ATP Synthase of Yeast Mitochondria

Arselin, Geneviève; Vaillier, Jacques; Graves, Pierre‐Vincent; Velours, Jean

doi:10.1074/jbc.271.34.20284

Cited by 87 publications

(58 citation statements)

References 36 publications

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“…SDS-PAGE was done according to Laemmli (32). Western blot analyses were performed as described previously (33). Polyclonal antibodies raised against yeast Atp4p, subunit ␦, Atp9p, and Atp6p were used after 1:10,000 dilutions, and those against Arg8p were used after a 1:2000 dilution.…”

Section: Construction Of a Strain Expressing Atp4p And The ␦ Subunit mentioning

confidence: 99%

A “Petite Obligate” Mutant of Saccharomyces cerevisiae

Duvezin-Caubet

Rak

Lefebvre-Legendre

et al. 2006

Journal of Biological Chemistry

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Within the mitochondrial F 1 F 0 -ATP synthase, the nucleus-encoded ␦-F 1 subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the ␦ subunit gene (⌬␦) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% ؊ /°petites (i.e. cells missing a large portion (>50%) of the mtDNA ( ؊ ) or totally devoid of mtDNA (°)). Previous work has suggested that the absence of complete mtDNA ( ؉ ) in ⌬␦ 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 ⌬␦ yeast. We further show that combined with a nuclear mutation (⌬atp4) preventing the ATP synthase proton channel assembly, a lack of ␦ subunit fails to destabilize the mtDNA, and ؉ ⌬␦ cells become viable. We conclude that ⌬␦ yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the ؊ /°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.The mitochondrial inner membrane contains the ATP synthase, which utilizes a transmembrane proton gradient to catalyze ATP synthesis from inorganic phosphate and ADP. The ATP synthase has two major structural domains, an F 0 component, which forms a protonpermeable pore across the membrane, and a peripheral, matrix-localized, F 1 component, where the ATP is synthesized (1).The F 1 domain comprises five different subunits, all nucleus-encoded, with ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 stoichiometry. The three ␣ subunits and the three ␤ subunits alternate in position within a hexamer that contains the adenine nucleotide processing sites (2-4). The ␥, ␦, and subunits form a subcomplex of F 1 , named the central stalk, linking the ␣ 3 ␤ 3 subcomplex to the ATP synthase proton channel (5). During catalysis, the central stalk rotates together with a transmembrane ring of 10 -12 c subunits in the F 0 (6 -12). In the course of this rotation, the ␥ subunit sequentially interacts with the three ␣␤ pairs in a way that favors ATP synthesis in the catalytic sites, as required by the binding change mechanism (1).In the yeast Saccharomyces cerevisiae, null mutations of the ␦ or ␥ subunit massively destabilize the mitochondrial genome in the form of 100% cytoplasmic petites, which are cells with a large (Ͼ50%) deletion in mitochondrial DNA (mtDNA) 6 ( Ϫ ) or completely lacking mtDNA (°) (13,14). This is an intriguing observation, since many other mutations impairing mitochondrial oxidative phosphorylation, including a null ...

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Section: Construction Of a Strain Expressing Atp4p And The ␦ Subunit mentioning

confidence: 99%

A “Petite Obligate” Mutant of Saccharomyces cerevisiae

Duvezin-Caubet

Rak

Lefebvre-Legendre

et al. 2006

Journal of Biological Chemistry

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“…It has been described as a supernumerary protein that, until this study, was not apparently related to any subunit described in other ATP synthases (14). Primary structural analysis of nucleotide data banks have identified the existence of an open reading frame in Schizosaccharomyces pombe (TrEMBL accession number 059673) that encodes an hypothetical protein 27% identical to subunit h and another in Botryotinia fuckeliana (GenBank TM accession number AL115386) that also encodes a hypothetical protein 38% identical to subunit h. Yeast subunit h is essential for yeast to grow on a nonfermentable carbon sources, and mitochondria isolated from a yeast strain with a null mutation in subunit h have an ATPase activity that is oligomycin-insensitive and the catalytic sector dissociated from the membrane components (14).…”

Section: Discussionmentioning

confidence: 99%

“…The strain with the null mutation in ATP14 (MAT␣, met6, ura3, his3, ATP14::URA3) has been described (14). The ⌬ATP14 strain containing the plasmid pbF 6 , was obtained by transformation of the null mutant in ATP14 gene by the nonintegrative single copy vector, pRS313, which contains the coding region of mature bF 6 , 1 the leader peptide of the ␤-subunit of the yeast ATP synthase, and the upstream and downstream transcriptional controlling elements of the ATP2 gene.…”

Section: Methodsmentioning

confidence: 99%

“…The strain containing the null mutation in the ATP14 gene (14), which encodes subunit h, was transformed by a single copy vector (pRS313) bearing the selection marker HIS3 and the DNA encoding the bovine F 6 , pbF 6 . The transformants were tested for growth on a complete medium containing glycerol as the sole carbon source, YPG (3% glycerol, 2% peptone, 1% yeast extract).…”

Section: The Coding Sequence Of the Bovine Coupling Factor 6 Complemementioning

confidence: 99%

“…Among the supernumerary subunits of the yeast ATP synthase F 0 , subunit h has been described as an essential component because inactivation of the ATP14 gene led to a lack of oxidative phosphorylations (14). Recently cross-linking experiments (15) have positioned this hydrophilic and acidic component of 10,408 Da close to subunit 4 (subunit b), a component of the second stalk of the ATP synthase.…”

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Bovine Coupling Factor 6, with Just 14.5% Shared Identity, Replaces Subunit h in the Yeast ATP Synthase

Velours

Vaillier

Paumard

et al. 2001

Journal of Biological Chemistry

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The mammalian mitochondrial ATP synthase is composed of at least 16 polypeptides. With the exception of coupling factor F 6 , there are likely yeast homologs for each of these polypeptides. There are no obvious yeast homologs of F 6 , as predicted from primary sequence comparison of the putative peptides encoded by the open reading frames in the yeast genome. In this manuscript, we demonstrate that expression of bovine F 6 complements a null mutant in ATP14 gene in yeast Saccharomyces cerevisiae. Subunit h of the yeast ATP synthase is encoded by ATP14 and is just 14.5% identical to bovine F 6 . Expression of bovine F 6 in an atp14 null mutant strain recovers oxidative phosphorylation, and the ATP synthase is active, although functioning with a lower efficiency than the wild type enzyme. Like subunit h, bovine F 6 is shown to interact mainly with subunit 4 (subunit b), a component of the second stalk of the enzyme. These data indicated the subunit h is the yeast homolog of mammalian coupling factor F 6 .

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Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

1998

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Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.

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ATP Synthase of Yeast Mitochondria

Cited by 87 publications

References 36 publications

A “Petite Obligate” Mutant of Saccharomyces cerevisiae

A “Petite Obligate” Mutant of Saccharomyces cerevisiae

Bovine Coupling Factor 6, with Just 14.5% Shared Identity, Replaces Subunit h in the Yeast ATP Synthase

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

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