Nuclear respiratory-deficient mutants of Saccharomyces cerevisiae (pet mutants) have been screened for defects in the mitochondrial ATPase. Mutants in two complementation groups were found to have 10% or less of wild-type ATPase activity. The two wild-type nuclear genes defined by the mutants have been designated ATPII and ATP12. The proteins encoded by the two genes are not subunits of the ATPase but rather appear to exercise an important function at a late stage in the synthesis of F1 after transport of the subunits into the internal compartment of mitochondria. Mitochondria ofatpll and atpl2 mutants have only marginally reduced levels of the a and ,B subunits of Fl. Both proteins are processed to their mature size but are not part of a native F1 structure or associated with the mitochondrial membrane. The most reasonable explanation for the mutant phenotype is a block in the assembly of the F1 oligomer.The F1-ATPase [ATP phosphohydrolase (H+-transporting), EC 3.6.1.34] of mitochondria is an oligomeric enzyme composed of five different subunit polypeptides (1). These proteins are synthesized on cytoplasmic ribosomes (2, 3) and transported into the matrix compartment of mitochondria (4,5) where they engage in an ordered set of physical interactions culminating in the formation of the enzymatically active oligomer. A question central to an understanding of the mechanics of assembly of subunit polypeptides into complex structures such as F1 is whether the process is guided by other proteins acting in a catalytic capacity, and if so at what stages is their intervention necessary.The refolding and acquisition of an assembly-competent conformation by some proteins after their translocation into mitochondria has recently been shown to be facilitated by the heat shock protein hsp60 (6-8), a member of the chaperonin family (9). The p subunit of F1 is one of the proteins whose tertiary structure depended on hsp60 (6, 7). These studies have provided direct evidence for a protein-directed step in F1 assembly. In the present communication we report evidence indicating that incorporation of the a and ,B subunits of yeast F1 into an active oligomer depends on at least two other proteins encoded by the ATPI I and ATP12 genes.MATERIALS AND METHODS Strains and Growth Media. The genotypes and origins of the yeast strains used in this study are described in (Miles) instead of Glusulase was used to digest cell walls. The postribosomal supernatant fraction was obtained by centrifugation of the postmitochondrial supernatant at 50,000 rpm for 20 min in a 5OTi Beckman rotor. This step removes the bulk of the cytoplasmic ribosomes and small membrane fragments. Mitochondria and postribosomal fractions were also prepared by mechanical disruption of cells with glass beads in a Braun cell homogenizer, as described (14). The same centrifugation schedules were used in both preparative procedures. To remove interfering inorganic phosphate, postribosomal supernatant fractions were dialyzed at room temperature against a buffer cont...
The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.
The F 1 component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, ␣, , ␥, ␦, and ⑀. In terms of mass, the bulk of the structure (ϳ90%) is provided by the ␣ and  subunits, which form an (␣ ) 3 hexamer with adenine nucleotide binding sites at the ␣/ interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the ␣ 3  3 oligomer, either because the ␣ or the  subunit is missing or because the cells are deficient for proteins that mediate F 1 assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F 1 ␣ and  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 ␣ and/or  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 ␣ 3  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.
Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.
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