Regulation of the Yeast Yap1p Nuclear Export Signal Is Mediated by Redox Signal-Induced Reversible Disulfide Bond Formation

AbstractYap1p, 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 H2O2 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 H2O2 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 H2O2 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 H2O2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H2O2-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys598) and second (Cys620) 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. ACKNOWLEDGMENTSWe thank Gigi Storz (National Institutes of Health, Bethesda, Md.) for her valuable advice and guidance in preparing the manuscript. We thank Masato Kobori and Ryuta Mizutani (Graduate School of Pharmaceutical Sciences, The University of Tokyo) for technical assistance with the MALDI-TOF (MS), the Human Genome Center of IMSUT for computer system as well as Internet service, and Gigi Storz and Orna Carmel-Harel for exchanging unpublished information.This work was supported by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan.