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Regulation Of The Yeast Yap1p Nuclear Export Signal Is Mediated By Redox Signal-Induced Reversible Disulfide Bond Formation

Shusuke Kuge, Minetaro Arita, Asako Murayama, Kazuhiro Maeta, Shingo Izawa, Yoshiharu Inoue, Akio Nomoto

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ABSTRACT Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae , is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H 2 O 2 or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H 2 O 2 and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H 2 O 2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H 2 O 2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H 2 O 2 -induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys 598 ) and second (Cys 620 ) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.