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Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the β-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100–500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b. © 1998 John Wiley & Sons, Ltd.

Cox17p was previously shown to be essential for the expression of cytochrome oxidase in Saccharomyces cerevisiae. In the present study COX17 has been placed under the control of the GAL10 promoter in an autonomously replicating plasmid. A yeast transformant harboring the high copy construct was used to purify Cox17p to homogeneity. Purified Cox17p contains 0.2–0.3 mol of copper per mol of protein. The molar copper content is increased to 1.8 after incubation of Cox17p in the presence of a 6-fold molar excess of cuprous chloride under reduced conditions. An antibody against Cox17p was obtained by immunization of rabbits with a carboxyl-terminal peptide coupled to bovine serum albumin. The antiserum detects Cox17p in both the mitochondrial and soluble protein fractions of wild type yeast and of the transformant overexpressing Cox17p. Exposure of intact mitochondria to hypotonic conditions causes most of Cox17p to be released as a soluble protein indicating that the mitochondrial fraction of Cox17p is localized in the intermembrane space. These results are consistent with the previously proposed function of Cox17p, namely in providing cytoplasmic copper for mitochondrial utilization.

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of a pet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme a biosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15 has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase in cox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombe and Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms.

The nuclear gene MRP10 of Saccharomyces cerevisiae was cloned by complementation of a respiratory deficient mutant N518/L1. This mutant is defective in mitochondrial translation and shows a tendency to accumulate deletions in mitochondrial DNA (rho-). Analysis revealed Mrp10p to be a component of the 37 S subunit of the mitochondrial ribosomes. Disruption of MRP10 in a haploid strain of yeast elicits a phenotype identical to that of the original mutant. The respiratory defect of the null mutant is rescued by re-introducing the MRP10 gene in a wild-type mitochondrial DNA background. These results indicate that Mrp10p belongs to the class of yeast mitochondrial ribosomal proteins that are essential for translation. Searches of current databases failed to reveal any homologs among known bacterial or eucaryotic cytoplasmic ribosomal proteins. Some sequence similarity, however, was detected between Mrp10p and Yml37p, previously identified as a component of the yeast mitochondrial 50 S ribosomal subunit.

Cox14p and Coa3p have been shown to regulate translation of the mitochondrial COX1 mRNA and to be required for assembly of cytochrome oxidase. We present evidence that Cox14p and Coa3p stabilize previously identified Cox1p intermediates and that in the absence of either protein, Cox1p aggregates with itself and other mitochondrial gene products, including cytochrome b, Var1p and Cox2p. Our evidence suggests that Cox1p assembly intermediates are in close proximity to other mitochondrially translated proteins and that an important function of Cox14p and Coa3p is to prevent Cox1 from entering into unproductive aggregation pathways.

Pulse-chase labeling of isolated yeast mitochondria identifies new assembly intermediates of Cox1p, characterizes their compositions, and orders them sequentially. The results indicate that cytochrome oxidase is assembled from separate modules, each consisting of different mitochondrial and nuclear gene products., Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high–molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

Mitochondria are subcellular organelles of eukaryotic cells responsible for conserving and converting the bulk of energy released during the oxidation of foodstuffs into ATP, the universal currency of biological energy. This organelle is the modern-day remnant of free-living bacteria that invaded and established themselves in a primitive nucleated cell with a less efficient mode of energy metabolism. Many genes of the invading bacteria have been lost but a substantial number were transferred to the nucleus of the host and only a few are still present in mitochondria. This article summarizes current knowledge about the PET genes that were incorporated during evolution into the host's genome and are now essential for maintaining the functional and structural intactness of mitochondria.

Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high–molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

Respiration-defective pet mutants of Saccharomyces cerevisiae, assigned to complementation group G25, are grossly deficient in mitochondrial respiratory and ATPase complexes. This phenotype is usually found in strains impaired in mitochondrial protein synthesis. The G25 mutants, however, synthesize all of the proteins encoded by mitochondrial DNA. The mutants are also able to import and process cytoplasmically derived subunits of these enzymes. These results are most compatible with the idea that the gene defined by G25 mutants (RCA1) codes for a protein essential for the assembly of functional respiratory and ATPase complexes. The RCA1 gene has been cloned by complementation of an rca1 mutant with a yeast genomic library. The sequence of the encoded product shows Rca1 protein to be a new member of a recently described family of ATPases. The Rca1 protein is a mitochondrial membrane protein and is the third known member of this family implicated to function in the biogenesis of mitochondria. The primary structure of Rca1 protein indicates several distinct domains in addition to the common purine nucleotide binding region shared by all members of this protein family. One, located in the amino-terminal half, contains two hydrophobic stretches of sufficient length to span a membrane lipid bilayer.

The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNA(Gln) to glutaminyl-tRNA(Gln). Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtf1 mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F(1)-F(0) ATP synthase that is degraded, thereby preventing assembly of the F(0) sector. Allotopic expression of recoded ATP8 and COX2 did not significantly improve growth of gtf1 mutants on respiratory substrates. However, ts gft1 mutants are partially rescued by overexpression of PET112 and HER2 that code for the yeast homologues of the catalytic subunits of bacterial AdT. Additionally, B66, a her2 point mutant has a phenotype similar to that of gtf1 mutants. These results provide genetic support for the essentiality, in vivo, of the GatF subunit of the heterotrimeric AdT that catalyzes formation of glutaminyl-tRNA(Gln) (Frechin, M., Senger, B., Brayé, M., Kern, D., Martin, R. P., and Becker, H. D. (2009) Genes Dev. 23, 1119-1130).

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) β subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F(1) α subunit. While yeast mutants lacking β subunit have stable aggregated F(1) α subunit in the mitochondrial matrix, the human α subunit is completely degraded in cells deficient in F(1) β subunit. These results are discussed in light of the different properties of the proteins and environments in which yeast and human mitochondria exist.

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.

We have cloned the nuclear gene MRP4 coding for a mitochondrial ribosomal protein of the yeast, Saccharomyces cerevisiae. The gene was isolated by complementation of a respiratory-deficient mutant with a pleiotropic defect in mitochondrial gene expression. The nucleotide sequence of MRP4 revealed that it has sequence similarity with Escherichia coli ribosomal protein S2 and related proteins of chloroplast ribosomes from different plants. Further characterization of the MRP4 protein revealed that it is a component of the 37 S subunit of mitochondrial ribosomes. Moreover, the phenotype of cells carrying a disrupted copy of MRP4 is consistent with the MRP4 protein being an essential component of the mitochondrial protein synthetic machinery. Finally, we note that the MRP4 protein and other members of the S2 family of ribosomal proteins have regions of sequence similarity with the mammalian 68-kDa high affinity laminin receptor.

Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes. In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively. Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway. We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain. Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment. Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons. The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains. Primer extension and Northern analyses indicate the gene is transcriptionally active. Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels. Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function. In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site. The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported.

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFMI gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain intiation in vivo.

The adenosine triphosphate (ATP) synthase (F1-F0 complex) of the mitochondrial inner membrane is responsible for making nearly all of the ATP utilized by eukaryotic organisms. The enzyme is an oligomer of more than 20 different subunits, 14 of which are essential for its catalytic activity. The other subunits function in the regulation and structure of the complex. Subunits essential for catalytic activity make up the proton pore, the bulk of the F1 headpiece, and the two stalks that physically and functionally couple the catalytic and proton-translocating activities of the ATP synthase. Saccharomyces cerevisiae provides an excellent model system for studying mutations that affect assembly of the complex because of the ability of this organism to survive on the ATP produced from fermentation in the absence of mitochondrial respiration or oxidative phosphorylation. Studies of such mutants have been instrumental in identifying novel molecular chaperones that act at discrete steps of F1-F0 assembly. Here, we describe some experimental approaches useful in assessing the status of F1-F0 assembly.

The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit. All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection. Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C. The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit. The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants. Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro. The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol. The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min. It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway. The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.

Atp6p (subunit 6) of the Saccharomyces cerevisiae mitochondrial ATPase is synthesized with an N-terminal 10-amino acid presequence that is cleaved during assembly of the complex. This study has examined the role of the Atp6p presequence in the function and assembly of the ATPase complex. Two mutants were constructed in which the codons for amino acids 2–9 or 2–10 of the Atp6p precursor were deleted from the mitochondrial ATP6 gene. The concentration of Atp6p and ATPase complex was approximately 2 times less in the mutants. The lower concentration of ATPase complex in the leaderless mutants correlated with less Atp6p complexed with the Atp9p ring of the F0 sector and with accumulation of an Atp6p-Atp8p complex that aggregated into polymers destined for eventual proteolytic elimination. We propose that the presequence either targets Atp6p to the Atp9p or signals insertion of the Atp6p precursor into a microcompartment of the membrane for more efficient interaction with the Atp9p ring. Despite the ATPase deficiency, growth of the leaderless atp6 mutants on respiratory substrates and the efficiency of oxidative phosphorylation were similar to that of wild type, indicating that the mutations did not affect the proton permeability of mitochondria.

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., National Institutes of Health Research Grants GM-071775A (to A.B.) and GM-50187 (to A.T.), a research grant from the Muscular Dystrophy Association (to A.B.), and Telethon-Italy fellowship GFP05008 (to F.F.)

In the January 3 issue of Cell Metabolism, Leary et al. (2007) report that the mitochondrial metallochaperones Sco1 and Sco2, essential for cytochrome c oxidase assembly, are also responsible for maintenance of cell copper homeostasis, thus showing a new function of mitochondria.

The adenosine triphosphate (ATP) synthase (F1-F0 complex) of the mitochondrial inner membrane is responsible for making nearly all of the ATP utilized by eukaryotic organisms. The enzyme is an oligomer of more than 20 different subunits, 14 of which are essential for its catalytic activity. The other subunits function in the regulation and structure of the complex. Subunits essential for catalytic activity make up the proton pore, the bulk of the F1 headpiece, and the two stalks that physically and functionally couple the catalytic and proton-translocating activities of the ATP synthase. Saccharomyces cerevisiae provides an excellent model system for studying mutations that affect assembly of the complex because of the ability of this organism to survive on the ATP produced from fermentation in the absence of mitochondrial respiration or oxidative phosphorylation. Studies of such mutants have been instrumental in identifying novel molecular chaperones that act at discrete steps of F1-F0 assembly. Here, we describe some experimental approaches useful in assessing the status of F1-F0 assembly.