The interactome of the yeast mitochondrial ribosome Organization of mitochondrial post-transcriptional regulation, membrane protein insertion and quality control Braulio Vargas Möller-Hergt Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 19 October 2018 at 13.00 in Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract The proteins found in mitochondria originate from two different genetic systems. Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into the organelle. However, a small subset of mitochondrial proteins is encoded in an organelle-resident genome. Mitochondria contain factors responsible for replication, transcription and, most important for this thesis, synthesis of the mitochondrially encoded proteins. In the course of evolution the mitochondria specific ribosomes were extensively remodeled. The reasons for many of these adaptations are currently not well understood. For example, the mitoribosome is less stable and abundant than its bacterial counterpart. Therefore, I contributed in the development of robust biochemical tools in order to isolate and analyze the intact yeast mitoribosome and interaction partners by mass spectrometry. The results revealed a higher order organization of mitochondrial gene expression in complexes that we termed MIOREX (mitochondrial organization of gene expression). Besides the mitoribosome, MIOREX complexes contain factors involved in all steps of gene expression. This study also established many new ribosomal interaction partners, among them some proteins that were previously completely uncharacterized. In order to study these proteins, I refined the mass spectrometry approach, allowing a subunit-specific assignment of ribosomal interaction partners. The Mrx15 protein was determined by this approach as an interactor of the large subunit. I established that Mrx15 has overlapping functions with the ribosome receptor Mba1. Both proteins are necessary for mitoribosome membrane attachment and co-translational Cox2 membrane insertion. In a subsequent study I found a functional interaction of MRX15 and MBA1 with the regulators of the membrane-bound AAA proteases of the mitochondrial quality control system. Furthermore, the absence of Mrx15 leads to increased, the absence of Mba1 to decreased proteotoxic stress resistance of yeast cells. These results demonstrate an interesting connection between the mitochondrial quality control and membrane insertion machineries, suggesting an early quality control step during the biogenesis of mitochondrially encoded proteins. In addition, we could reveal a subunit-specific interaction of translational activators and client mRNAs with the mitochondrial ribosome. This organization demonstrated how cytochrome b synthesis is pre-organized by specific translational activators independently of the COB mRNA. In summary, the work in this thesis showed how the vast and diverse interactome of the yeast mitoribosome organizes and regulates mitochondrial translation. These regulation mechanisms highlighted many organelle specific features. The work presented here will serve as starting point to design future studies aimed at a better understanding on how mitochondria adapted to organize gene expression inside the organelle. Keywords: Mitochondria, mitochondrial post-transcriptional regulation, mitochondrial ribosome, membrane protein insertion, mitochondrial quality control. Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-159455 ISBN 978-91-7797-404-8 ISBN 978-91-7797-405-5 Department of Biochemistry and Biophysics Stockholm University, 106 91 Stockholm
Transcript
The interactome of the yeast mitochondrialribosomeOrganization of mitochondrial post-transcriptional regulation,membrane protein insertion and quality controlBraulio Vargas Möller-Hergt
Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at StockholmUniversity to be publicly defended on Friday 19 October 2018 at 13.00 in MagnélisalenKemiska övningslaboratoriet, Svante Arrhenius väg 16 B.
AbstractThe proteins found in mitochondria originate from two different genetic systems. Most mitochondrial proteins aresynthesized in the cytosol and post-translationally imported into the organelle. However, a small subset of mitochondrialproteins is encoded in an organelle-resident genome. Mitochondria contain factors responsible for replication, transcriptionand, most important for this thesis, synthesis of the mitochondrially encoded proteins. In the course of evolution themitochondria specific ribosomes were extensively remodeled. The reasons for many of these adaptations are currently notwell understood. For example, the mitoribosome is less stable and abundant than its bacterial counterpart. Therefore, Icontributed in the development of robust biochemical tools in order to isolate and analyze the intact yeast mitoribosomeand interaction partners by mass spectrometry. The results revealed a higher order organization of mitochondrialgene expression in complexes that we termed MIOREX (mitochondrial organization of gene expression). Besides themitoribosome, MIOREX complexes contain factors involved in all steps of gene expression. This study also establishedmany new ribosomal interaction partners, among them some proteins that were previously completely uncharacterized. Inorder to study these proteins, I refined the mass spectrometry approach, allowing a subunit-specific assignment of ribosomalinteraction partners. The Mrx15 protein was determined by this approach as an interactor of the large subunit. I establishedthat Mrx15 has overlapping functions with the ribosome receptor Mba1. Both proteins are necessary for mitoribosomemembrane attachment and co-translational Cox2 membrane insertion. In a subsequent study I found a functional interactionof MRX15 and MBA1 with the regulators of the membrane-bound AAA proteases of the mitochondrial quality controlsystem. Furthermore, the absence of Mrx15 leads to increased, the absence of Mba1 to decreased proteotoxic stressresistance of yeast cells. These results demonstrate an interesting connection between the mitochondrial quality controland membrane insertion machineries, suggesting an early quality control step during the biogenesis of mitochondriallyencoded proteins. In addition, we could reveal a subunit-specific interaction of translational activators and client mRNAswith the mitochondrial ribosome. This organization demonstrated how cytochrome b synthesis is pre-organized by specifictranslational activators independently of the COB mRNA. In summary, the work in this thesis showed how the vastand diverse interactome of the yeast mitoribosome organizes and regulates mitochondrial translation. These regulationmechanisms highlighted many organelle specific features. The work presented here will serve as starting point to designfuture studies aimed at a better understanding on how mitochondria adapted to organize gene expression inside theorganelle.
Mitochondria, a host for cellular energy metabolism
Mitochondria are the host for many catabolic biochemical processes. The
TCA cycle, degradation of amino acids and the β-oxidation of fatty acids
generate reduction equivalents in the form of NADH and FADH2 in the
organelle. These reduction equivalents are used to synthesize ATP by oxida-
tive phosphorylation (OXPHOS). Mitochondria are compartmentalized by
two lipid bilayers. The outer and inner mitochondrial membranes surround
the matrix of the organelle (Figure 1A). The inner mitochondrial membrane
houses the respiratory chain, which is responsible for OXPHOS. The
OXPHOS system generally consists of four respiratory chain complexes
and the ATP synthase: (a) the NADH dehydrogenase (complex I); (b) the
succinate dehydrogenase (complex II); (c) the cytochrome c reductase
(complex III); (d) the cytochrome c oxidase (complex IV); (e) the ATP syn-
thase (complex V).
During OXPHOS electrons from NADH and FADH2 oxidation are fed
into the respiratory chain and employed to reduce molecular oxygen to wa-
ter. The electrons are channeled through the different respiratory complexes
by membrane-bound electron carriers in a series of exergonic redox reac-
tions [1]. In complex I, III and IV these reactions are coupled to an ender-
gonic transfer of protons across the inner mitochondrial membrane [2]. The
energy from the generated proton gradient between the mitochondrial ma-
trix and the intermembrane space is used by the ATP synthase to generate
ATP from ADP and inorganic phosphate (Figure 1B). This process is the
main source of chemical energy in the form of ATP in aerobic cells.
OXPHOS complexes are a mosaic from two genetic systems
Each of the OXPHOS complexes consists of several protein subunits and
prosthetic groups. Most protein subunits, like the other roughly 1000 mito-
chondrial proteins [3], are encoded in the nucleus, synthesized in the cytosol
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and post-translationally imported into the organelle (Figure 1A) [4,5]. How-
ever, a subset of the OXPHOS subunits is encoded in an organelle-resident
mitochondrial genome [6,7]. The organelle contains a full genetic system to
replicate, transcribe and translate the genes encoded in this genome.
Figure 1. (A) Mitochondria contain different membrane surrounded compartments. The inner mitochondrial membrane houses the OXPHOS system. The matrix contains an or-ganelle-resident genome and a full genetic system for transcription and mRNA translation. Mitochondrial proteins are either imported from the cytosol through TOM (translocase of the outer membrane) and TIM (translocase of the inner membrane) complexes or synthe-sized inside the matrix (adapted from [8]). (B) The mitochondrial OXPHOS system. Elec-trons released from NADH and succinate oxidation in complex I (CI) and complex II (CII) are transferred via complex III (CIII) to complex IV (CIV). Here the electrons are used to reduce molecular oxygen to water. The electron transfer is coupled to an endergon-ic proton transfer in CI, CIII and CIV from the matrix to the intermembrane space (IMS). The energy of the generated proton gradient is used by complex (CV) to generate ATP.
