Studies on Energy‐Linked Reactions: Modified Mitochondrial ATPase of Oligomycin‐Resistant Mutants of Saccharomyces cerevisiae

Abstract

Oligomycin resistance is expressed at the mitochondrial and submitochondrial levels in oligomycin-resistant mutants of Saccharomyces cerevisiae. Studies of the mitochondrial ATPase and Pi-ATP exchange reaction indicate that a modification of the mitochondrial ATPase (ATP-synthetase) complex is involved. The specific resistance at the mitochondrial level to oligomycin and related inhibitors such as ossamycin, rutamycin and peliomycin and the lack of resistance to other inhibitors of mitochondrial ATP synthetase (triethyltin sulphate, venturicidin, aurovertin, Dio-9) correlate with whole cell studies and provide further evidence for a specific modification of an inhibitor site on the mitochondrial inner membrane.Mitochondrial mutants which map at the OL I and OL I1 loci can be differentiated biochemically by their sensitivity to oligomycin of mitochondrial ATPase and the pH 9.5/pH 6.5 ATPase activity ratios.Studies utilising F1-ATPase from wild type and oligomycin-resistant mutants indicate that oligomycin resistance is due to a modification of a membrane-bound component of the mitochondrial ATP-synthetase complex and not to a modification of F1-ATPase or the oligomycin-sensitivityconferring protein.Partially purified oligomycin-sensitive ATPase preparations (Triton X-100 extracts) retain the sensitivity (resistance) and specificity of the original membrane-bound system in mitochondria. Purified oligomycin-sensitive ATPase preparations also retain the specificity of the original membrane preparation but the sensitivity to inhibitors is partially modified. The results support the conclusion that oligomycin resistance is due to modification of mitochondrially coded and mitochondrially synthesised subunits of the oligomycin-sensitive ATPase (ATP synthetase) complex. There is no correlation of oligomycin resistance with the ergosterol concentration of yeast cells, yeast mitochondria or mitochondrial ATPase.The isolation of yeast mitochondrial mutants resistant to inhibitors and uncouplers of mitochondrial energy conservation systems provides a genetic probe of the inhibitor binding sites of the membranebound ATP-synthetase complex and studies of these mutants may provide information as to the components and the organisation of the mitochondrial energy conservation system [l].Previous studies [2,3] have described the isolation of oligomycin-resistant mutants of Saccharomyces cerevisiae and their phenotypic subdivision into two Abbreviations. mtDNA, mitochondrial DNA; OL I and OL I1 mutants, mitochondrial mutants which map at the OL I (01) and OL I1 (0x1) loci on mtDNA, respectively [4,51.Enzyme. ATPase or ATP phosphohydrolase (EC 3.6.

Biogenesis of mitochondrial ATPase

“…Genetic analysis of numerous oligomycin-resistant strains revealed the presence of two genetically unlinked loci, OLll and OLl2 [101,102]. It was tentatively inferred that the two loci represent two distinct genes [102,104]. Later on, a third locus was identified, OLl 3, which is closely linked to OLl 1.…”

“…In these mutants, ATPase activity of submitochondrial particles or even (·f the solubilized enzyme was found to be resistant to oligomycin [99,[103][104][105] indicating that the ATPase complex has been modified by the mutation of mitochondrial DNA. By in vitro reconstitution experiments in reciprocal systems (F 1, OSCP and membrane factor from either wild-type or mutant) this alteration was found to reside in the membrane factor [103,104].…”

“…antimycin-resistant mutants) and for the ATPase complex. Oligomycin-resistant mutants with a cyto-plasmic mode of inheritance have been isolated by different laboratories [95][96][97][98][99][100][101][102][103][104]. Genetic analysis of numerous oligomycin-resistant strains revealed the presence of two genetically unlinked loci, OLll and OLl2 [101,102].…”

Association of pantothenic acid with a protein subunit of yeast mitochondrial ATPase

“…The labeled yeast cells were harvested, washed and broken with glass beads in a Braun homogenizer. Mitochondria were prepared by differential sedimentation [5,6] and submitochondrial particles were prepared by sonication of the purified mitochondrial preparation [6]. Oligomycin-sensitive ATPase was then prepared by density gradient purification of Triton X-100 extracts of submitochondrial particles [7].…”

“…Oligomycin sensitive ATPase was aaso purifed by immunoprecipitation as described [9]. ATPase activities were measured using the coupled spectrophotometric method [10] or by release of inorganic phosphate [6]. Protein was analyzed by the Lowry method.…”