Research Archive

The mitochondrial genome (mtDNA) encodes essential subunits of the electron transport chain and ATP synthase. Mutations in these genes impair oxidative phosphorylation, compromise mitochondrial ATP production and cellular energy supply, and can cause mitochondrial diseases. These consequences highlight the importance of mtDNA quality control (mtDNA-QC), the process by which cells selectively maintain intact mtDNA to preserve respiratory function. Here, we developed a high-throughput flow cytometry assay for Saccharomyces cerevisiae to track mtDNA segregation in cell populations derived from heteroplasmic zygotes, in which wild-type (WT) mtDNA is fluorescently labeled and mutant mtDNA remains unlabeled. Using this approach, we observe purifying selection against mtDNA lacking subunits of complex III (COB), complex IV (COX2) or the ATP synthase (ATP6), under fermentative conditions that do not require respiratory activity. By integrating cytometric data with growth assays, qPCR-based mtDNA copy-number measurements, and simulations, we find that the decline of mtDNAΔatp6 in populations derived from heteroplasmic zygotes is largely explained by the combination of its reduced mtDNA copy number—biasing zygotes toward higher contributions of intact mtDNA—and the proliferative disadvantage of cells carrying this variant. In contrast, the loss of mtDNAΔcob and mtDNAΔcox2 cannot be explained by growth defects and copy-number asymmetries alone, indicating an additional intracellular selection against these mutant genomes when intact mtDNA is present. In heteroplasmic cells containing both intact and mutant mtDNA, fluorescent reporters revealed local reductions in ATP levels and membrane potential near mutant genomes, indicating spatial heterogeneity in mitochondrial physiology that reflects local mtDNA quality. Disruption of the respiratory chain by deletion of nuclear-encoded subunits (RIP1, COX4) abolished these physiological gradients and impaired mtDNA-QC, suggesting that local bioenergetic differences are required for selective recognition. Together, our findings support a model in which yeast cells assess local respiratory function as a proxy for mtDNA integrity, enabling intracellular selection for functional

Mitochondrial DNA (mtDNA) copy number regulation remains incompletely understood, despite its importance in cellular function. In Saccharomyces cerevisiae, Mrx6 belongs to the Pet20-domain-containing protein family, consisting of Mrx6, Pet20, and Sue1. Notably, absence of Mrx6 leads to increased mtDNA copy number. Here, we identify the C-terminus of Mrx6 as essential for its stability and interaction with the mitochondrial matrix protein Mam33. Deletion of Mam33 mimics the effect of Mrx6 loss, resulting in elevated mtDNA copy number. Bioinformatics, mutational analyses, and immunoprecipitation studies corroborate that a subcomplex of Mam33 and Mrx6 trimers interacts with the substrate recognition domain of the conserved mitochondrial Lon protease Pim1 through a bipartite domain in the Pet20 domain of Mrx6. Loss of Mrx6, its paralog Pet20, Mam33, or mutations disrupting the interaction between Mrx6 and Pim1 stabilize key proteins required for mtDNA maintenance, the RNA polymerase Rpo41 and the HMG-box-containing protein Cim1. We propose that Mrx6, alongside Pet20 and Mam33, regulates mtDNA copy number by modulating substrate degradation through Pim1. Additionally, Mrx6 loss alters Cim1's function, preventing the detrimental effect on mtDNA maintenance observed upon Cim1 overexpression. The presence of three Pet20-domain proteins in yeast implies broader roles of Lon protease substrate recognition beyond mtDNA regulation.

The inner membrane of mitochondria contains many membrane-embedded carrier proteins of the SLC25 family to facilitate the exchange of metabolites between the cytosol and mitochondria. These carriers use a specific import route for their biogenesis that relies on the TIM22 complex as an inner membrane translocase. The molecular details of carrier biogenesis are not well understood. Using an improved, desthiobiotin-based proximity labeling approach called Destni in living yeast cells, we identified the mitochondrial protein Aim11 as an interactor of newly imported carrier proteins. Aim11 forms a 160 kDa complex together with Iai11, Gep7 and Mtc1 in the mitochondrial inner membrane that we named the comrade-of-prohibitin (cohibitin) complex owing to its genetic interaction with prohibitins. Deletion of Aim11 impairs the import of carrier proteins into the inner membrane and renders cells hypersensitive to carrier overexpression. Our data suggest that the cohibitin complex plays a quality control function that supports the TIM22-mediated insertion of carrier proteins into the inner membrane of mitochondria.

Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.

Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. In this study, we took advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. Combining behavioral, population genomic, and expression analyses, we show that two Heliconius species have evolved the same preferences for red patterns by exchanging genetic material through hybridization. Neural expression of regucalcin1 correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR-Cas9 impairs courtship toward conspecific females, providing a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution and show how visually guided behaviors contributing to adaptation and speciation are encoded within the genome.

The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals are critical for mtDNA maintenance and packaging and have been linked to mtDNA copy number control. Here, we discover the previously unrecognized mitochondrial HMG-box protein Cim1 (copy number influence on mtDNA) in Saccharomyces cerevisiae, which exhibits metabolic state dependent mtDNA association. Surprisingly, in contrast to Abf2's supportive role in mtDNA maintenance, Cim1 negatively regulates mtDNA copy number. Cells lacking Cim1 display increased mtDNA levels and enhanced mitochondrial function, while Cim1 overexpression results in mtDNA loss. Intriguingly, Cim1 deletion alleviates mtDNA maintenance defects associated with loss of Abf2, while defects caused by Cim1 overexpression are mitigated by simultaneous overexpression of Abf2. Moreover, we find that the conserved LON protease Pim1 is essential to maintain low Cim1 levels, thereby preventing its accumulation and concomitant repressive effects on mtDNA. We propose a model in which the protein ratio of antagonistically acting Cim1 and Abf2 determines mtDNA copy number.

To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell-cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear-encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

The MS2 and MS2-coat protein (MS2-MCP) imaging system is widely used to study messenger RNA (mRNA) spatial distribution in living cells. Here, we report that the MS2-MCP system destabilizes some tagged mRNAs by activating the nonsense-mediated mRNA decay pathway. We introduce an improved version, which counteracts this effect by increasing the efficiency of translation termination of the tagged mRNAs. Improved versions were developed for both yeast and mammalian systems.

The mitochondrial respiratory chain is composed of nuclear as well as mitochondrial-encoded subunits. A variety of factors mediate co-translational integration of mtDNA-encoded proteins into the inner membrane. In Saccharomyces cerevisiae, Mdm38 and Mba1 are ribosome acceptors that recruit the mitochondrial ribosome to the inner membrane, where the insertase Oxa1, facilitates membrane integration of client proteins. The protein Yme2 has previously been shown to be localized in the inner mitochondrial membrane and has been implicated in mitochondrial protein biogenesis, but its mode of action remains unclear. Here, we show that multiple copies of Yme2 assemble into a high molecular weight complex. Using a combination of bioinformatics and mutational analyses, we find that Yme2 possesses an RNA recognition motif (RRM), which faces the mitochondrial matrix and a AAA+ domain that is located in the intermembrane space. We further show that YME2 genetically interacts with MDM38, MBA1 and OXA1, which links the function of Yme2 to the mitochondrial protein biogenesis machinery.

Here, we introduce YeastMate, a user-friendly deep learning-based application for automated detection and segmentation of Saccharomyces cerevisiae cells and their mating and budding events in microscopy images. We build upon Mask R-CNN with a custom segmentation head for the subclassification of mother and daughter cells during lifecycle transitions. YeastMate can be used directly as a Python library or through a standalone application with a graphical user interface (GUI) and a Fiji plugin as easy-to-use frontends.

Mitochondrial genomes (mtDNA) encode essential subunits of the mitochondrial respiratory chain. Mutations in mtDNA can cause a shortage in cellular energy supply, which can lead to numerous mitochondrial diseases. How cells secure mtDNA integrity over generations has remained unanswered. Here, we show that the single-celled yeast Saccharomyces cerevisiae can intracellularly distinguish between functional and defective mtDNA and promote generation of daughter cells with increasingly healthy mtDNA content. Purifying selection for functional mtDNA occurs in a continuous mitochondrial network and does not require mitochondrial fission but necessitates stable mitochondrial subdomains that depend on intact cristae morphology. Our findings support a model in which cristae-dependent proximity between mtDNA and the proteins it encodes creates a spatial “sphere of influence,” which links a lack of functional fitness to clearance of defective mtDNA.

