ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.     

Assembly factors of F1FO-ATP synthase across genomes

Work with respiration-deficient strains of Saccharomyces cerevisiae has provided evidence that assembly of the mitochondrial ATP synthase is dependent on proteins that serve substrate-specific, chaperone-type functions: Atp10p, Atp11p, Atp12p, Atp22p, and Fmc1p. Atp11p and Atp12p mediate the formation of the F1 moiety via interaction with subunits F1-beta and F1-alpha, respectively. The role of Fmc1p is less clear. Atp10p and Atp22p are essential for the formation of the F(O) part, during which Atp10p assists in the incorporation of the F(O)-a subunit. Here we present a comprehensive analysis of ATP synthase assembly factors from all available genomes. The mechanism of the F1 assembly is preserved in all eukaryotic lineages that are capable of ATP synthesis via oxidative phosphorylation and requires Atp11p and Atp12p. Conversely, composition of the F(O) part as well as its assembly is more versatile. We found two distinct subtypes of the F(O)-a subunit, one of which seems to be dependent on the action of Atp10p while the other does not. Restricted occurrence of Fmc1p and Atp22p suggests the existence of lineage-specific assembly factors. Our phylogenetic data served as a source for comparative sequence analysis, which identified evolutionarily conserved residues, putative functional domains and their basic structural features for Atp10p, Atp11p, and Atp12p orthologs. These results provide the basis for detailed molecular analysis of the ATP synthase-specific chaperones.     

Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.     

The metalloprotease encoded by ATP23 has a dual function in processing and assembly of subunit 6 of mitochondrial ATPase

In the present study we have identified a new metalloprotease encoded by the nuclear ATP23 gene of Saccharomyces cerevisiae that is essential for expression of mitochondrial ATPase (F(1)-F(O) complex). Mutations in ATP23 cause the accumulation of the precursor form of subunit 6 and prevent assembly of F(O). Atp23p is associated with the mitochondrial inner membrane and is conserved from yeast to humans. A mutant harboring proteolytically inactive Atp23p accumulates the subunit 6 precursor but is nonetheless able to assemble a functional ATPase complex. These results indicate that removal of the subunit 6 presequence is not an essential event for ATPase biogenesis and that Atp23p, in addition to its processing activity, must provide another important function in F(O) assembly. The product of the yeast ATP10 gene was previously shown to interact with subunit 6 and to be required for its association with the subunit 9 ring. In this study one extra copy of ATP23 was found to be an effective suppressor of an atp10 null mutant, suggesting an overlap in the functions of Atp23p and Atp10p. Atp23p may, therefore, also be a chaperone, which in conjunction with Atp10p mediates the association of subunit 6 with the subunit 9 ring.     

Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells

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.     

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.     

Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called "peripheral stalk" that connects the enzyme catalytic F(1) component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (deltah) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of deltah-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane.     

Binding of subunit E into the A-B interface of the A(1)A(O) ATP synthase

Two of the distinct diversities of the engines A(1)A(O) ATP synthase and F(1)F(O) ATP synthase are the existence of two peripheral stalks and the 24kDa stalk subunit E inside the A(1)A(O) ATP synthase. Crystallographic structures of subunit E have been determined recently, but the epitope(s) and the strength to which this subunit does bind in the enzyme complex are still a puzzle. Using the recombinant A(3)B(3)D complex and the major subunits A and B of the methanogenic A(1)A(O) ATP synthase in combination with fluorescence correlation spectroscopy (FCS) we demonstrate, that the stalk subunit E does bind to the catalytic headpiece formed by the A(3)B(3) hexamer with an affinity (K(d)) of 6.1±0.2μM. FCS experiments with single A and B, respectively, demonstrated unequivocally that subunit E binds stronger to subunit B (K(d)=18.9±3.7μM) than to the catalytic A subunit (K(d)=53.1±4.4). Based on the crystallographic structures of the three subunits A, B and E available, the arrangement of the peripheral stalk subunit E in the A-B interface has been modeled, shining light into the A-B-E assembly of this enzyme.     

Modular assembly of yeast mitochondrial ATP synthase

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.     