How did the organelle acquire its own genome? According to the now
widely accepted endosymbiont hypothesis mitochondria evolved from an α-
proteobacterium that was incorporated into an α-archaeal cell [9,10]. This
endosymbiotic event was a key step in the evolution of aerobic eukaryotes
[11]. During the course of evolution most of genes of the α-proteo-
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bacterium were lost, due to a lack of evolutionary pressure or transferred to
the nucleus, leaving behind the genes which constitute the current mito-
chondrial genome [12]. Interestingly, the genetic code was slightly altered
after the endosymbiotic event [13]. In Saccharomyces cerevisiae (S. cerevisiae) six
codons were reassigned and differ from the universal genetic code. In the
human mitochondrial genome four codons are also used differently. Codon
reassignments together with high hydrophobicity of mitochondrially encod-
ed proteins were suggested as reasons why some genes resisted transfer to
the nucleus [14].
The mitochondrial genome is organized and condensed within the matrix in
a so called nucleoid [15,16]. The specific mitochondrial genes vary between
organisms, but generally mitochondrial DNA encodes membrane protein
subunits of the OXPHOS system, rRNA of the mitochondria specific ribo-
somes (mitoribosomes) and tRNAs necessary for translation. Therefore, the
mitochondrial genome is dedicated towards the production of OXPHOS
subunits (Figure 2A). The genome size can vary from 15 to 75 kb [17]. The
size is not directly correlated to the number of genes that are encoded in the
respective genome. For example, the 75 kb S. cerevisiae mitochondrial ge-
nome encodes 8 polypeptides [18]. All are membrane protein subunits of
the OXPHOS system, with the exception of Var1, which is part of the small
subunit of the mitoribosome (Figure 2B). On the other hand, the human
mitochondrial genome is only 16.5 kb long and encodes 13 polypeptides, all
of which are OXPHOS subunits [19]. The size of the mitochondrial ge-
nome is therefore dependent on the presence and constitution of non-
coding regions. Long introns in the yeast mitochondrial genome account for
the larger size, whereas the human mitochondrial DNA is condensed and
contains only a small portion of non-coding sequences.
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Figure 2. (A) OXPHOS complexes of S. cerevisiae containing nuclear and mitochondrially encoded subunits. (B) Mitochondrial genomes of yeast and human (adapted from [20]).
The expression of mitochondrial genes is not self-sufficient and requires
nuclear gene products. Components necessary for transcription, RNA pro-
cessing and translation are synthesized in the cytosol and post-
translationally imported into the organelle (Figure 1A). Roughly 250 nuclear
genes are required for mitochondrial gene expression [21]. How the
OXPHOS subunits encoded in the mitochondrial genome are expressed,
inserted into the membrane and assembled into functional complexes, will
be discussed in the following chapters.
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The mitoribosome
According to the endosymbiont hypothesis the mitoribosome evolved from
an α-bacterial ancestor [9,10]. The bacterial ancestry of the mitoribosome is
reflected in a few similarities between the bacterial and mitochondrial ribo-
somes. The overall organization into a large and small subunit, consisting of
enzymatic rRNA and protein subunits, is similar between both systems. The
rRNA and protein domains involved in decoding and peptidyl transferase
catalysis show a high degree of conservation [20]. Additionally, both ribo-
somes are sensitive against the same groups of antibiotics [22,23].
Nevertheless, the mitoribosome has been extensively remodeled in the
course of evolution. The rRNA sequences of bacterial and mitoribosomes
differ significantly in length. The S. cerevisiae rRNA increased in size, by ac-
quiring mitochondria specific expansion segments (ES). In the mammalian
system the rRNA portion is dramatically reduced [24], leading to smaller
rRNAs with lower sedimentation coefficients [25]. The rRNA evolution was
accompanied in yeast and mammals by recruitment of additional mitochon-
dria-specific ribosomal proteins. The protein content of the mitoribosome
is further increased by N- and C-terminal extensions of the conserved pro-
tein subunits [26]. The addition of new protein subunits and extension of
conserved ribosomal proteins, accompanied with the loss of rRNA seg-
ments in mammals, lead to a shift in the rRNA to protein ratio in mitoribo-
somes compared to the bacterial ribosome (Figure 3A).
The identity and exact number of mitoribosomal proteins was long under
debate [27–30]. Due to the low abundancy of mitoribosomes crystal struc-
tures could not be obtained, therefore mitoribosome subunits were identi-
fied by mass spectrometry [31–36] or inferred from homology [37]. Only
the recent advancement in high resolution cryo-electron microscopy (EM)
[38,39] allowed the determination of molecular models for the yeast and
mammalian mitoribosomes [40–46]. In the structures the identities and po-
sitions of all mitoribosomal proteins were determined.
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Figure 3. (A) Protein and RNA content of the bacterial ribosome, yeast and human mi-toribosome (adapted from [25]). (B) Comparison of the central protuberance (red) of the bacterial ribosome with the yeast and human mitoribosomes (PDB 5DOX, 5MRC and 3J9M).
The mitoribosome structures revealed an architecture that differs substan-
tially from the bacterial ribosome (Figure 3B). The 11 rRNA expansion
segments in the large subunit of the yeast mitoribosome act as scaffold for
the mitochondria-specific proteins and spread over the whole secondary
structure [45]. These new elements occupy unique positions in the mitori-
bosome, restructuring for example intersubunit bridges [40]. In mammals
the rRNA loss is not completely compensated by the newly recruited ribo-
somal proteins, giving the mammalian mitoribosome a more porous struc-
ture when compared to its bacterial counterpart [20]. The different protein
and rRNA contents of the mammalian and yeast mitoribosomes become
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also apparent in their structures. The mitoribosomal proteins occupy differ-
ent positions, giving the different mitoribosomes distinct shapes (Figure
3B).
Mitochondria lack the 5S rRNA, which is part of the bacterial central pro-
tuberance in the large ribosomal subunit. In S. cerevisiae mitochondria the
central protuberance is restructured by expansion segments and newly re-
cruited mitoribosomal proteins. These new elements occupy the same posi-
tion as the bacterial 5S rRNA, but give the central protuberance a different
shape (Figure 3B)[45]. In mammals the missing 5S rRNA is compensated by
recruiting a mitochondrial tRNA into the large ribosomal subunit [43,44].
Although the central protuberance was remodeled its functions in transla-
tion fidelity and inter-subunit communication seem to be conserved [44].
The small mitoribosomal subunit was also restructured after the endosym-
biotic event. Because mitochondrial transcripts lack Shine-Dalgarno se-
quences, the 3´-end of the 15S and 12S rRNAs occupy different positions
in the yeast and mammalian mitoribosomes [40–42].
After decades of studying the components and architecture of the mitoribo-
some, only in recent years the determination of high resolution cryo-EM
structures solved some long standing questions in the field. These structures
revealed surprising differences between the mitochondrial and bacterial
translation machineries. Furthermore, species specific adaptions altered the
structures of mitoribosomes from mammals and yeast. For this reason, it
was proposed that the mitoribosomes co-evolved with the OXPHOS genes
encoded in the mitochondrial genome [47]. Accordingly, the evolution hap-
pened in two phases, the rRNA expansion, witnessed in the yeast mitoribo-
some, preceded the reduction of the rRNA content in the current mamma-
lian mitoribosome. During the rRNA reduction the recruitment of addition-
al proteins continued, accounting for the higher protein content of the
mammalian mitoribosome. However, the structures of the mitoribosomes
are just the starting point for detailed molecular studies of translation in
mitochondria. These will help in the future understanding processes like the
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evolution of different mitoribosomes, mechanisms of membrane protein
insertion and translation initiation or antibiotic side effects.
Translational activators
The concept of translational activators was proposed by Fox and Costanzo
in 1990 [48]. Accordingly, in S. cerevisiae the translation of the eight mito-
chondrially encoded mRNAs is dependent on a set of transcript specific
translational activators. These factors organize and regulate the translation
for their respective client mRNAs. Translational activators are characterized
by a functional [49] or physical interaction [50] with the 5´-untranslated
region (UTR) of their client transcript and defective mitochondrial transla-
tion in their absence. Mature mitochondrial mRNAs are different from their
cytosolic or bacterial counterparts. In mitochondria of S. cerevisiae the ma-
ture mRNAs do not contain a CAP structure or a 3´-end poly A tail. Instead
the 5´- and 3´-ends of all mature transcripts contain long (54-954 bp) UTRs,
that are required for their translation [51–57].
The 5´-UTR regulates the translation of the downstream coding sequence
by a functional interaction with a specific translational activator. The ab-
sence of this translational activator can be compensated by exchanging the
5´-UTR with the 5´-UTR of another mRNA. In this case the authentic
translational activator is replaced by the translational activator of the other
mRNA [58]. Many translational activators were identified by fusing 5´-
UTRs to reporter genes through biolistic transformation and screening for
required nuclear gene products [59]. The exact molecular function and role
in mitochondrial translation of the found translational activators have been
studied in some cases. The following table summarizes the known transla-
tion activators and their proposed functions:
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Table 1. Functions of S. cerevisiae translational activators (transl. act.).