Maintaining organelle function in the face of stress is known to involve organelle-specific retrograde signaling. Using Caenorhabditis elegans, we present evidence of the existence of such retrograde signaling for peroxisomes, which we define as the peroxisomal retrograde signaling (PRS). Specifically, we show that peroxisomal import stress caused by knockdown of the peroxisomal matrix import receptor prx-5/PEX5 triggers NHR-49/peroxisome proliferator activated receptor alpha (PPARα)- and MDT-15/MED15-dependent upregulation of the peroxisomal Lon protease lonp-2/LONP2 and the peroxisomal catalase ctl-2/CAT. Using proteomic and transcriptomic analyses, we show that proteins involved in peroxisomal lipid metabolism and immunity are also upregulated upon prx-5(RNAi). While the PRS can be triggered by perturbation of peroxisomal β-oxidation, we also observed hallmarks of PRS activation upon infection with Pseudomonas aeruginosa. We propose that the PRS, in addition to a role in lipid metabolism homeostasis, may act as a surveillance mechanism to protect against pathogens.

Mitochondria supply virtually all eukaryotic cells with energy through ATP production by oxidative phosphoryplation (OXPHOS). Accordingly, maintenance of mitochondrial function is fundamentally important to sustain cellular health and various diseases have been linked to mitochondrial dysfunction. Biogenesis of OXPHOS complexes crucially depends on mitochondrial DNA (mtDNA) that encodes essential subunits of the respiratory chain and is distributed in multiple copies throughout the mitochondrial network. During cell division, mitochondria, including mtDNA, need to be accurately apportioned to daughter cells. This process requires an intimate and coordinated interplay between the cell cycle, mitochondrial dynamics and the replication and distribution of mtDNA. Recent years have seen exciting advances in the elucidation of the mechanisms that facilitate these processes and essential key players have been identified. Moreover, segregation of qualitatively distinct mitochondria during asymmetric cell division is emerging as an important quality control step, which secures the maintenance of a healthy cell population.

Mitochondrial function depends crucially on the maintenance of multiple mitochondrial DNA (mtDNA) copies. Surprisingly, the cellular mechanisms regulating mtDNA copy number remain poorly understood. Through a systematic high-throughput approach in Saccharomyces cerevisiae, we determined mtDNA–to–nuclear DNA ratios in 5148 strains lacking nonessential genes. The screen revealed MRX6, a largely uncharacterized gene, whose deletion resulted in a marked increase in mtDNA levels, while maintaining wild type–like mitochondrial structure and cell size. Quantitative superresolution imaging revealed that deletion of MRX6 alters both the size and the spatial distribution of mtDNA nucleoids. We demonstrate that Mrx6 partially colocalizes with mtDNA within mitochondria and interacts with the conserved Lon protease Pim1 in a complex that also includes Mam33 and the Mrx6-related protein Pet20. Acute depletion of Pim1 phenocopied the high mtDNA levels observed in Δmrx6 cells. No further increase in mtDNA copy number was observed upon depletion of Pim1 in Δmrx6 cells, revealing an epistatic relationship between Pim1 and Mrx6. Human and bacterial Lon proteases regulate DNA replication by degrading replication initiation factors, suggesting a model in which Pim1 acts similarly with the Mrx6 complex, providing a scaffold linking it to mtDNA.

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

Mitochondrial DNA (mtDNA) encodes essential subunits of respiratory complexes, which are responsible for the generation of ATP through oxidative phosphorylation in mitochondria. Copies of mtDNA are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. We have developed a novel tool in Saccharomyces cerevisiae that allows us to watch mtDNA dynamics in living cells and to characterize its distribution and inheritance. We show that, surprisingly, mitochondrial fusion and fission are dispensable for both processes. The absence of fusion and fission events, however, leads to the accumulation of rearranged and dysfunctional mitochondrial genomes. These results reveal crucial roles of mitochondrial fusion and fission in maintaining the quality and integrity of the mitochondrial genome.

Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.

Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER-mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER-mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER-mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells.

Mitochondria are connected to the endoplasmic reticulum (ER) through specialized protein complexes. We recently identified the ER-mitochondria encounter structure (ERMES) tethering complex, which plays a role in phospholipid exchange between the two organelles. ERMES also has been implicated in the coordination of mitochondrial protein import, mitochondrial DNA replication, and mitochondrial dynamics, suggesting that these interorganelle contact sites play central regulatory roles in coordinating various aspects of the physiology of the two organelles. Here we purified ERMES complexes and identified the Ca²⁺-binding Miro GTPase Gem1 as an integral component of ERMES. Gem1 regulates the number and size of the ERMES complexes. In vivo, association of Gem1 to ERMES required the first of Gem1's two GTPase domains and the first of its two functional Ca²⁺-binding domains. In contrast, Gem1's second GTPase domain was required for proper ERMES function in phospholipid exchange. Our results suggest that ERMES is not a passive conduit for interorganellar lipid exchange, but that it can be regulated in response to physiological needs. Furthermore, we provide evidence that the metazoan Gem1 ortholog Miro-1 localizes to sites of ER-mitochondrial contact, suggesting that some of the features ascribed to Gem1 may be evolutionarily conserved.

