The yeast transcription factor Yap1 activates expression of antioxidant genes in response to oxidative stress. Yap1 regulation involves nuclear accumulation, but the mechanism sensing the oxidative stress signal remains unknown. We provide biochemical and genetic evidence that upon H 2 O 2 treatment, Yap1 is activated by oxidation and deactivated by enzymatic reduction with Yap1-controlled thioredoxins, thus providing a mechanism for autoregulation. Two cysteines essential for Yap1 oxidation are also essential for its activation by H 2 O 2 . The data are consistent with a model in which oxidation of Yap1 leads to disul®de bond formation with the resulting change of conformation masking recognition of the nuclear export signal by Crm1/Xpo1, thereby promoting nuclear accumulation of the protein. In sharp contrast to H 2 O 2 , diamide does not lead to the same Yap1 oxidized form and still activates mutants lacking cysteines essential for H 2 O 2 activation, providing a molecular basis for differential activation of Yap1 by these oxidants. This is the ®rst example of an H 2 O 2 -sensing mechanism in a eukaryote that exploits the oxidation of cysteines in order to respond rapidly to stress conditions.

IntroductionAerobic organisms are exposed to the toxicity of reactive oxygen species (ROS) produced during respiration, the boxidation of fatty acids, the exposure to radiation, light, metals and redox cycling drugs (Storz and Imlay, 1999). To protect against oxidative stress and to maintain a A. Exponential cell cultures grown in yeast extract peptone dextrose (YEPD) were diluted to OD 600 = 0.2 and 3 ml of three serial dilutions (10-fold) were patched on YEPD plates that contained H 2 O 2 at the indicated concentration. Plates were incubated at 30°C for 2 d and analysed. B. Wild-type and isogenic mutant strains cultures (as indicated) that were untreated or pretreated with H 2 O 2 (0.3 mM) were challenged during 2 h with H 2 O 2 at the indicated concentration, as described in Experimental procedures. The multicopy plasmid used for overexpression of PDE2 under its own promoter is pGR103 (Geymonat et al., 1998).