Client Transl. act. Function(s)
COX1
Mss51
Peripheral membrane protein [60] that functionally and physically interacts with the COX1 mRNA 5´-UTR and coding region [61]. Regulates Cox1 synthesis through feedback loop [60].
Pet309
Integral membrane protein [62] that functionally [49] and physically [50] interacts with the COX1 mRNA 5´-UTR. Contains PPR domains, that are necessary for COX1 translation [63].
Mam33 Functional interaction with the COX1 mRNA 5´- or 3´-UTR, that is necessary for Cox1
synthesis [64].
Mss116 Interacts with Pet309 and is required for Cox1 synthesis. Also is involved in transcrip-tion and splicing [65].
COX2
Pet111
Firmly bound to the membrane [66]. Functionally interacts with predicted stem-loop structure [67] in the 5´-UTR of the COX2 mRNA [68,69]. Contains PPR domains [70] and is rate limiting for Cox2 synthesis [66]. Overexpression inhibits COX1 translation, probably by interacting with the translational activators of COX1 [71].
COX3
Pet54
Peripheral membrane protein [72], that functionally interacts with the COX3 mRNA 5´-UTR [73]. Physically interacts with Pet122 and Pet494 [74].
Pet122 Integral inner membrane protein [72], that functionally interacts with the COX3 mRNA 5´-UTR [73]. Functionally interacts with mS26 (Pet123) of the SSU [75].
Pet494 Integral inner membrane protein [72], that functionally interacts with the COX3 mRNA
5´-UTR [76] and is rate limiting for Cox3 synthesis [77]
COB
Cbp1
PPR protein [70], that stabilizes the COB mRNA by a functional interaction with the 5´-UTR [78,79]. Suggested to be necessary for COB mRNA translation in the absence of Pet127 [80].
Cbs1 Integral inner membrane protein [81] that functionally interacts with the 5´-UTR of the COB mRNA [82,83]. Interacts with actively translating ribosomes [84].
Cbs2 Peripheral membrane protein [81] that functionally interacts with the 5´-UTR of the
COB mRNA [82]. Binds to mitoribosomes [85].
Cbp3/Cbp6 Bind ribosomes at tunnel exit and functionally interact with the 5´-UTR of COB mRNA [86]. Bind newly synthesized cytochrome b for feedback regulation [87].
ATP8/6
Aep3
Peripheral membrane protein that stabilizes the ATP8/6 mRNA [88].
Smt1 Represses translation of the ATP8/6 mRNA [89].
ATP6 Atp22 Functional interaction with the 5´-UTR of the ATP6 mRNA [90].
ATP8 Aep3 PPR protein that is required for Atp8 expression [91]. Required for mIF2-dependent initiation of mitochondrial mRNAs with unformylated initiator tRNAs [92]
ATP9
Aep1 Aep2
PPR proteins [70], that functionally interact with the ATP9 mRNA and are required for Atp9 expression [93,94].
VAR1
Sov1
PPR protein [70], that functionally interacts with the 5´-UTR of the VAR1 mRNA [95]. Belongs to group of mitochondrial translation control proteins that extend life span upon deletion [96].
- 24 -
Post-transcriptional regulation by translational activators
Apart from the interaction with the 5´-UTR, the functions of translational
activators are diverse (Table 1). Therefore, different roles in post-
transcriptional regulation by translational activators have been found and
proposed:
(A) mRNA-mitoribosome interaction: As many translational activators
directly interact with the ribosome [85] and in some cases the mRNA
[50] it was proposed that they aid the mRNA-mitoribosome interac-
tion, specifically for start site selection during initiation. Mature mito-
chondrial mRNAs lack Shine-Dalgarno sequences, which are important
for translation initiation in bacteria [97]. Translational activators can
compensate this loss by playing an active role in translation initiation.
This hypothesis was supported by the presence of an unidentified elec-
tron density in the structure of the yeast mitoribosome [40]. The densi-
ty contacts the mRNA exit canyon, a part of the ribosome that has
been shown to functionally interact with 5´-UTRs [98]. Therefore, the
authors proposed that the density represents a mixture of co-purified
translational activators and the mRNA exit canyon acts as platform for
the binding of translational activators to the yeast mitoribosome.
(B) Localization of the mRNA to the inner mitochondrial membrane: Al-
most all translational activators are integral membrane proteins or pe-
ripherally associated with the inner mitochondrial membrane (Table 1).
Therefore, translational activators could recruit mRNAs to the mem-
brane by binding the 5´-UTR of their client transcript. However, it is
not clear at the moment if all translational activators physically interact
with their client mRNA.
(C) Stabilization of mRNA: Cbp1 is necessary for cytochrome b (COB)
mRNA stabilization [78,79]. Many other translational activators are re-
quired for accumulation of their client transcript. Pet309 is necessary
for accumulation of the COX1 mRNA [49]. However, Pet309 is only
required in the presence of introns in the COX1 mRNA. Since the
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splicing of mitochondrial transcripts depends on translation by the mi-
toribosome, it is not clear if this destabilization originates from the ab-
sence of the translational activator or a splicing defect. The intronless
COX2 transcript is destabilized in the absence of Pet111 [68]. But, in
this case it is not clear if the translational activator directly stabilizes the
mRNA or the translation inhibition generally destabilizes mitochondrial
transcripts. It remains to be elucidated, if somer translational activators
directly stabilize their client mRNA, like the Cbp1 protein.
(D) Coordination of nuclear and mitochondrial gene expression: The trans-
lational activator Mss51 binds to the newly produced Cox1 after syn-
thesis [61]. Mss51 stays bound to its client protein until a certain inter-
mediate in the assembly of complex IV is formed. The formation of
the assembly intermediate depends on the import of nuclear imported
subunits. Until the intermediate is assembled the translational activator
is sequestered in the assembly process and not free to induce new
rounds of Cox1 synthesis [60]. This type of regulation represents an el-
egant way of coordinating nuclear and mitochondrial gene expression
during the assembly of OXPHOS complexes and will be discussed in
detail on the example of cytochrome b biogenesis.
(E) Rate limit for mitochondrial translation: Some translational activators
are rate limiting for the translation of their client mRNA [77]. There-
fore, the availability of a translational activator represents an important
part of post-transcriptional gene regulation in mitochondria. Further-
more, it was shown that yeast mitochondrial transcript levels are not
changing dramatically in response to a shift to a non-fermentable car-
regulation and translational activators seem to be more important for
respiratory growth than transcriptional activation of OXPHOS subu-
nits.
(F) Organization of respiratory chain assembly: The translational activators
of the COX1, 2 and 3 mRNAs have been shown to interact physically
- 26 -
on the inner mitochondrial membrane [100]. On this basis, it was hy-
pothesized that they organize complex IV assembly prior synthesis of
mitochondrially encoded complex IV subunits. Through the physical
interaction of the COX mRNA translational activators, the synthesis of
complex IV subunits is guided to the same compartment. Interestingly,
the COB and COX1 mRNA translational activators Cbp1 and Pet309
were found together in a large complex [101]. As complex III and IV
build higher order assemblies in the form of supercomplexes, this in-
teraction could direct the translation of both complexes of the same
compartment for efficient complex formation [102]. Furthermore,
Pet309 interacts directly with the mitochondrial transcription and mat-
uration machinery, suggesting a direct link of transcription, maturation
and translation of mitochondrial mRNAs [100,101].
Cytochrome b biogenesis
Cytochrome b synthesis illustrates several features by which translation acti-
vators regulate mitochondrial protein synthesis and will be therefore dis-
cussed in detail. The translation of the COB mRNA depends on five tran-
script specific translational activators. Cbp1 was shown to stabilize the ma-
ture COB mRNA by interacting with the 5´-UTR, prior to translation
[78,79]. The translational activators Cbs1 and Cbs2 are integral and periph-
eral membrane proteins, respectively [81]. Both proteins functionally inter-
act with the COB 5´-UTR [82,83] and the mitoribosome [84,85]. Cbs1 and
Cbs2 probably recruit the COB mRNA to the ribosome and the inner mito-
chondrial membrane.