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis.

Cardiolipin (CL), a unique dimeric phosphoglycerolipid predominantly present in mitochondrial membranes, has pivotal functions for the cellular energy metabolism, mitochondrial dynamics and the initiation of apoptotic pathways. Perturbations in the mitochondrial CL metabolism cause cardiomyopathy in Barth syndrome. Here, we identify a novel phosphatase in the mitochondrial matrix space, Gep4, and demonstrate that it dephosphorylates phosphatidylglycerolphosphate to generate phosphatidylglycerol, an essential step during CL biosynthesis. Expression of a mitochondrially targeted variant of Escherichia coli phosphatase PgpA restores CL levels in Gep4-deficient cells, indicating functional conservation. A genetic epistasis analysis combined with the identification of intermediates of CL biosynthesis allowed us to integrate Gep4 in the CL-biosynthetic pathway and assign an essential function during early steps of CL synthesis to Tam41, which has previously been shown to be essential for the maintenance of normal CL levels. Our experiments provide the framework for the further dissection of mechanisms that are required for accumulation and maintenance of CL levels in mitochondria.

The mitochondrial phospholipid metabolism critically depends on members of the conserved Ups1/PRELI-like protein family in the intermembrane space. Ups1 and Ups2 (also termed Gep1) were shown to regulate the accumulation of cardiolipin (CL) and phosphatidylethanolamine (PE), respectively, in a lipid-specific but coordinated manner. It remained enigmatic, however, how the relative abundance of both phospholipids in mitochondrial membranes is adjusted on the molecular level. Here, we describe a novel regulatory circuit determining the accumulation of Ups1 and Ups2 in the intermembrane space. Ups1 and Ups2 are intrinsically unstable proteins, which are degraded by distinct mitochondrial peptidases. The turnover of Ups2 is mediated by the i-AAA protease Yme1, whereas Ups1 is degraded by both Yme1 and the metallopeptidase Atp23. We identified Mdm35, a member of the twin Cx₉C protein family, as a novel interaction partner of Ups1 and Ups2. Binding to Mdm35 ensures import and protects both proteins against proteolysis. Homologues to all components of this pathway are present in higher eukaryotes, suggesting that the regulation of mitochondrial CL and PE levels is conserved in evolution.

Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

Prohibitins constitute an evolutionarily conserved and ubiquitously expressed family of membrane proteins that are essential for cell proliferation and development in higher eukaryotes. Roles for prohibitins in cell signaling at the plasma membrane and in transcriptional regulation in the nucleus have been proposed, but pleiotropic defects associated with the loss of prohibitin genes can be largely attributed to a dysfunction of mitochondria. Two closely related proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), form large, multimeric ring complexes in the inner membrane of mitochondria. The absence of prohibitins leads to an increased generation of reactive oxygen species, disorganized mitochondrial nucleoids, abnormal cristae morphology and an increased sensitivity towards stimuli-elicited apoptosis. It has been found that the processing of the dynamin-like GTPase OPA1, which regulates mitochondrial fusion and cristae morphogenesis, is a key process regulated by prohibitins. Furthermore, genetic analyses in yeast have revealed an intimate functional link between prohibitin complexes and the membrane phospholipids cardiolipin and phosphatidylethanolamine. In light of these findings, it is emerging that prohibitin complexes can function as protein and lipid scaffolds that ensure the integrity and functionality of the mitochondrial inner membrane.

The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F(1)F(O)-ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial-encoded F(O)-subunit Atp6 after its insertion into the inner membrane. On the other hand and independent of its proteolytic activity, Atp23 promotes the association of mature Atp6 with Atp9 oligomers. This assembly step is thus under the control of two substrate-specific chaperones, Atp10 and Atp23, which act on opposite sides of the inner membrane. Strikingly, both ATP10 and ATP23 were found to genetically interact with prohibitins, which build up large, ring-like assemblies with a proposed scaffolding function in the inner membrane. Our results therefore characterize not only a novel processing peptidase with chaperone activity in the mitochondrial intermembrane space but also link the function of prohibitins to the F(1)F(O)-ATP synthase complex.