CLONING, SEQUENCING, AND MUTATION OF THIOL-SPECIFIC ANTIOXIDANT GENE OF SACCHAROMYCES-CEREVISIAE

We have previously shown that the yeast Saccharomyces cerevisiae conta ins an antioxidant enzyme that can provide protection against a thiol- containing oxidation system but not against an oxidation system withou t thiol. This 25-kDa enzyme was thus named thiol-specific antioxidant (TSA). We have now isolated and sequenced a yeast genomic DNA fragment that encodes TSA. Comparison of the predicted amino acid sequence of TSA with those of conventional antioxidant enzymes, including catalase s, peroxidases, and superoxide dismutases, revealed no sequence homolo gy. The 195-amino acid TSA sequence contains 2 cysteine residues. Sout hern blot analysis of petite yeast DNA, studies with protein synthesis inhibitors, and protein immunoblot analyses of cytosolic and mitochon drial proteins suggest that TSA is a cytosolic protein encoded by nucl ear DNA (chromosome XIII). The yeast TSA gene was selectively disrupte d by homologous recombination. The haploid tsa mutant was viable under air, suggesting that TSA is not essential for cell viability. The gro wth rates of the tsa mutant and wild-type strains were identical under anaerobic conditions. However, under aerobic conditions, especially i n the presence of methyl viologen or a peroxide (t-butyl hydroperoxide or H2O2), the growth rate of the mutant was significantly less than t hat of wild-type cells. This result suggests that TSA is a physiologic ally important antioxidant.