The translational activators Cbp3 and Cbp6 build a complex that interacts
with the COB 5´-UTR and the mitoribosome [86]. The Cbp3/6 complex
interacts with the newly produced cytochrome b after synthesis [87]. Upon
hemylation and Cbp4 interaction with cytochrome b, Cbp3/6 dissociates
from the complex III assembly intermediate [103]. If assembly of cyto-
chrome b is arrested the Cbp3/6 complex is sequestered in the assembly
intermediate and consequently cytochrome b synthesis is downregulated
- 27 -
(Figure 4). Therefore, the Cbp3/6 complex is not only required for transla-
tion of the COB mRNA, but also for regulating cytochrome b synthesis ac-
cording to the efficiency of complex III assembly. Since the assembly of
complex III depends on nuclear encoded subunits and assembly factors,
this feedback loop couples mitochondrial to nuclear gene expression.
Figure 4. Cytochrome b synthesis is regulated by the Cbp1,3,6 and Cbs1,2 translational activators. After protein synthesis Cbp3/6 binds to cytochrome b until hemylation is com-pleted and Cbp4 binds to cytochrome b. As long as Cbp3/6 is sequestered in the assembly process COB mRNA translation is downregulated.
Translation activators in mammals
Mammalian mitochondrial transcripts differ from the described yeast
mRNAs. They lack long 5´-UTRs and contain 3´-end poly-A tails [104,105].
As the interaction with the 5´- UTR is one of the defining characteristics of
translational activators, it is not surprising that only a few homologs have
been found in the mammalian system. The LRPPRC (Leucine-rich PPR
motif-containing) protein is a distant homolog of Pet309 and mutations
cause French-Leigh syndrome, which is characterized by complex IV defi-
ciency [106]. It was shown that the LRPPRC protein is involved in post-
transcriptional gene regulation in mammalian mitochondria by influencing
mRNA stability, polyadenylation and translation [107,108]. Contrary to the
yeast translation activators, the LRPPRC protein is not client specific. The
TACO1 (Translational activator of Cox1) protein was proposed to be a
human translation activator of Cox1 [109]. No yeast homolog of TACO1
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was found so far, but unlike the LRPPRC protein it seems to be specific for
the synthesis of Cox1. How TACO1 modulates Cox1 synthesis is currently
not well understood. The human homologs of Cbp3 (UQCC1) and Cbp6
(UQCC2) seem to play a similar role as their yeast counterparts. UQCC1
and UQCC2 form a stable complex that interacts with newly synthesized
cytochrome b [110]. Additionally, in the absence of UQCC2 cytochrome b
synthesis or stability is decreased.
The different evolution of yeast and mammalian mitochondria is reflected
in different genomes, transcripts and ribosome structures. Therefore, alt-
hough some proteins with similarities to yeast translational activators have
been described, it is likely that mitochondrial translation is regulated differ-
ently in both systems.
Mitochondrial membrane protein insertion
Mitoribosome membrane attachment
In bacteria ribosomes are targeted to the membrane for protein insertion by
a dedicated process [111]. Nascent chains destined for co-translational
membrane insertion contain N-terminal signal sequences. These sequences
are recognized by the signal recognition particle (SRP) which targets the
ribosome nascent chain complex to the membrane by interacting with the
SRP receptor. Homologs for all these key components are absent in mito-
chondria [112]. Additionally, mitoribosomes seem to be permanently at-
tached to the inner mitochondrial membrane [113–116] and contrary to
bacteria, the interaction with the membrane is independent of the nascent
chain [117]. Since mitochondrial ribosomes produce almost exclusively
membrane proteins, this adaptation of the ribosome membrane interaction
probably occurred to accommodate the membrane insertion of mitochon-
drially encoded proteins [118].
- 29 -
Figure 5. (A) Structures of the large ribosomal subunit of yeast (PDP 3J6B) and human (PDB 3J7Y) mitochondria. Points of membrane attachment are the rRNA expansion seg-ment (96-ES1, red) or mL45 (blue). The path of the tunnel and the protein domains form-ing the tunnel exit are highlighted in both structures. (B) Cryo-electron tomography struc-ture of the S. cerevisiae mitoribosome (EMD 2826), showing two domains of membrane attachment (96-ES1 and Mba1; adapted from [116]). (C) Hypothetical model of membrane protein insertion in yeast mitochondria. Mba1 and Mdm38 interact with the mitoribosome ensuring correct alignment of the nascent chain for membrane insertion. Oxa1 translocates the nascent polypeptide chain across the membrane.
How is the mitoribosome attached to the inner mitochondrial membrane in
the absence of the SRP and SRP receptor? The high resolution structure of
the yeast large ribosomal subunit revealed a membrane facing protuberance,
consisting of an mitochondria specific rRNA expansion segment (Figure
5A)[45]. Cryo-electron tomography of yeast mitochondria showed that this
expansion segment (96-ES1) is one of two contact sites of the mitoribo-
- 30 -
some with the inner mitochondrial membrane [116]. The second contact
site is not part of the mitoribosome, but the peripheral membrane protein
Mba1 (Figure 5B). Biochemical evidence showed that Mba1 acts as a mi-
toribosome membrane receptor [113,114]. On this basis, it was proposed
that the interaction with Mba1 tethers the mitoribosome to the inner mito-
chondrial membrane and aligns the tunnel exit for insertion into the mem-
brane. The mL45 protein is the human homolog of Mba1. It is a permanent
ribosomal subunit and occupies a similar site on the mitoribosome (Figure
5A) [46]. The mammalian mitoribosome lacks the yeast rRNA expansion
segment (96-ES1). Tomography studies of human mitochondria confirmed
that the mL45 protein is the only membrane contact site of the human mi-
toribosome [119]. As the overall orientation of the large ribosomal subunit
was similar in yeast and human, the Mba1/Mrpl45 proteins seem to play a
general role in the mitoribosome membrane attachment. However, the sec-
ond contact point of the yeast mitoribosome and the dynamic mitoribo-
some association of Mba1 indicate different adaptations in both systems.
Additionally, it seems that the position of the tunnel exit was altered in the
yeast mitoribosome (Figure 5A). Accordingly, the path of the tunnel is dif-
ferent in the LSU of the yeast mitoribosome.
Mitochondrial membrane protein insertion
Mitochondria not only lack the membrane targeting machinery, but also the
protein conducting Sec channel [112]. Instead, insertion of S. cerevisiae mito-
chondrially encoded proteins into the inner mitochondrial is carried out by
Oxa1 [120]. This protein is universally conserved and belongs to the
YidC/Oxa1/Alb3 family, with members in bacteria, mitochondria and chlo-
roplasts [121]. All members are characterized by five transmembrane seg-
ments responsible for membrane protein insertion [122]. The soluble C-
terminal α-helical domain of Oxa1 binds to the mitoribosome and is re-
quired for membrane protein insertion [123]. Crosslinking studies mapped
the interaction site close to the tunnel exit proteins uL23 and uL24
[26,124,125]. This biochemical data is in line with a cryo-EM structure of
Oxa1 reconstituted with the Escherichia coli ribosome, in which Oxa1 binds
- 31 -
to the bacterial homologs of uL23 and uL24 [126]. Furthermore, Oxa1 in-
teracts directly with nascent polypeptide chains emerging from the tunnel
exit, probably to support co-translational membrane protein insertion [113].
The previously discussed ribosome receptor Mba1 is also a component of
the mitochondrial membrane protein insertion machinery. Mba1 interacts
directly with nascent chains and is required for efficient membrane insertion
of Cox2 [113]. Genetic evidence also points to a role of MBA1 in mem-
brane insertion, as MBA1 functionally interacts with the C-terminal domain
of Oxa1[114]. Mba1 forms a complex with Mdm38 [127], another inner
membrane protein that binds to the yeast mitoribosome and Oxa1 (Figure
5C) [128]. The molecular mechanism by which Oxa1 and Mba1 insert mito-
chondrially encoded proteins into the inner membrane is currently not fully
understood, but will be discussed in the following chapter on the example
of Cox2.
Cox2 membrane insertion
A stem loop structure in the 5´-UTR of the S. cerevisiae COX2 mRNA is
recognized by Pet111 [68,69]. This translational activator is membrane-
bound and recruits the COX2 mRNA to the inner mitochondrial membrane
[67–69]. The newly synthesized Cox2 protein (pCox2) consists of an N-
terminal signal sequence, two transmembrane domains and a C-terminal tail.
After successful translocation across the inner mitochondrial membrane the
N-terminal sequence is cleaved off by the inner membrane peptidase com-
plex, consisting of Imp1/2 and Som1 [129–131]. The processed Cox2
(mCox2) is then assembled into complex IV. During and after synthesis
Oxa1 is required for efficient export of the soluble N- and C-terminal tails
across the membrane to the intermembrane space [132]. The group of
Thomas Fox established by an elegant reporter system that the Oxa1 de-
pendent export of the N-terminus is independent of membrane potential,
but the C-terminus requires it, suggesting that both domains are exported
by a distinct mechanism [133].