NMR solution structure of subunit F of the methanogenic A1AO adenosine triphosphate synthase and its interaction with the nucleotide-binding subunit B

The A1AO adenosine triphosphate (ATP) synthase from archaea uses the ion gradients generated across the membrane sector (AO) to synthesize ATP in the A3B3 domain of the A1 sector. The energy coupling between the two active domains occurs via the so-called stalk part(s), to which the 12 kDa subunit F does belong. Here, we present the solution structure of the F subunit of the A1AO ATP synthase from Methanosarcina mazei G?1. Subunit F exhibits a distinct two-domain structure, with the N-terminal having 78 residues and residues 79-101 forming the flexible C-terminal part. The well-ordered N-terminal domain is composed of a four-stranded parallel beta-sheet structure and three alpha-helices placed alternately. The two domains are loosely associated with more flexibility relative to each other. The flexibility of the C-terminal domain is further confirmed by dynamics studies. In addition, the affinity of binding of mutant subunit F, with a substitution of Trp100 against Tyr and Ile at the very C-terminal end, to the nucleotide-binding subunit B was determined quantitatively using the fluorescence signals of natural subunit B (Trp430). Finally, the arrangement of subunit F within the complex is presented.     

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.     

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria

The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.     

On the structure of the stator of the mitochondrial ATP synthase

The structure of most of the peripheral stalk, or stator, of the F-ATPase from bovine mitochondria, determined at 2.8 A resolution, contains residues 79-183, 3-123 and 5-70 of subunits b, d and F6, respectively. It consists of a continuous curved alpha-helix about 160 A long in the single b-subunit, augmented by the predominantly alpha-helical d- and F6-subunits. The structure occupies most of the peripheral stalk in a low-resolution structure of the F-ATPase. The long helix in subunit b extends from near to the top of the F1 domain to the surface of the membrane domain, and it probably continues unbroken across the membrane. Its uppermost region interacts with the oligomycin sensitivity conferral protein, bound to the N-terminal region of one alpha-subunit in the F1 domain. Various features suggest that the peripheral stalk is probably rigid rather than resembling a flexible rope. It remains unclear whether the transient storage of energy required by the rotary mechanism takes place in the central stalk or in the peripheral stalk or in both domains.     

The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F(1)-F(0) interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F(1)F(0)-ATP synthase.     

Folding and stability of the b subunit of the F(1)F(0) ATP synthase

The F(1)F(0) ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F(0)) and soluble (F(1)) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F(1)F(0) complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T(m) values between 30 and 40 degrees C. CD spectra for the region comprising residues 53-122 (b(53-122)) showed theta;(222)/theta;(208) = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b(53-122) (DeltaG, DeltaH and DeltaC(p)) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.     

Genetic complementation between mutant b subunits in F1F0 ATP synthase

In Escherichia coli, a parallel homodimer of identical b subunits constitutes the peripheral stalk of F(1)F(0) ATP synthase. Although the two b subunits have long been viewed as a single functional unit, the asymmetric nature of the enzyme complex suggested that the functional roles of each b subunit should not necessarily be considered equivalent. Previous mutagenesis studies of the peripheral stalk suffered from the fact that mutations in the uncF(b) gene affected both of the b subunits. We developed a system to express and study F(1)F(0) ATP synthase complexes containing two different b subunits. Two mutations already known to inactivate the F(1)F(0) ATP synthase complex have been studied using this experimental system. An evolutionarily conserved arginine, b(Arg-36), was known to be crucial for F(1)F(0) ATP synthase function, and the last four C-terminal amino acids had been shown to be important for enzyme assembly. Experiments expressing one of the mutants with a wild type b subunit demonstrated the presence of heterodimers in F(1)F(0) ATP synthase complexes. Activity assays suggested that the heterodimeric F(1)F(0) complexes were functional. When the two defective b subunits were expressed together and in the absence of any wild type b subunit, an active F(1)F(0) ATP synthase complex was assembled. This mutual complementation between fully defective b subunits indicated that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking.     

Functional analysis of subunit e of the F1Fo-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region

Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.     

Oxa1 directly interacts with Atp9 and mediates its assembly into the mitochondrial F1Fo-ATP synthase complex

The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F(1)F(o)-ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded F(o)-sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F(1)-subunits, but its further assembly with subunit 6 (Atp6) of the F(o)-sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F(1)-subcomplex for its association with Atp6.     

A "petite obligate" mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.