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

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.

Identification of a quinone-sensitive redox switch in the ArcB sensor kinase

“…Despite that the cytosol of both prokaryotic and eukaryotic cells provides a highly reducing environment, which is almost untenable for disulfide bond formation (30,31), a number of cytoplasmic proteins that are regulated by reversible disulfide bond formation are known. Some examples include the transcriptional factors Yap1p in Saccharomyces cerevisiae, OxyR and the chaperone Hsp33 in E. coli, and the CtrJ and the membrane-bound kinase RegB in Rhodobacter capsulatus (31)(32)(33)(34)(35)(36). However, our in vivo and in vitro analyses indicate that disulfide bond formation in ArcB may differ from that of the other redox regulators in that it appears to respond specifically to the oxidized forms of quinones rather than to molecular oxygen or to reactive-oxygen species such as H 2 O 2 .…”

Yap8p activation in Saccharomyces cerevisiae under arsenic conditions

“…C 132 , C 137 and C 274 are essential for Yap8p nuclear translocation In analogy to Yap1, which can respond to cysteine-reactive chemicals through its modification at specific cysteine residues [12,23,24], we investigated the requirement of Yap8 cysteine residues in Yap8 activation by arsenic compounds. Yap8p and Yap1p share 19% identity with two domains of higher homology, one being in the C-terminus of these proteins (Fig.…”

A systems approach to delineate functions of paralogous transcription factors: Role of the Yap family in the DNA damage response

“…The Yap family belongs to the bZIP superfamily of TFs that is widely conserved from yeast to human. Specificity of gene regulation among paralogous Yaps has been attributed to two mechanisms: (i) slight but important differences in the DNA binding motifs targeted by different Yap TFs, as in the case of Yap1 versus Yap2 (6), and (ii) Variation in the regulatory domains present within each Yap protein that are modulated by specific upstream activation signals, i.e., Yap1 is modulated by intramolecular disulfide bond formation (7) and Yap4 is modulated by protein phosphorylation (8). A variety of other mechanisms for achieving specificity are also likely, although they have not yet been systematically demonstrated for the Yap proteins.…”