- 32 -
Figure 6. Cox2 translation is regulated by the translational activator Pet111. The Oxa1 and Mba1 proteins are necessary for the export of the N-tail across the membrane. After suc-cessful translocation the N-terminal signal sequence is delivered to the Imp1/Imp2/Som1 complex by Mba1 and Cox20. The C-tail is translocated subsequently across the mem-brane. This process is dependent on Cox18, Mss2, Pnt1, Cox20 and Oxa1. Cox2 biogenesis is completed by the formation of CuA center.
While Oxa1 is required for N-and C-terminal translocation of Cox2, the
Cox18 protein is specifically necessary for the export of the C-tail [134].
Cox18 is an integral membrane protein with a role in Cox2 accumulation
and complex IV assembly [135]. It was shown that Cox18 interacts with
Mss2 and Pnt1 [134]. Both proteins are necessary for the export of the
Cox2 C-tail [136,137]. A role of the Cox18/Mss2/Pnt1 complex in protein
translocation is supported by the fact that Cox18 is a member of the
YidC/Alb3/Oxa1 family [138]. Interestingly, Cox18 lacks the C-terminal
ribosomal binding site of Oxa1. Translocation of the C-tail was suggested to
happen post-translationally and in this case the interaction with the mitori-
bosome would not be required [138].
Efficient translocation of Cox2 across the inner mitochondrial membrane
also depends on Mba1. Upon deletion of MBA1 the pCox2 form accumu-
lates and cells grow slower on respiratory media [114,127]. As discussed
before, Mba1 interacts directly with the large ribosomal subunit aligning the
tunnel exit for membrane protein insertion [116].
- 33 -
The Cox20 protein is another factor that is required for Cox2 biogenesis
[139]. This membrane-bound chaperone binds to the newly synthesized
pCox2 and is required for N-terminal processing as well as C-tail export
[140]. Furthermore, Cox20 interacts with the mitoribosome, Mba1 and
Cox18 in a Cox2 dependent manner [141]. Therefore, it was suggested that
Mba1 together with Cox20 plays an active role in delivering pCox2 to the
Afterwards Mba1 dissociates from pCox2 and the inner membrane pepti-
dase (Imp1/2, Som1) complex cleaves of the N-terminal signal sequence.
The Cox18/Mss2/Pnt1 complex together with Cox20 exports the C-tail
into the intermembrane space and biogenesis of Cox2 is completed by for-
mation of the CuA center by the metallochaperone Sco1 [142].
Degradation of mitochondrially encoded membrane proteins
Mitochondrial quality control
The mitochondrial quality control system is responsible for protein biogen-
esis and homeostasis in the organelle. The degradation of mitochondrially
encoded proteins is carried out by a set of organelle-specific chaperones and
proteases [143]. The proteases of the mitochondrial quality control system
belong to the AAA+ (ATPases associated with various cellular activities)
superfamily of proteins [144]. Members are characterized by a 200-250 ami-
no acids long ATP-binding (AAA) domain consisting of a Walker A and B
motif and other conserved domains [145]. The AAA domain provides the
energy for conformational changes (unfolding and membrane extraction) of
substrate proteins, which are degraded by a proteolytic domain. Mitochon-
drial AAA proteases are classified into three families, according to their
bacterial homologs:
(A) The Lon/Pim1 proteases degrade oxidatively damaged proteins in
the mitochondrial matrix [146,147].
(B) The mtClpXP protease is only present in mammals with no homo-
logs in yeast mitochondria. The exact function of the protease
complex is currently not understood [148].
- 34 -
(C) Mitochondrial FtsH homologs are membrane-bound proteases,
which are divided into m-AAA and i-AAA proteases, dependent on
the localization of their active site in the matrix or intermembrane
space [149].
Membrane-bound mitochondrial AAA proteases
In yeast mitochondria non-assembled inner membrane proteins are degrad-
ed by the hetero-oligomeric m-AAA protease complex, that consists of
Afg3 (or Yta10) and Yta12 (or Rca1) complex [150]. Degradation of mito-
chondrially encoded membrane proteins by the m-AAA protease is nega-
tively regulated by the Phb1 and Phb2 proteins [151]. Both proteins form a
complex that has been suggested to function as membrane-bound chaper-
one of newly synthesized mitochondrial translation products, protecting
them for pre-mature degradation [152]. On the other hand, the Phb1/Phb2
complex was suggested to act as membrane scaffold, defining functional
subcompartments at the inner mitochondrial membrane [153]. How this
function alters the degradation of proteins at the inner mitochondrial mem-
brane by the m-AAA protease is currently not understood.
The i-AAA protease is a homo-oligomeric complex, consisting of the Yme1
membrane protein [154,155]. Yme1 is positively regulated by the Mgr1 and
Mgr3 complex [156,157]. The complex binds directly to the i-AAA protease
and probably aids substrate binding. Since the function of Mgr1 and Mgr3
was only studied on model substrates, it was not known which proteins in
the inner mitochondrial membrane depend for their proteolysis on these
regulators of the i-AAA protease. However, a recent study found that deg-
radation of mitochondrial outer membrane proteins depends on the
Mgr1/Mgr3 complex and the i-AAA protease [158]. Substrates at the inner
mitochondrial membrane are to this date not identified.
- 35 -
Figure 7. The i-AAA and m-AAA proteases, their regulators and substrates
Since the catalytic domains of the AAA proteases are on opposite sides of
the inner mitochondrial membrane, it was assumed that the substrates of
the proteases are recognized either in the intermembrane space or the ma-
trix. However, the group of Thomas Langer showed that the m-AAA and i-
AAA protease were able to degrade the same model substrate [159]. Ac-
cordingly, substrate transmembrane segments need to be extracted by the
proteases prior or during degradation [160]. The substrate recognition by
both membrane-bound AAA proteases is degenerate and seems to be only
dependent on the presence of unfolded soluble domains in the intermem-
brane space or the matrix [161]. The Pim1/Lon protease is a high copy
number suppressor of the m-AAA protease, suggesting that in the absence
of membrane-bound proteases the soluble AAA protease can degrade
membrane-bound substrates [162]. If this is happening in vivo or how the
different substrates are delivered to the proteases is currently not known.
Several substrates of the i-AAA protease have been determined so far. A
genetic study found YME1 (the i-AAA protease) among other genes to be
necessary for Cox2 degradation, when assembly of complex IV is inhibited
[163]. Degradation of Cox2 by the i-AAA protease requires the previously
- 36 -
discussed chaperone Cox20 [164]. In the absence of cytochrome c, Cox2
and Cox3 cannot be assembled into complex IV and their degradation de-
pends on the i-AAA protease [165]. Interestingly, Cox3 was also described
as substrate for the m-AAA protease, together with Cox1 and cytochrome b
[165]. Furthermore, the m-AAA protease is required for degradation of
non-assembled Atp9 and carries out a role in the assembly of the ATP syn-
thase (Figure 7) [150].
The m-AAA complex also controls respiratory chain formation [166]. Fur-
thermore, processing of the intron containing COB and COX1 transcripts
seems to be dependent on the presence of the m-AAA protease. It is cur-
rently not known how the m-AAA protease influences splicing of theses
mitochondrial transcripts. The dual role in degradation and assembly of
respiratory chain subunits is interesting but also complicates the studies of
mitochondrial membrane protein degradation. The absence of the m-AAA
protease can be compensated by high copy numbers of the mitochondrial
membrane insertion machinery, Oxa1 and Mba1 [162]. But, as both pro-
teins are also involved in respiratory chain assembly it is not clear at the
moment which function the high copy numbers are suppressing
[141,167,168]. The bacterial homologs of the m-AAA protease, Oxa1 and
the regulators Phb1/Phb2 are forming a complex [169]. The Oxa1 homolog
YidC apparently acts as a chaperone aiding degradation of misassembled
membrane proteins. Therefore, bacterial quality control appears to happen
very early during or after membrane protein insertion. If the mitochondrial
quality control system and membrane protein insertion machinery also in-
teract physically is currently not known. However, the discussed genetic
evidence points to a functional interaction. It remains an exciting task to
study how mitochondrial protein synthesis, membrane insertion, respiratory
chain assembly and quality control are connected and regulated.
- 37 -
Aims
Mitochondria are responsible for the expression of a handful of OXPHOS
subunits. Transcription and translation of the genetic information in the
mitochondrial genome is therefore critical for ATP production by
OXPHOS. The mitoribosome was extensively remodeled in the course of
evolution. All adaptions lead to a translation machinery dedicated towards
production of a small amount of hydrophobic proteins in an oxidatively
challenging environment. The organelle-specific translation regulation
mechanisms are currently not well understood, as they are not strongly re-
lated to the bacterial system.
