Glyceraldehyde-3-phosphate dehydrogenase is largely unresponsive to low regulatory levels of hydrogen peroxide in Saccharomyces cerevisiae

BACKGROUND: The reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H(2)O(2)) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H(2)O(2 )in a variety of cellular systems. In yeast, when using a high initial H(2)O(2 )dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H(2)O(2 )functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H(2)O(2 )doses on GAPDH activity and expression in Saccharomyces cerevisiae. RESULTS: A calibrated and controlled method of H(2)O(2 )delivery - the steady-state titration - in which cells are exposed to constant, low, and known H(2)O(2 )concentrations, was used in this study. This technique, contrary to the common bolus addition, allows determining which H(2)O(2 )concentrations trigger specific biological responses. This work shows that both in exponential- and stationary-phase cells, low regulatory H(2)O(2 )concentrations induce a large upregulation of catalase, a fingerprint of the cellular oxidative stress response, but GAPDH oxidation and the ensuing activity decrease are only observed at death-inducing high H(2)O(2 )doses. GAPDH activity is constant upon incubation with sub-lethal H(2)O(2 )doses, but in stationary-phase cells there is a differential response in the expression of the three GAPDH isoenzymes: Tdh1p is strongly upregulated while Tdh2p/Tdh3p are slightly downregulated. CONCLUSIONS: In yeast GAPDH activity is largely unresponsive to low to moderate H(2)O(2 )doses. This points to a scenario where (a) cellular redoxins efficiently cope with levels of GAPDH oxidation induced by a vast range of sub-lethal H(2)O(2 )concentrations, (b) inactivation of GAPDH cannot be considered a sensitive biomarker of H(2)O(2)-induced oxidation in vivo. Since GAPDH inactivation only occurs at cell death-inducing high H(2)O(2 )doses, GAPDH-dependent rerouting of carbohydrate flux is probably important merely in pathophysiological situations. This work highlights the importance of studying H(2)O(2)-induced oxidative stress using concentrations closer to the physiological for determining the importance of protein oxidation phenomena in the regulation of cellular metabolism.