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M G Douglas

Publications and source records attributed to M G Douglas.

68 records · Page 4Linked to original sources
PubMed1982

Selection of the nuclear gene for the mitochondrial adenine nucleotide translocator by genetic complementation of the op1 mutation in yeast.

Oxidative phosphorylation 1 (OP1), a nuclear gene of yeast Saccharomyces cerevisiae which is required for the expression of a functional mitochondria, has been isolated on a recombinant plasmid. The gene was selected from a recombinant plasmid pool which contained wild type yeast genomic DNA by transformation of the yeast nuclear mutant (op1) followed by a two-stage screening procedure. A recombinant plasmid containing a 2.6 kilobase Bam HI fragment of genomic yeast DNA inserted in either orientation into the single Bam HI site of yeast vector YEp 13 could complement the op1 mutation. Analysis of the gene product of this inserted DNA by three independent methods, 1) in vivo expression in Escherichia coli maxicells, 2) cell-free translation of plasmid selected RNA, and 3) expression analysis in yeast, revealed that its gene product is a protein of Mr = 30,000-32,000, which cross-reacts with specific anti-serum to the adenine nucleotide translocator of the mitochondrial inner membrane. The selection procedure is efficient and can be used for the isolation of any defined yeast nuclear gene which participates in mitochondrial development.

Cell Nucleus↗
PubMed1981

Localization of unassembled subunits of the mitochondrial ATPase in an assembly-defective yeast nuclear mutant.

The yeast nuclear mutant, pet 936, has previously been shown to be defective in the assembly of a functional mitochondrial ATPase (Todd, R. D., McAda, P. C., and Douglas, M. G. (1979) J. Biol. Chem. 254, 11134-11141). In the present report, trypsin degradation and subunit-specific antibody binding have been used to localize subunits 1, 2, and 3 external to or associated with the outer aspect of the inner mitochondrial membrane in the mutant strain. A similar population of unassembled subunits was found in the parental strain as well. Isotope dilution experiments are compatible with those unassembled subunits being normal intermediates in the assembly pathway of the ATPase complex which are blocked from transport across the inner mitochondrial membrane in the mutant, pet 936.

Adenosine Triphosphatases↗
PubMed1981

A model for the structure of the yeast mitochondrial adenosine triphosphatase complex.

Monodisperse, Triton-solubilized ATPase complex was treated with the reversible protein-protein cross-linker dithiobis(succinimidyl)propionate under conditions where only intracomplex cross-links were formed. The resulting products were analyzed by two-dimensional sodium dodecyl sulfate-acrylamide slab gel electrophoresis under reducing and oxidizing conditions. Using the observed major subunit-subunit cross-links and the previously published subunit stoichiometries (Todd, R., Griesenbeck, T., and Douglas, M. (1980) J. Biol. Chem. 255, 5461-5467), a model of the ATPase complex was constructed. The model is a closed structure which could be formed by self-limited assembly of the subunits. The model also has the novel features of a pseudo-mirror symmetry and clustering of mitochondrially and nuclearly coded subunits into two different domains.

Adenosine Triphosphatases↗
PubMed1981

Structure of the yeast mitochondrial adenosine triphosphatase. Results of trypsin degradation.

A comparison was made of the subunit sensitivities of the F1-ATPase and the Triton-solubilized ATPase complex to trypsin degradation. The dissociation of the F1-ATPase from ATPase complex increased the trypsin sensitivity of subunit 3 by a factor of 2 and increased the sensitivity of a particular trypsin site (or group of sites) on subunit 1 by 7-fold. The overall degradation of subunits 1 and 2 appears to be the same in solubilized ATPase complex and the F1-ATPase. Implications of these findings for structural models of the ATPase complex are discussed.

Adenosine Triphosphatases↗
PubMed1981

Binding of citreoviridin to the beta subunit of the yeast F1-ATPase.

Citreoviridin, a nonfluorescent inhibitor of bovine and bacterial ATPases, also inhibits the yeast F1 (K1 = 2 microM). The beta subunit-specific fluorescent ligand, aurovertin, has been used to report the interaction of citreoviridin with the yeast F1-ATPase and the isolated beta subunit. Citreoviridin caused a marked decrease in the fluorescence increment associated with the binding of aurovertin to either intact F1 or the isolated beta subunit. Three lines of evidence indicate that citreoviridin and aurovertin bind to nonidentical sites on the beta subunit: 1) the binding of citreoviridin to the F1 or isolated beta subunit is noncompetitive with respect to aurovertin; 2) the number of aurovertin binding sites (Kd = 0.2 to 0.6 microM) per F1-ATPase molecule remains the same (1.89 +/- 0.6 mol of aurovertin bound per mol of F1) in the presence or absence of citreoviridin; 3) the F1-ATPase obtained from the aurovertin-resistant mutant aur-1 is partly inhibited by citreoviridin.

Adenosine Triphosphatases↗
PubMed1980

The yeast mitochondrial adenosine triphosphatase complex. Subunit stoichiometry and physical characterization.

Immunoprecipitation of uniformly labeled yeast submitochondrial preparations using a subunit-specific or a holoenzyme antiserum has been employed to determine the subunit stoichiometry of the oligomycin-sensitive ATPase complex. The Triton-solubilized enzyme consists of 10 types of subunits. The number of copies of each subunit, in order of decreasing molecular weight, is 3:3:1:2:1:2:2:1:2:3. on the basis of the stoichiometry data, the ATPase complex has a molecular weight of 5.8 x 10(5) and contains a minimum of 20 polypeptide chains. Analysis of water-soluble ATPase (F1-ATPase) indicates that the stoichiometry of the three largest subunits of the enzyme is preserved in the absence of the other subunits. The molecular weights of both forms of the ATPase, derived from stoichiometry data, agree well with measurements obtained from gel filtration and sedimentation studies. The implications of these data for the structure, function, and assembly of the complex are discussed.

Adenosine Triphosphatases↗
PubMed1979

A nuclear mutation conferring aurovertin resistance to yeast mitochondrial adenosine triphosphatase.

A single gene nuclear yeast mutant was isolated whose mitochondrial F1-ATPase was resistant to the specific F1 inhibitor aurovertin. The mutant enzyme was not cross-resistant to other F1 inhibitors. The binding of aurovertin to F1 and to the two largest F1 subunits (alpha and beta) was measured by enhancement of aurovertin fluorescence. Aurovertin bound to wild type F1-ATPase and to its monomeric beta subunit with about the same binding constant. It failed to bind to wild type alpha subunit or to either F1 or F1 subunits from the mutant. The aurovertin-resistant mutant thus contains an altered nuclear gene which specifies the structure of the beta subunit of F1.

Adenosine Triphosphatases↗
PubMed1977

Aurovertin binds to the beta subunit of yeast mitochondrial ATPase.

Isolated beta subunit of ATPase (F1) from yeast mitochondria does not catalyze an ATPase reaction but still binds the specific F1 inhibitor aurovertin. Binding was measured by enhancement of aurovertin fluorescence; it was as tight as that to F1-ATPase. No binding was observed with F1 or with isolated beta subunit from a single-gene nuclear yeast mutant whose F1-ATPase was resistant to aurovertin.

Adenosine Triphosphatases↗
PubMed1976

Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.

Chloramphenicol↗