The studies presented in this thesis focused on the yeast mitoribosome and
protein interaction partners. Despite many efforts and decades of research
in mitochondrial translation, to this date no in vitro translation system could
be established. This causes a number of open questions concerning the ex-
act molecular function of proteins that interact with the mitoribosome. Fur-
thermore, the mitoribosome is less stable and abundant compared to bacte-
rial counterparts, making biochemical studies of the mitochondrial transla-
tion apparatus challenging. Therefore, this thesis was aimed at establishing
robust biochemical tools for the analysis of the yeast mitoribosome. These
tools were designed in order to find and study the molecular functions of
mitoribosome interaction partners and consequently understand the mito-
chondria specific adaptations of the translation machinery. Working with
the facultative anaerobe S. cerevisiae allowed me to study the function of
components that are essential for respiratory growth.
- 38 -
- 39 -
Summary of papers
Paper I: Organization of mitochondrial gene expression in two
distinct ribosome-containing assemblies
Mitochondria contain an organelle specific genome and house all compo-
nents necessary for transcription, RNA maturation and translation of the
encoded genetic information. In this study we established conditions that
allowed the efficient isolation of intact mitoribosomes and interacting pro-
teins.
Figure 8. Experimental procedure for the purification of the mitoribosome and interac-tors. Proteins were identified after immunoprecipitation (IP) by label free quantitative mass spectrometry and protein classes represented in a pie chart (LS, low salt; HS, high salt; LSU, large subunit; SSU, small subunit; CR, chaperones; DNA, proteins involved in DNA metabolism; ME, metabolic enzymes; OXPHOS, components of the oxidative phosphory-lation system; PA, proteases; RNA, proteins involved in RNA metabolism; TL, translation; UC, uncharacterized proteins)
- 40 -
We found a vast and diverse interactome of the S. cerevisiae mitoribosome
organized in large assemblies (Figure 8). These assemblies probably escaped
detection so far, as the mitoribosome is very unstable during isolation and
native interactions were lost in the process. We determined that factors in-
volved in mRNA metabolism, mitoribosome assembly, DNA metabolism
together with translational activators, chaperones and proteases are physi-
cally associated with the mitoribosome. Accordingly, we termed these as-
semblies MIOREX, for mitochondrial organization of gene expression.
STED microscopy of yeast mitochondria visualized two different subsets of
MIOREX complexes. One subset is very close to the mitochondrial nucle-
oid, organizing all steps of mitochondrial gene expression in close proximi-
ty. The exact role of the different MIOREX complexes is currently not
known. They could serve as platforms in order to channel mRNAs from
transcription to translation within one complex. The organization into high-
er order complexes is a common theme in mitochondria. Respiratory chain
complexes, ER-mitochondria contact sites and protein import machineries
are all organized in large assemblies. The exact necessity of these organelle
specific adaptations remains to be elucidated but demonstrate the striking
differences between the mitochondrial and bacterial evolution.
Paper II: The ribosome receptors Mrx15 and Mba1 jointly or-
ganize cotranslational insertion and protein biogenesis in mito-
chondria
The yeast mitoribosome almost exclusively produces membrane protein
subunits of the OXPHOS complexes. This specialization allowed many
mitochondria specific adaptations of the membrane protein insertion mech-
anism. First, the mitoribosome is permanently attached and not targeted to
the inner mitochondrial membrane. The interaction with the membrane
depends on the mitoribosome receptor Mba1. This protein interacts with
the mitoribosome and is in direct contact with nascent polypeptide chains.
Second, protein translocation in mitochondria is independent of the Sec
translocon. Instead proteins are inserted into the membrane by Oxa1.
- 41 -
Figure 9. (A) Procedure to isolate MIOREX complexes and separate mitoribosomal subu-nits. (B) Sedimentation profiles of proteins from the large and small ribosomal subunit. Interactors of the large and small ribosomal subunit were identified by determining sedi-mentation profiles of individual proteins and comparing them to the ribosomal subunits (LSU, large subunit; SSU, small subunit; qMS, label free quantitative mass spectrometry).
In this study we refined the biochemical analysis of mitoribosome interac-
tors, initiated in paper I (Figure 9A). This allowed us to establish subunit-
specific interactomes of the large and small ribosomal subunits (Figure 9B).
Among the interactors of the large subunit we found a previously uncharac-
terized protein, that we renamed Mrx15. We showed that Mrx15 is a mem-
brane protein in the inner mitochondrial membrane and interacts with the
ribosome via a soluble C-terminal domain. Furthermore, chemical cross-
linking established that Mrx15 contacts nascent polypeptides chains. The
MRX15 gene is dispensable for respiratory growth, but required upon sim-
ultaneous deletion with MBA1. In the absence of Mrx15 and Mba1 the mi-
toribosome interacts less efficiently with the inner mitochondrial membrane
and the precursor form of Cox2 accumulates. This leads to a global com-
plex IV deficiency causing the respiratory deficient phenotype. On this ba-
sis, we proposed an overlapping role of Mba1 and Mrx15 in mitoribosome
membrane attachment and co-translational membrane protein insertion.
This study also allowed us to assign the interacting subunits of known in-
teractors of the ribosome. As the function of most ribosomal interactors is
poorly understood, this will help future studies in establishing the exact
molecular function of these proteins.
- 42 -
Paper III: Insertion defects of mitochondrially encoded proteins
burden the mitochondrial quality control system
Mitochondrially encoded proteins are degraded by AAA proteases. These
proteases are part of the mitochondrial quality control system, which is re-
sponsible for proteostasis and degradation of non-native polypeptides.
Membrane-bound AAA proteases are large oligomeric complexes and char-
acterized by the position of their active site in the intermembrane space (i-
AAA) or matrix (m-AAA). The activity of either AAA- protease is modulat-
ed by a class of protease specific regulators. In this study, we combined
yeast genetics with disk diffusion assays and in organello translation to study
the mitoribosome receptors Mba1 and Mrx15. We found that in the ab-
sence of Mrx15, yeast cells become more resistant against proteotoxic
stress. Mba1 was shown to have overlapping functions with Mrx15, but
yeast cells become hypersensitive against proteotoxic stress in the absence
of Mba1. This result implied different functions of Mba1 and Mrx15, apart
from their role in membrane protein insertion. To further characterize the
role of the ribosome receptors in stress tolerance we tested functional inter-
actions of MRX15 and MBA1 with components of the mitochondrial quali-
ty control system. We found a mild growth retardation on respiratory media
upon simultaneous absence of Mrx15 and either of the m-AAA protease
regulators Mgr1 and Mgr3. However, analogous to cells lacking Mba1, yeast
cells became hypersensitive against proteotoxic stress upon deletion of
MGR1 or MGR3. On the other hand, MBA1 showed functional interac-
tions with the regulators of the i-AAA and m-AAA proteases, MGR1,
MGR3 and PHB1. The functional connection between the mitochondrial
membrane protein insertion and quality control machineries suggests an
early regulatory mechanism, directing mitochondrially encoded proteins
during or shortly after synthesis towards OXPHOS complex assembly or
degradation.
- 43 -
Paper IV: Organization of cytochrome b synthesis in yeast mi-
tochondria
In paper I we identified the vast and diverse protein interactome of the
yeast mitoribosome in the MIOREX complex. Among the interactors we
found almost all known translational activators. We therefore investigated
subsequently how the translational activator-mitoribosome interaction is
modulated by the presence or absence of their client mRNA. For this pur-
pose we split the ribosomal subunits by magnesium depletion and separated
them subsequently on a sucrose gradient. We checked by northern and
western blotting, if mRNAs and translational activators co-migrated with
the small or the large subunit (Figure 10).
Figure 10. Sucrose gradient analysis of ribosomal interaction partners. Isolated mitochon-dria were lysed in the presence of EDTA to split ribosomal subunits. The subunits were separated from each other and the rest of the lysate by a continuous sucrose gradient. Af-terwards individual fractions of the gradient were analyzed by SDS PAGE and western blotting or an agarose gel with subsequent northern blotting.
Surprisingly, first we discovered that not all mitochondrial mRNAs are in-
teracting with the small ribosomal subunit. As translational initiation usually
begins by loading the mRNA on the small ribosomal subunit we expected
- 44 -
all mitochondrial transcripts to be associated with the small subunit. How-
ever, we demonstrated that the COB mRNA co-sediments with the large
subunit in a sucrose gradient. On the other hand the ATP9 mRNA co-
sedimented with the small ribosomal subunit. Next we tested how the trans-
lational activators of the COB and ATP9 mRNAs interact with the mitori-
bosome. We showed that all known cytochrome b translational activators
interacted with the large subunit, the ATP9 mRNA translational activators
with the small subunit. To test if the interaction with the large subunit is
mediated by the mRNA or the translational activators we studied the trans-
lational activator-mitoribosome interaction in the absence of the COB
mRNA. All translational activator remained bound to the large subunit, with
the exception of Cbs1. In order to gain an understanding how Cbs1 is local-
ized on the mitoribosome we employed a proximity based labeling ap-
proach. The experiment revealed that Cbs1 is closely localized to proteins
of the small subunit and the tunnel exit. All these results implied a dynamic
interaction of Cbs1 with the mitoribosome. On this basis, we suggested a
subunit-specific interaction of translational activators with either the small
or large subunit of the mitoribosome. This interaction is, in the case of cy-
tochrome b synthesis, independent of the mRNA and therefore pre-
organizes the translation of one specific transcript, prior loading onto the
small ribosomal subunit.
- 45 -
Conclusions and outlook
Mitochondria contain organelle-resident ribosomes, which are responsible
for the translation of mitochondrially encoded mRNAs. The mitoribosome
was adapted in the course of evolution to optimize the expression of mito-
chondrially encoded membrane proteins. The adaptations of the protein
and rRNA content were accompanied by new protein interaction partners
of the mitoribosome. The identity and function of the mitoribosomal inter-
action partners was the main subject of the thesis. The work presented here
revealed an overall organization of gene expression in mitochondria in the
MIOREX complex, new components responsible for membrane protein
insertion and mitoribosome membrane attachment, a functional link of
membrane protein insertion and protein degradation and the regulation
mechanism of cytochrome b synthesis by translational activators.
Organization of mitochondrial gene expression by the MIOREX
complex
The MIOREX complex represents a new conceptual framework of gene
expression organization. Transcription and translation of nuclear encoded
genes are spatially separated. After transcription the mRNA is exported out
of the nucleus and translation is carried out in the cytosol. The MIOREX
complex suggests that freely diffusing mRNAs do not exist in mitochondria.
Instead, mRNAs are channeled after transcription to the mitoribosome.
This hypothesis is supported by a study in which mRNAs were artificially
introduced into mitochondria, failed to be translated [170]. We propose that
these mRNA fail to be translated, because they are not channeled to the
mitoribosome within the MIOREX complex.
Fluorescent microscopy studies in mammalian cells identified granules in
mitochondria that are located closely to the mitochondrial genome. Because
these granules are enriched in newly synthesized RNA and factors for RNA
processing and decay, they were named mitochondrial RNA granules [171–
- 46 -
174]. Components responsible for mRNA maturation and mitoribosome
assembly are also part of the mitochondrial RNA granules [175–177]. A
subset of ribosomal proteins was also found within the mitochondrial RNA
granules. It was suggested that ribosome assembly occurs inside the gran-
ules. This is different from the MIOREX complex, where we find fully in-
tact, assembled mitoribosomes. Due to the inherent instability of the mi-
toribosome it is not clear at the moment, if the entire mitoribosome is pre-
sent in the granules or the found ribosomal subunits are only engaged in
assembly. There is a striking functional overlap among the other compo-
nents found in the MIOREX complex and mitochondrial RNA granules.
Nevertheless, it is not known if the mitochondrial granules fulfill the same
function as the MIOREX complexes in mitochondrial gene expression.
Further studies employing the biochemical tools developed for the MI-
OREX isolation are necessary to determine if the mammalian mitochondria
also employ a higher order organization of all steps of gene expression.
How MIOREX complexes are internally organized is completely unknown.
Work presented in this thesis already started determining the role of some
uncharacterized MIOREX components (paper II and III), determining the
function of the other MIOREX parts remains an exciting task for the fu-
ture.
Functions of Mrx15 and Mba1 in protein biogenesis
The MIOREX component Mrx15 was shown to interact with the large ri-
bosomal subunit. Mrx15 is an integral membrane protein and interacts with
the mitoribosome through a soluble C-terminal domain. Genetic evidence
confirmed overlapping functions of Mrx15 with the ribosome receptor
Mba1. We showed that in the absence of Mrx15 and Mba1 the mitoribo-
some interacts less efficiently with the inner mitochondrial membrane.
Nevertheless, a significant portion of the mitoribosome is still attached to
the membrane. The high resolution structure and cryo-electron tomography
of the yeast mitoribosome showed that one point of membrane attachment
- 47 -
consists of an mitochondria-specific rRNA expansion segment [45,116].
Our results suggest that this segment is sufficient to attach a certain portion
of the large ribosomal subunit to the inner mitochondrial membrane. This
hypothesis was supported by a study showing that the large subunit is al-
ready membrane-bound during mitoribosome assembly [178]. Membrane
attachment is only dependent on the incorporation of the 16S rRNA, where
the previously mentioned expansion segment is located. This suggests a
more specific role of Mba1 and Mrx15 in mitoribosome membrane attach-
ment. This role is probably related to membrane protein insertion, as indi-
cated by the interaction with nascent chain of both proteins.
The functions of Mba1 and Mrx15 are not completely redundant. For ex-
ample Mrx15 cannot fully compensate the absence of Mba1. Furthermore,
our experiments showed that Mrx15 is quantitatively attached to the large
ribosomal subunit, whereas Mba1 dissociates from the mitoribosome even
under mild salt conditions. This is in line with a recently discovered func-
tion of Mba1 in Cox2 maturation [141]. The authors showed that Mba1
escorts the newly synthesized Cox2 from the ribosome to the inner mem-
brane peptidase for proteolytic cleavage. It is tempting to speculate that
Mrx15 replaces Mba1 upon dissociation form the mitoribosome for effi-
cient membrane protein insertion. However, it is currently not clear if both
proteins are able to bind simultaneously to the mitoribosome.
The absence of Mba1 and Mrx15 mostly affected Cox2 biogenesis. We
showed that the Cox2 processing is defective when both proteins are miss-
ing. Processing depends on the co-translational export of the Cox2 N-tail
by Mba1 [114,141]. Therefore, Mrx15 is probably necessary for co-
translational export of Cox2. However, a role of Mrx15 in post-translational
export of the C-tail by the Cox18/Mss2/Pnt1 cannot be excluded at this
stage [134,136–138]. Furthermore, if the functions of Mrx15 and Mba1 are
limited to Cox2 biogenesis, or if both proteins play a more general role in
mitochondrial membrane protein insertion is currently not known.
- 48 -
We tested the sensitivity of yeast cells against proteotoxic stress in the ab-
sence of either Mrx15 or Mba1. We found opposite effects on growth in
their absence. Cells lacking Mba1 became hypersensitive against the induced
stress, whereas cells lacking Mrx15 could grow at all tested concentrations
of the employed antibiotic. This result confirmed independent functions of
Mba1 and Mrx15. Interestingly, Mba1 was initially discovered as a multi
copy bypass of the m-AAA protease [162]. The m-AAA protease is a com-
ponent of the mitochondrial quality control system and responsible for the
degradation of non-native polypeptides [144]. Additionally we showed that
MBA1 functionally interacts with regulators of the i-AAA and m-AAA pro-
teases. All this data is consistent with a role of Mba1 in the turnover of mi-
tochondrially encoded proteins.
Subsequently, we tested functional interactions between Mrx15 and regula-
tors of the mitochondrial AAA proteases. In contrast to MBA1, we found
only interactions with the regulators of the i-AAA protease, MGR1 and
MGR3. Both Mgr1 and Mgr3 promote turnover of non-native mitochondri-
al proteins by the i-AAA protease [156,157]. As Mrx15 is responsible for
Cox2 biogenesis, this functional interaction indicates turnover of Cox2 is
dependent on Mgr1 and Mgr3. This is supported by several studies, in
which Cox2 was shown to be degraded by the i-AAA protease [163–165].
How the functions of Mba1 and Mrx15 in protein turnover relate to their
roles in membrane protein insertion is not understood. However, it is
tempting to speculate that nascent chains are directed towards biogenesis or
degradation during or shortly after synthesis. The mitochondrial quality
control system could act during this early stage to avoid the accumulation
and aggregation of hydrophobic proteins. In the MIOREX complex we
found the m-AAA and i-AAA proteases together with the above mentioned
regulatory proteins, suggesting that the mitochondrial quality control system
is in close proximity to the mitoribosome. Additionally, the bacterial homo-
logs of the m-AAA protease and Oxa1 interact [169], showing that quality
control of bacterial inner membrane proteins occurs during translocation.
