FIG. 3.

Correlation between oxidation of c-CRD in vivo and the nuclear localization of Yap1p. (A) The cysteine residues in the c-CRD are reduced in vivo under unstressed conditions. TW (yap1) cells carrying pRS HA GFP-c-CRD (lanes 1 and 2) or pRS HA GFP-c-CRD^(TAT) (lanes 3 and 4) were cultured until exponential phase and were analyzed for free and oxidized thiols using AMS (with or without pretreatment with IAA) as described in Materials and Methods. Eight-centimeter SDS-PAGE gels were used. (B) Time-dependent oxidation of cysteine residues in the c-CRD in response to H₂O₂ (0.5 mM) or diamide (1.5 mM). Free and oxidized thiols were analyzed as described for panel A at the indicated times (min) after imposition of oxidative stress or without stress (−; lanes 1 and 7). Thirteen-centimeter SDS-PAGE gels were used. Arrows, migration of the reduced (Red) and oxidized (Ox) forms after AMS treatment. (C) Time-dependent nuclear accumulation of Yap1p in response to H₂O₂ or diamide. TW (yap1) cells carrying pRS-HA-GFP-YAP1 were cultured until exponential phase, collected, and resuspended in medium containing 0.5 mM H₂O₂ or 1.5 mM diamide. GFP fluorescence was observed by confocal microscopy before oxidant treatment (−) and after the indicated times of treatment (min). After 15 min of oxidant treatment, cells were collected, washed, and resuspended in the same medium without oxidants, and the GFP fluorescence was observed 5 (w5) and 10 (w10) min later.