Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress.

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeast Saccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.