteins present in the Glc7-Ypi1-Sds22
complex have well conserved orthologues throughout evolu-tion raises the interesting possibility that the function of eachcomponent and/or the function of the complex as a wholeshould be also conserved. We are currently investigatingwhether inhibitor 3 (PPP1R11; human orthologue of yeastYpi1) and hSds22 (PPP1R7; human orthologue of yeast Sds22)form a complex with PP1 (human orthologue of yeast Glc7),similar to the one we have described in yeast.
Acknowledgments—We thank Dr. Mike Stark and Dr. Kelly Tatchellfor strains, Dr. Jesus Avila for the anti-tubulin antibody, and Dr.Lynne Yenush for critical reading of the manuscript. We also thankDr. Ester Desfilis (Instituto Cabanilles, Valencia) for help with theYpi1 nuclear localization experiments.
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