MUTATIONS IN THE CYS4 GENE PROVIDE EVIDENCE FOR REGULATION OF THE YEAST VACUOLAR H-ATPASE BY OXIDATION AND REDUCTION IN-VIVO()

The vma41-1 mutant was identified in a genetic screen designed to iden tify novel genes required for vacuolar H+-ATPase activity in Saccharom yces cerevisiae. The VMA41 gene was cloned and shown to be allelic to the CYS4 gene, The CYS4 gene encodes the first enzyme: in cysteine bio synthesis, and in addition to cysteine auxotrophy, cys4 mutants have m uch lower levels of intracellular glutathione than wild-type cells. cy s4 mutants display the pH-dependent growth phenotypes characteristic o f vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidification in vivo. The vacuolar proton -translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific a ctivity is reduced (47% of wild type) and the activity is unstable. Ad dition of reduced glutathione to the growth medium complements the pH- dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. The CYS4 gene was d eleted in a strain in which the catalytic site cysteine residue implic ated in oxidative inhibition of the yeast V-ATPase has been mutagenize d (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P. M., and F orgac, M. (1997) J. Biol. Chem. 272, 11750-11756). This catalytic site point mutation suppresses the effects of the cys4 mutation. The data indicate that the acidification defect of cys4 mutants arises from ina ctivation of the vacuolar ATPase in the less reducing cytosol resultin g from loss of Cys4p activity and provide the first evidence for the m odulation of V-ATPase activity by the redox state of the environment i n vivo.