The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.
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The mitochondrial respiratory chain (MRC) is a key energy transducer in eukaryotic cells. Four respiratory chain complexes cooperate in the transfer of electrons derived from various metabolic pathways to molecular oxygen, thereby establishing an electrochemical gradient over the inner mitochondrial membrane that powers ATP synthesis. This electron transport relies on mobile electron carries that functionally connect the complexes. While the individual complexes can operate independently, they are in situ organized into large assemblies termed respiratory supercomplexes. Recent structural and functional studies have provided some answers to the question of whether the supercomplex organization confers an advantage for cellular energy conversion. However, the jury is still out, regarding the universality of these claims. In this review, we discuss the current knowledge on the functional significance of MRC supercomplexes, highlight experimental limitations, and suggest potential new strategies to overcome these obstacles.
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Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded.
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The complexes mediating oxidative phosphorylation (OXPHOS) in the inner mitochondrial membrane consist of proteins encoded in the nuclear or the mitochondrial DNA. The mitochondrially encoded membrane proteins (mito-MPs) represent the catalytic core of these complexes and follow complicated pathways for biogenesis. Owing to their overall hydrophobicity, mito-MPs are co-translationally inserted into the inner membrane by the Oxa1 insertase. After insertion, OXPHOS biogenesis factors mediate the assembly of mito-MPs into complexes and participate in the regulation of mitochondrial translation, while protein quality control factors recognize and degrade faulty or excess proteins. This review summarizes the current understanding of these early steps occurring during the assembly of mito-MPs by concentrating on results obtained in the model organism baker's yeast.
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Aim: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.
Methods: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.
Results: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.
Conclusion: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.
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Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.
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Cytochrome c oxidase is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of cytochrome c oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here, experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of cytochrome c oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.
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Motivation: Cells respond to environments by regulating gene expression to exploit resources optimally. Recent advances in technologies allow measuring the abundances of transcripts, proteins, lipids and metabolites. These highly complex datasets reflect the state of the different layers in a biological system. Multi-omics is the integration of these disparate methods and data to gain a clearer picture of the biological state. Multi-omic studies of the proteome and metabolome are becoming more common as mass spectrometry technology continues to be democratized. However, knowledge extraction through integration of these data remains challenging.
Results: Connections between molecules in different omic layers were discovered through a combination of machine learning and model interpretation. Discovered connections reflected protein control over metabolites. Proteins discovered to control citrate were mapped onto known genetic and metabolic networks, revealing that these protein regulators are novel. Further, clustering the magnitudes of protein control over all metabolites enabled prediction of five gene functions, each of which was validated experimentally. Two uncharacterized genes, YJR120W and YLD157C, were accurately predicted to modulate mitochondrial translation. Functions for three incompletely characterized genes were also predicted and validated, including SDH9, ISC1, and FMP52. A website enables results exploration and also MIMaL analysis of user-supplied multi-omic data.
Availability: The website for MIMaL is at https://mimal.appCode for the website is at https://github.com/qdickinson/mimal-websiteCode to implement MIMaL is at https://github.com/jessegmeyerlab/MIMaL.
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Many aspects of mitochondrial gene expression are still unknown, which can be attributed to limitations in molecular tools. Here, we present a protocol to introduce reporter genes into the mitochondrial genome of budding yeast, Saccharomyces cerevisiae. Mitochondrially encoded reporter constructs can be used to interrogate various aspects of mitochondrial gene expression. The power of this technique is exemplified by a mitochondrially encoded nanoluciferase, which allows to monitor levels of mitochondrial translation under a variety of growth conditions.
10.1016/j.xpro.2022.101359

A key function of mitochondria consists of energy conversion, performed with the help of the respiratory chain and the ATP synthase. Biogenesis of these essential molecular machines requires expression of nuclear and mitochondrially encoded genes. We describe our current understanding how these processes are coordinated and how they are organized in specific areas of the inner membrane to facilitate the assembly of these sophisticated complexes.
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Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3-Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains: an N-terminal domain present in mitochondrial Cbp3 homologs and a highly conserved C-terminal domain comprising a ubiquinol-cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-cross-linking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.
10.1074/jbc.RA119.010483

The mitochondrial proteome contains proteins from two different genetic systems. Proteins are either synthesized in the cytosol and imported into the different compartments of the organelle or directly produced in the mitochondrial matrix. To ensure proteostasis, proteins are monitored by the mitochondrial quality control system, which will degrade non-native polypeptides. Defective mitochondrial membrane proteins are degraded by membrane-bound AAA-proteases. These proteases are regulated by factors promoting protein turnover or preventing their degradation. Here we determined genetic interactions between the mitoribosome receptors Mrx15 and Mba1 with the quality control system. We show that simultaneous absence of Mrx15 and the regulators of the i-AAA protease Mgr1 and Mgr3 provokes respiratory deficiency. Surprisingly, mutants lacking Mrx15 were more tolerant against proteotoxic stress. Furthermore, yeast cells became hypersensitive against proteotoxic stress upon deletion of MBA1. Contrary to Mrx15, Mba1 cooperates with the regulators of the m-AAA and i-AAA proteases. Taken together, these results suggest that membrane protein insertion and mitochondrial AAA-proteases are functionally coupled, possibly reflecting an early quality control step during mitochondrial protein synthesis.
10.3390/cells7100172

