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.     

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.     

The alpha-subunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F(1) component of the mitochondrial F(1)F(0) ATP synthase. In this report, we show that the F(1) alpha-subunit co-precipitates and co-purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F(1) alpha-subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the alpha-subunit. Yeast two-hybrid screens confirm the direct association of Atp12p with the alpha-subunit and indicate that the binding site for the assembly factor lies in the nucleotide-binding domain of the alpha-subunit, between Asp133 and Leu322. These studies provide the basis for a model of F(1) assembly in which Atp12p is released from the alpha-subunit in exchange for a beta-subunit to form the interface that contains the non-catalytic adenine nucleotide-binding site.     

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200–400 kDa that co-immunopurify with Cox6p. Pulse-labeling analysis disclosed a time-dependent reduction of radiolabeled Atp9p in the complexes and an increase of Atp9p in the ring form of wild type yeast and of mss51, pet111, and pet494 mutants lacking Cox1p, Cox2p, and Cox3p, respectively. Ring formation was not significantly different from wild type in an mss51 or atp10 mutant. The atp10 mutation blocks the interaction of the Atp9p ring with other modules of the ATP synthase. In contrast, ring formation was reduced in a cox6 mutant, consistent with a role of Cox6p in oligomerization of Atp9p. Cox6p involvement in ATP synthase assembly is also supported by studies showing that ring formation in cells adapting from fermentative to aerobic growth was less efficient in mitochondria of the cox6 mutant than the parental respiratory-competent strain or a cox4 mutant. We speculate that the constitutive and Cox6p-independent rate of Atp9p oligomerization may be sufficient to produce the level of ATP synthase needed for maintaining a membrane potential but limiting for optimal oxidative phosphorylation.     

The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins

Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.     

The assembly factor Atp11p binds to the beta-subunit of the mitochondrial F(1)-ATPase

Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the F(1) component of the mitochondrial F(1)F(0)-ATP synthase. This study presents evidence that Atp11p binds selectively to the beta-subunit of F(1). Under conditions in which avidin-Sepharose beads specifically adsorbed biotinylated Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated with the tagged Atp11p protein. Binding interactions between Atp11p and the entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding domain of the beta-subunit located between Gly(114) and Leu(318). Certain elements of this sequence that would be accessible to Atp11p in the free beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme. This observation suggests that the alpha-subunits may exchange for bound Atp11p during the process of F(1) assembly.     

A purified subfragment of yeast Atp11p retains full molecular chaperone activity

Atp11p is a molecular chaperone of the mitochondrial matrix that participates in the biogenesis pathway to form F1, the catalytic unit of the ATP synthase. Affinity tag pull-down assays and yeast two-hybrid screens have shown that Atp11p binds to free beta subunits of F1 (Wang, Z. G., and Ackerman, S. H. (2000) J. Biol. Chem. 275, 5767-5772). This binding action prevents the beta subunit from associating with itself in non-productive complexes and fosters the formation of a (alpha beta)3 hexamer. Following the premise that Atp11p action is mediated primarily through a surface (as opposed to specific amino acids, as in an enzyme active site), solving its three-dimensional structure so that we may learn how the shape of the protein influences its function is a high priority. Recombinant yeast Atp11p has proven refractory for such analysis because of the presence of a disordered region in the protein. In this article, we show that removal of 67 residues from the amino terminus of recombinant Atp11p yields a subfragment of the protein (called Atp11pTRNC) that retains molecular chaperone function as determined in vitro with both a surrogate substrate (reduced insulin) and the natural substrate (F1 beta). Moreover, preliminary 15N-1H heteronuclear single quantum coherence spectra obtained with Atp11pTRNC indicate that the truncated protein is well ordered and amenable to structure determination by nuclear magnetic resonance.     

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.     

Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 degrees C) for the formation/stability of the F(1) sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the alpha-F(1) and beta-F(1) proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F(1) oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F(1) sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986--4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28--30 degrees C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.     

The leader peptide of yeast Atp6p is required for efficient interaction with the Atp9p ring of the mitochondrial ATPase

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.     

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in <Emphasis Type="BoldItalic">Saccharomyces cerevisiae</Emphasis>

ATP1-111, a suppressor of the slow-growth phenotype of yme1Δ lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.     

Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase

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

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.     

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.     

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.     

The Drosophila gene 2A5 complements the defect in mitochondrial F1-ATPase assembly in yeast lacking the molecular chaperone Atp11p.

Assembly of mitochondrial F1-ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p. Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S. cerevisiae Atp11p. A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele. Furthermore, the product of this Drosophila gene was shown to interact with the S. cerevisiae F1 beta subunit in the yeast two-hybrid assay. These results indicate that Atp11p function is conserved in higher eukaryotes.     

Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae

The F(1) component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, alpha, beta, gamma, delta, and epsilon. In terms of mass, the bulk of the structure ( approximately 90%) is provided by the alpha and beta subunits, which form an (alphabeta)(3) hexamer with adenine nucleotide binding sites at the alpha/beta interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the alpha(3)beta(3) oligomer, either because the alpha or the beta subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F(1) alpha(1) and beta subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F(1) alpha and/or beta subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the alpha(3)beta(3) oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner 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.