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Human neurodegenerative proteinopathies are disorders associated with abnormal protein depositions in brain neurons. They include polyglutamine (polyQ) conditions such as Huntington's disease (HD) and α-synucleinopathies such as Parkinson's disease (PD). Overexpression of NMNAT/Nma1, an enzyme in the NAD+ biosynthetic salvage pathway, acts as an efficient suppressor of proteotoxicities in yeast, fly and mouse models. Screens in yeast models of HD and PD allowed us to identify three additional enzymes of the same pathway that achieve similar protection against proteotoxic stress: Npt1, Pnc1 and Qns1. The mechanism by which these proteins maintain proteostasis has not been identified. Here, we report that their ability to maintain proteostasis in yeast models of HD and PD is independent of their catalytic activity and does not require cellular protein quality control systems such as the proteasome or autophagy. Furthermore, we show that, under proteotoxic stress, the four proteins are recruited as molecular chaperones with holdase and foldase activities. The NAD+ salvage proteins act by preventing misfolding and, together with the Hsp90 chaperone, promoting the refolding of extended polyQ domains and α-synuclein (α-Syn). Our results illustrate the existence of an evolutionarily conserved strategy of repurposing or moonlighting housekeeping enzymes under stress conditions to maintain proteostasis. We conclude that the entire salvage NAD+ biosynthetic pathway links NAD+ metabolism and proteostasis and emerges as a target for therapeutics to combat age-associated neurodegenerative proteotoxicities., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis., (© 2017 International Federation for Cell Biology.)

Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.

A network of conserved proteases known as the intramitochondrial quality control (IMQC) system is central to mitochondrial protein homeostasis and cellular health. IMQC proteases also appear to participate in establishment of signaling cues for mitochondrion-to-nucleus communication. However, little is known about this process. Here, we show that in Saccharomyces cerevisiae, inactivation of the membrane-bound IMQC protease Oma1 interferes with oxidative-stress responses through enhanced production of reactive oxygen species (ROS) during logarithmic growth and reduced stress signaling via the TORC1-Rim15-Msn2/Msn4 axis. Pharmacological or genetic prevention of ROS accumulation in Oma1-deficient cells restores this defective TOR signaling. Additionally, inactivation of the Oma1 ortholog in the human fungal pathogen Candida albicans also alters TOR signaling and, unexpectedly, leads to increased resistance to neutrophil killing and virulence in the invertebrate animal model Galleria mellonella Our findings reveal a novel and evolutionarily conserved link between IMQC and TOR-mediated signaling that regulates physiological plasticity and pancellular oxidative-stress responses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aim: Mitochondrial cytochrome c oxidase (COX), the last enzyme of the respiratory chain, catalyzes the reduction of oxygen to water and therefore is essential for cell function and viability. COX is a multimeric complex, whose biogenesis is extensively regulated. One type of control targets cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. In this study, we investigated whether Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment., Results: We report that Cox1 synthesis is attenuated under oxidative stress conditions and have identified one of the underlying mechanisms. We show that in vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation., Innovation: H2O2-induced downregulation of mitochondrial translation has so far not been reported. We show that high H2O2 concentrations induce a global attenuation effect, but milder concentrations specifically affect COX1 mRNA processing and translation in an Mss51-dependent manner., Conclusion: The redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production.

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity., (© FASEB.)

Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell., (© 2013 John Wiley & Sons Ltd.)

Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae, a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Mia40 import pathway facilitates the import and oxidative folding of cysteine-rich protein substrates into the mitochondrial intermembrane space. Here we describe the in vitro and in organello oxidative folding of Cmc1, a twin CX(9)C-containing substrate, which contains an unpaired cysteine. In vitro, Cmc1 can be oxidized by the import receptor Mia40 alone when in excess or at a lower rate by only the sulfhydryl oxidase Erv1. However, physiological and efficient Cmc1 oxidation requires Erv1 and Mia40. Cmc1 forms a stable intermediate with Mia40 and is released from this interaction in the presence of Erv1. The three proteins are shown to form a ternary complex in mitochondria. Our results suggest that this mechanism facilitates efficient formation of multiple disulfides and prevents the formation of non-native disulfide bonds.

