Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase

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.     

A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.     

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.     

A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10

In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A. (1990) J. Biol. Chem. 265, 9952-9959). To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence. The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene. The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase. The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins. Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane. Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant. The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor. Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation. These data argue against a role of Atp10p in post-translational modification of subunit 6. Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly.     

Distinct domains of small Tims involved in subunit interaction and substrate recognition

Tim9 and Tim10 belong to the small Tim family of mitochondrial ATP-independent chaperones. They are organised in a specific hetero-oligomeric complex (TIM10) that escorts polytopic proteins into the mitochondrial inner membrane. The contributions of the individual subunits to the assembly and function of the TIM10 complex remain poorly understood. Here, we show that substrate recognition and assembly of the complex are mediated by distinct domains of the subunits. These are unrelated to the characteristic "twin CX3C" motif that is present in all small Tims and ensures proper folding of the unassembled subunits. Specifically, we show that substrate recognition is achieved by the Tim10 subunit, whilst Tim9 serves a more structural role. The N-terminal domain of Tim10 is a substrate sensor whilst its C-terminal part is essential for complex formation. By contrast, both N and C-terminal domains of Tim9 are involved in the stability of the complex.     

The rate of import and assembly of F1-ATPase in Saccharomyces cerevisiae

Subunit specific antiserum can be employed to study the course of ATPase assembly in mitochondria isolated from bakers' yeast. Comparing rates of subunit import with rates of enzyme assembly indicated that no substantial pool of unassembled subunits exists for the three largest ATPase peptides (alpha, beta, and gamma). Blocking import of specific ATPase subunits, however, did reveal a possible accumulation of unassembled alpha and gamma subunits in isolated mitochondria. The kinetic experiments also revealed a lag in the import of beta subunit relative to the uptake of alpha and gamma precursors. Experiments conducted in yeast cells confirmed that beta subunit is assembled soon after it is imported, but did not indicate a delay in import relative to the other subunits of F1.     

Cytochrome c oxidase biogenesis: new levels of regulation

Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are posttranslationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed.     

Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Deltaoxa1, Deltaoxa1Deltaafg3, and Deltaoxa1Deltayme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F(0) subunits Atp4p, Atp6p, and Atp17p are stabilized in the Deltaoxa1Deltayme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.     

The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.     

Biosynthesis of mitochondrial membrane proteins: Co-ordination with special reference to cytochrome c oxidase

Summary This paper reviews mechanisms by which the rate of synthesis of subunits of mitochondrial inner membrane protein complexes and the assembly of these subunits are co-ordinated. Current models are evaluated and critically discussed in the light of some recent evidences. The focus is on the incorporation of cytoplasmically-synthesized cytochrome c oxidase subunits in the development of a newer model, which introduces some twists into a combination of several current ideas. A mechanism which governs both organized assembly and the co-ordination of rates of polypeptide synthesis is illustrated and the principles of the model are applied to the elucidation of some odd features of certain mutants. The possibilities that mitochondrial ATPase and cytochrome c reductase may also be synthesized and assembled according to this model are discussed.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex.

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.     

Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.     

DNA-dependent RNA polymerase of thermoacidophilic archaebacteria

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.

Mutants of Saccharomyces cereviaiae showing defects in cytochrome oxidase, coenzyme QH2-cytochrome c reductase, and rutamycin-sensitive ATPase are described. The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 2:2 segregation of tetrads. Genetic analysis indicate the coenzyme QH2-cytochrome c reductase and cytochrome oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F1 ATPase, while the third synthesizes F1 but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex.     

ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase

We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F(0). Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure.