Abstract
In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of Deltagsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of Deltagsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that gamma-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a Deltagsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions.
Highlights
In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of ⌬gsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis
This observation and the inability of anaerobic conditions to rescue the GSH auxotrophy strongly suggest that the essential requirement of GSH is not related to ribonucleotide reduction or to preventing a toxic accumulation of nonnative protein disulfides
We found that mutations in the proline biosynthetic pathway could suppress this requirement but did so by restoring the synthesis of GSH
Summary
MUTATIONS IN THE PROLINE BIOSYNTHESIS PATHWAY ARE THE ONLY SUPPRESSORS OF GLUTATHIONE AUXOTROPHY IN YEAST*. A mutagenesis of a ⌬gsh strain lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Our data indicate that this requirement is not related to ribonucleotide reduction or to preventing toxic accumulation of nonnative protein disulfide, leaving open this question We used this strain as a model to study the contribution of GSH to oxidative stress tolerance
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