Here we show that yeast strains with reduced target of rapamycin (TOR) signaling have greater overall mitochondrial electron transport chain activity during growth that is efficiently coupled to ATP production. This metabolic alteration increases mitochondrial membrane potential and reactive oxygen species (ROS) production, which we propose supplies an adaptive signal during growth that extends chronological life span (CLS). In strong support of this concept, uncoupling respiration during growth or increasing expression of mitochondrial manganese superoxide dismutase significantly curtails CLS extension in tor1Δ strains, and treatment of wild-type strains with either rapamycin (to inhibit TORC1) or menadione (to generate mitochondrial ROS) during growth is sufficient to extend CLS. Finally, extension of CLS by reduced TORC1/Sch9p-mitochondrial signaling occurs independently of Rim15p and is not a function of changes in media acidification/composition. Considering the conservation of TOR-pathway effects on life span, mitochondrial ROS signaling may be an important mechanism of longevity regulation in higher organisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx(9)C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis.

Alterations in mitochondrial metabolism have been associated with age-related neurodegenerative disorders. This is seen in diseases caused by misfolding of proteins with expanded polyglutamine (polyQ) tracts, such as Huntington's disease. Although evidence of mitochondrial impairment has been extensively documented in patients and disease models, the mechanisms involved and their relevance to the initiation of polyQ cytotoxicity and development of clinical manifestations remain controversial. We report that in yeast models of polyQ cytotoxicity, wild-type and mutant polyQ domains might associate early with the outer mitochondrial membrane. The association of mutant domains with mitochondrial membranes could contribute to induce significant changes in mitochondrial physiology, ultimately compromising the cell's ability to respire. The respiratory defect can be fully prevented by enhancing mitochondrial biogenesis by overexpression of Hap4p, the catalytic subunit of the transcriptional activator Hap2/3/4/5p complex, the master regulator of the expression of many nuclear genes encoding mitochondrial proteins in yeast. Protecting cellular respiratory capacity in this way ameliorates the effect of expanded polyQ on cellular fitness. We conclude that mitochondrial dysfunction is an important contributor to polyQ cytotoxicity. Our results suggest that therapeutic approaches enhancing mitochondrial biogenesis could reduce polyQ toxicity and delay the development of clinical symptoms in patients.

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.

E153 is a respiratory deficient mutant of Saccharomyces cerevisiae with a mutation in the active site of the Sit4p protein phosphatase. Measurements of mitochondrial respiration and cytochromes indicate that the mutation suppresses glucose repression. The escape from catabolite repression is accompanied by a marked reduction of the transcriptional repressor Mig1p. The presence of normal levels of MIG1 mRNA in the mutant and its association with the polysome fraction suggests that depletion of Mig1p is the result of protein degradation. This study shows that in addition to phosphorylation by Snf1p, the transcriptional repressor activity of Mig1p is also regulated by a post-transcriptional Sit4p-dependent pathway. Our evidence suggests that this pathway involves turnover of Mig1p.

Expression of yeast mitochondrial genes depends on specific translational activators acting on the 5'-untranslated region of their target mRNAs. Mss51p is a translational factor for cytochrome c oxidase subunit 1 (COX1) mRNA and a key player in down-regulating Cox1p expression when subunits with which it normally interacts are not available. Mss51p probably acts on the 5'-untranslated region of COX1 mRNA to initiate translation and on the coding sequence itself to facilitate elongation. Mss51p binds newly synthesized Cox1p, an interaction that could be necessary for translation. To gain insight into the different roles of Mss51p on Cox1p biogenesis, we have analyzed the properties of a new mitochondrial protein, mp15, which is synthesized in mss51 mutants and in cytochrome oxidase mutants in which Cox1p translation is suppressed. The mp15 polypeptide is not detected in cox14 mutants that express Cox1p normally. We show that mp15 is a truncated translation product of COX1 mRNA whose synthesis requires the COX1 mRNA-specific translational activator Pet309p. These results support a key role for Mss51p in translationally regulating Cox1p synthesis by the status of cytochrome oxidase assembly.

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.

The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.

We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies.

SHY1 codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX) in Saccharomyces cerevisiae. Mutations in the homologous human gene (SURF1) have been reported to cause Leigh's syndrome, a neurological disease associated with COX deficiency. The function of Shy1p/Surf1p is poorly understood. Here we have characterized revertants of shy1 null mutants carrying extragenic nuclear suppressor mutations. The steady-state levels of COX in the revertants is increased by a factor of 4-5, accounting for their ability to respire and grow on non-fermentable carbon sources at nearly wild-type rates. The suppressor mutations are in MSS51, a gene previously implicated in processing and translation of the COX1 transcript for subunit 1 (Cox1) of COX. The function of Shy1p and the mechanism of suppression of shy1 mutants were examined by comparing the rates of synthesis and turnover of the mitochondrial translation products in wild-type, mutant and revertant cells. We propose that Shy1p promotes the formation of an assembly intermediate in which Cox1 is one of the partners.