The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.
10.1038/s41598-018-29567-4

Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2-/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.
10.1038/s41419-017-0144-y

Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles' morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization.
10.1038/cddis.2017.448

Mitochondria are organelles critical for the functionality of eukaryotic cells. One of their most prominent functions is energy conversion, thereby producing most of the cellular ATP. Energy conversion relies on the oxidative phosphorylation system, an ensemble of large protein complexes that include the respiratory chain and the ATP synthase. Biogenesis of this machinery requires the coordination of two separate genetic systems, namely nuclear and mitochondrial gene expression. Recent research into the molecular causes of aging have revealed a prominent contribution of mitochondrial gene expression on many aspects of degenerative processes that typically involve cellular stress signaling pathways. In this review, we summarize recent developments in attempting to identify the molecular mechanism by which dysfunction of mitochondrial gene expression activates cellular stress signaling pathways and how this affects organismal aging. By comparing data obtained in various model organisms, we identify conserved and species-specific aspects of this mitochondria-to-nucleus signaling.
10.1007/s00441-016-2460-4

The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinol-cytochrome c reductase; cyt. bc1) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc1 In the present study, we have investigated the kinetics of ligand binding, the single-turnover reaction of CytcO with O2, and the linked cyt. bc1-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1Δ and rcf2Δ, respectively). The data show that in the rcf1Δ and rcf2Δ strains, in a significant fraction of the population, ligand binding occurs over a time scale that is ∼100-fold faster (τ ≅ 100 μs) than observed with the wild-type mitochondria (τ ≅ 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc1-CytcO supercomplex. Furthermore, in the rcf1Δ and rcf2Δ strains, the single-turnover reaction of CytcO with O2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electron-transfer bridge from cyt. bc1 to CytcO via a tightly bound cyt. c We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc1 and CytcO.
10.1073/pnas.1601196113

The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.
10.1091/mbc.E15-12-0811

The mitochondrial citric acid cycle is a central hub of cellular metabolism, providing intermediates for biosynthetic pathways and channeling electrons to the respiratory chain complexes. In this study, we elucidated the composition and organization of the multienzyme complex α-ketoglutarate dehydrogenase (α-KGDH). In addition to the three classical E1-E3 subunits, we identified a novel component, Kgd4 (Ymr31/MRPS36), which was previously assigned to be a subunit of the mitochondrial ribosome. Biochemical analyses demonstrate that this protein plays an evolutionarily conserved role in the organization of mitochondrial α-KGDH complexes of fungi and animals. By binding to both the E1-E2 core and the E3 subunit, Kgd4 acts as a molecular adaptor that is necessary to a form a stable α-KGDH enzyme complex. Our work thus reveals a novel subunit of a key citric acid-cycle enzyme and shows how this large complex is organized.
10.1091/mbc.E14-07-1178

Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.
10.1083/jcb.201401009

Significance: The mitochondrial genetic system is responsible for the production of a few core-subunits of the respiratory chain and ATP synthase, the membrane protein complexes driving oxidative phosphorylation (OXPHOS). Efficiency and accuracy of mitochondrial protein synthesis determines how efficiently new OXPHOS complexes can be made.
Recent advances: The system responsible for expression of the mitochondrial-encoded subunits developed from that of the bacterial ancestor of mitochondria. Importantly, many aspects of genome organization, transcription, and translation have diverged during evolution. Recent research has provided new insights into the architecture, regulation, and organelle-specific features of mitochondrial translation. Mitochondrial ribosomes contain a number of proteins absent from prokaryotic ribosomes, implying that in mitochondria, ribosomes were tailored to fit the requirements of the organelle. In addition, mitochondrial gene expression is regulated post-transcriptionally by a number of mRNA-specific translational activators. At least in yeast, these factors can regulate translation in respect to OXPHOS complex assembly to adjust the level of newly synthesized proteins to amounts that can be successfully assembled into respiratory chain complexes.
Critical issues: Mitochondrial gene expression is determining aging in eukaryotes, and a number of recent reports indicate that efficiency of translation directly influences this process.
Future directions: Here we will summarize recent advances in our understanding of mitochondrial protein synthesis by comparing the knowledge acquired in the systems most commonly used to study mitochondrial biogenesis. However, many steps have not been understood mechanistically. Innovative biochemical and genetic approaches have to be elaborated to shed light on these important processes.
10.1089/ars.2012.4896

Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc(1) complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc(1) complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc(1) complex assembly.
10.1083/jcb.201206040

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady-state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.
10.1016/j.jmb.2012.07.018

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.
10.1038/cdd.2011.180

Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript [corrected]. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.
10.1083/jcb.201103132

Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.
10.1515/BC.2011.013

The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co-translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co-factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially-encoded proteins.
10.1002/bies.201000081

Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.
10.1091/mbc.e10-02-0101

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.
10.1074/jbc.M110.113837

The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.
10.1091/mbc.e08-12-1162