Further studies will be necessary to test if mitochondrial quality control
- 49 -
system directly interacts with the mitoribosome in order to probe the integ-
rity of nascent polypeptides.
Control of cytochrome b synthesis by translational activators
In paper IV we found a subunit-specific interaction of translational activa-
tors and the mitoribosome. As this interaction is independent of the
mRNA, we suggest that the synthesis of mitochondrially encoded protein is
pre-organized on the mitoribosome by the respective translational activa-
tors. This would create a subset of specialized ribosomes that are dedicated
towards the synthesis of one specific protein. This hypothesis was already
proposed in 2010 and is supported by the data presented in paper IV [8].
As discussed previously, the role of the COB mRNA translational activators
Cbp3 and Cbp6 is understood very well. Both proteins form a complex that
regulates cytochrome b synthesis through a feedback loop [87]. But, how
the Cbp3/6 complex initiates the translation of COB mRNA is still not
clear. Cbp3 was shown to bind to the tunnel exit of the large ribosomal
subunit [86]. Initiation of translation is executed on the small subunit. It is
therefore puzzling how the Cbp3/6 complex can induce translation of the
COB mRNA. By determining how the COB mRNA and translational activa-
tors interact with the mitoribosome we discovered a subunit specific organ-
ization of cytochrome b synthesis. Most importantly Cbp3 and Cbs1 were
not able to bind simultaneously to the large ribosomal subunit in the ab-
sence of the COB mRNA. These results indicate that binding of Cbp3 to
the tunnel exit of the large ribosomal subunit causes a rearrangement of
Cbs1 on the mitoribosome to the small subunit. This rearrangement would
allow Cbs1 to place the COB mRNA onto the small subunit for translation
initiation. Our results could finally explain how Cbp3 triggers the initiation
of cytochrome b synthesis. Additionally, we provide for the first time evi-
dence that translational activators play an active role in mitochondrial trans-
lation initiation as was suggested earlier (see Post-transcriptional regulation
- 50 -
by translational activators). However, the role of Cbs2 in this process is not
clear at the moment and will have to be studied in more detail.
- 51 -
Sammanfattning på svenska
Proteinerna som finns i mitokondrier härrör från två olika genetiska system.
De flesta mitokondriella proteiner produceras i cytosolen och importeras
post-translationellt till organellen. En liten del mitokondriella proteiner
kodas emellertid i ett organellspecifikt genom. Mitokondrier innehåller
faktorer som är ansvariga för replikation, transkription och translation.
Mitoribosomerna är mindre stabila och rikligare än dess bakteriella förfäder.
Därför bidrog jag till utvecklingen av robusta biokemiska verktyg för att
isolera och analysera intakta jäst mitoribosomer och dess
interaktionspartner genom masspektrometri. Resultaten avslöjade en högre
organisation av mitokondriellt genuttryck i ett komplex som vi betecknade
MIOREX (mitochondrial organization of gene expression). Förutom
mitoribosomen innehåller MIOREX-komplexet faktorer som är
involverade i alla steg av genuttryck. Denna studie upptäckte många nya
partnerproteiner till mitroribosomer, varav några var tidigare helt
okarakteriserade. För att studera dessa proteiner, förfinade jag
masspektrometri-metoden, vilket möjliggjorde en identifiering av
interaktionen mellan den specifika subenheten och partnerproteinerna.
Proteinet Mrx15 fastställdes genom detta tillvägagångssätt som en interaktör
till den stora subenheten. Jag visade att Mrx15 är nödvändigt för
interaktionen mellan mitoribosomerna och membranet, membran-
insättningen av Cox2, samt har överlappande funktioner med
mitoribosomens receptor Mba1. I en efterföljande studie hittade jag en
funktionell interaktion av MRX15 och MBA1 med regulatorer för det
mitokondriella kvalitetskontrollen AAA-proteaser. Dessutom leder
frånvaron av Mrx15 till en ökad proteotoxisk stressresistens hos jästceller.
Frånvaron av Mba1 leder till en minskat proteotoxisk stressresistens. Dessa
resultat visar intressanta samband mellan den mitokondriella
kvalitetskontrollen och membranproteininsättningen. Detta tyder på en
tidig kvalitetskontroll i biogenesen av mitokondriellt kodade proteiner.
Vidare kunde vi avslöja en subenhetsspecifik interaktion mellan
translationaktivatorer och dess klient mRNA med mitoribosomer. Denna
organisation visade hur syntesen av cytokrom b är förorganiserad av
- 52 -
specifika ”translational activators” oberoende av COB mRNA.
Sammanfattningsvis visade arbetet i denna doktorsavhandling hur den stora
och varierande interaktionen av jästmitoribosomen organiserar och reglerar
mitokondriell translation. Dessa regleringsmekanismer visade många
organellspecifika särdrag. Arbetet som presenteras här kommer att användas
som utgångspunkt för att utveckla framtida studier som syftar till en bättre
förståelse för hur mitokondrier är anpassade för att organisera genuttryck i
organellen.
- 53 -
Acknowledgments
Many people contributed to the work presented here. All the great scien-
tists, whose shoulders this thesis stands on are acknowledged in the refer-
ence list. Here I want to express my gratitude to the people that worked
directly with me and helped me over the course of my doctoral thesis. First
I would like to thank Martin Ott, for giving me the opportunity to work in
his group. His passion for science and introduction into the fascinating
world of mitochondrial biology were greatly appreciated. Furthermore, I am
thankful for the co-supervision of Gunnar von Heijne and the mentorship
of Dan Daley.
I would like to thank all my co-authors, for contributing to our research and
stimulating discussions. Kirsten, your dedication and seemingly endless
knowledge about mitochondrial biology was a tremendous help and missed
once you finished your thesis. Sharing your brilliance with everyone in the
lab was one of your many great qualities. Additionally, the groups of Thom-
as Langer and Axel Imhoff helped me with the mass spectrometry, that was
a big part of my thesis.
The “Ott group” changed considerably over the course of my thesis, from
being an exclusively German group, to having members from Sweden,
Cameroon, Italy, Spain, Poland and India. The help in the beginning of the
“Germans on the fifth floor”, in the persons of Manfred, Kirsten, Steffi,
Markus and Kathi was greatly appreciated. Tamara, you started your PhD in
the same month as me and I enjoyed sharing this rollercoaster ride with
you. I also want to thank our first non-german group members Lorena,
Mama, Alex and Roger for the good times in the lab and apologize for the
terrible German radio we had to listen to in the beginning (it was not up to
me).
I want to thank the colleagues from DBB who were responsible for creating
a great work environment. Jacob (J-bird; owner of a green hat), we pretty
much instantly became friends and had a lot of great times and beers over
- 54 -
the last years. Povilas and Aziz, it was great sharing an office with you, hear-
ing Povilas with his headphones on in the background singing some rock
song and discussing life with Aziz. I want to thank Rickard, Kiavash and
Johan for teaching together with me and helping out when my limited Swe-
dish did not suffice. I am also thankful to have met and discussed with
I want to thank the people in the cryo-EM facility at the science for life
laboratory. Martha and Julian, although my cryo-EM studies did not make it
into the thesis I learned a lot from you guys. During the long cryo-EM ses-
sion late into the night Jens stayed to the end. Thank you for the ballerina
cookies you shared to give us the extra energy in order to set up data collec-
tions.
Many thanks go to the students that worked with me over the years. Espe-
cially Andreas, it was a pleasure accompanying you from your master thesis
to becoming a PhD student. I am sure many great discoveries wait in the
future of your scientific career.
I want to thank Doris, Jacob, Hannah and Martin for proofreading this the-
sis. Caro, Andreas and Jens, thank you for helping with the sammanfattning
på svenska.
My family made me who I am today. For this I have to thank mostly two
people, my mother and my sister. Mama, although being a biologist your-
self, you were surprised when I came home one day from school, fascinated
by the picture of an eukaryotic cell and switched my interests from politics
and history to science. As always, you helped and encouraged me on this
path. Carolina, I admire your passion and integrity. Our discussions about
politics, ethics and scrubs will always inform my decisions. I also want to
thank my extended family, Agneta and Kjell. When we relocated to Sweden
you helped us tremendously.
- 55 -
Last but not least I want to thank Caro. As with anything in my Life since I
met you, this journey to Sweden and my PhD was more fun and easier with
you by my side. I cannot express how much you helped me and I do not
know where I would be without you. You accompanied me through every
step in this thesis, picked me up when I was down and we celebrated to-
gether the good results. Now I am excited and cannot wait to begin a new
chapter in our lives.
- 56 -
- 57 -
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