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.     

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.     

DNA sequence analysis of the oli1 gene reveals amino acid changes in mitochondrial ATPase subunit 9 from oligomycin-resistant mutants of Saccharomyces cerevisiae

The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin-resistant mutants of Saccharomyces cerevisiae. Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds. Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane. The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane-spanning arms of subunit 9, is considered to constitute an oligomycin-binding domain. By consideration of the amino acid substitutions in those mutants cross-resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin-binding domain, also centered on residues 23 and 59. These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross-resistant to venturicidin). The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.     

Biogenesis of mitochondria. oli2 Mutations affecting the coupling of oxidation to phosphorylation in Saccharomyces cerevisiae

1. Two oligomycin-resistant strains of Saccharomyces cerevisiae have been isolated and shown to have mutations in the oli2 region of the mitochondrial DNA. On solid media containing a non-fermentable energy source, the mutant strains were able to grow only slowly at 28 degrees C and not at all at 18 degrees C or 36 degrees C. 2. When grown in a glucose-limited chemostat at 28 degrees C, the mutant strains were almost completely defective in oxidative metabolism. The mutant mitochondria contained significant levels of all respiratory enzymes, and an active, oligomycin-sensitive ATPase, but the ATP-32Pi exchange activity and P : O ratio were very low. 3. The mutations in these strains are genetically closely linked to mit mutations which have been shown to affect a 20 000-dalton ATPase subunit (Roberts, H., Choo, W.M., Murphy, M., Marzuki, S., Lukins, H.B. and Linnane, A.W. (1979) FEBS Lett. 108, 501-504). Since the mitochondrial ATPase in these mutant strains appears to be fully assembled, the defect in the coupling mechanism is probably a result of a small alteration in the structure of the 20 000-dalton ATPase subunit. 4. When the mutant strains were grown at 18 degrees C, the mitochondria had very low cytochrome oxidase activities, and reduced levels of cytochrome aa3. The largest subunit (Mr 40 000) of this enzyme was not synthesized.     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor

Mutations in the Saccharomyces cerevisiae ATP22 gene were previously shown to block assembly of the F0 component of the mitochondrial proton-translocating ATPase. Further inquiries into the function of Atp22p have revealed that it is essential for translation of subunit 6 of the mitochondrial ATPase. The mutant phenotype can be partially rescued by the presence in the same cell of wild-type mitochondrial DNA and a rho- deletion genome in which the 5'-UTR, first exon, and first intron of COX1 are fused to the fourth codon of ATP6. The COX1/ATP6 gene is transcribed and processed to the mature mRNA by splicing of the COX1 intron from the precursor. The hybrid protein translated from the novel mRNA is proteolytically cleaved at the normal site between residues 10 and 11 of the subunit 6 precursor, causing the release of the polypeptide encoded by the COX1 exon. The ability of the rho- suppressor genome to express subunit 6 in an atp22 null mutant constitutes strong evidence that translation of subunit 6 depends on the interaction of Atp22p with the 5'-UTR of the ATP6 mRNA.     

Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5′ end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes.     

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.     

DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.     

Biogenesis of mitochondria: a mutation in the 5''-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.     

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

Summary The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit ^- mutants. These mutants were isolated in an op1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1—cytochrome c oxidase subunit 2, OXI2 — subunit 3, OXI3 — subunit 1, and COB — cytochrome b.The ability of diploid mit ^- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit ⁺ tetrads, both mit ⁺ and mit ^- tetrads, or only mit ^- tetrads. Mutants forming mit ^- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit ^- mutants have retained the ability to perform meiosis and sporulation.     

Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance

The amino acid substitutions in subunit 6 of the mitochondrial ATPase complex have been determined for 4 oligomycin resistant mutants of Saccharomyces cerevisiae. The data were obtained for each mutant by nucleotide sequence analysis of the mitochondrial oli2 gene. Amino acid substitutions conferring oligomycin resistance in subunit 6 are located in two conserved regions that are thought to form domains which span the inner mitochondrial membrane. The disposition of these amino acid substitutions is consistent with the view that these two membrane-spanning domains interact structurally and functionally with the DCCD-binding proteolipid subunit 9 in the Fo-sector.