Mitochondrial H+-ATPase in mutants of Saccharomyces cerevisiae with defective subunit 8 of the enzyme complex.
Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.
Marzuki
S
S
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Watkins
L C
LC
Choo
W M
WM
D000595:Amino Acid Sequence; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009154:Mutation; D010101:Oxygen Consumption; D014176:Protein Biosynthesis; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases