Molecular mechanism for activation of yeast transcription factor Yap1 by post-translational modification
| Project/Area Number |
13460041
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
INOUE Yoshiharu Kyoto Univ., Graduate School of Agriculture, Associate Professor, 農学研究科, 助教授 (70203263)
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| Co-Investigator(Kenkyū-buntansha) |
IZAWA Shingo Kyoto Univ., Graduate School of Agriculture, Instructor, 農学研究科, 助手 (10273517)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥16,100,000 (Direct Cost: ¥16,100,000)
Fiscal Year 2002: ¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 2001: ¥9,500,000 (Direct Cost: ¥9,500,000)
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| Keywords | Yap1 / Saccharomyces cerevisiae / glyoxalase I / disulfide bond / glutathione / oxdative stress / transcription factor / チオレドキシン |
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| Research Abstract |
Yap1, 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_2O_2 or the thiol oxidant diamide, resulting in Yap1 nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H_2O_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_2O_2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1 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 Yap1 (n-CRD) are required for prolonged nuclear localization of Yap1 in response to H_2O_2 and are thus also required for m
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aximum transcriptional activity. Using mass spectrometry analysis, the H_2O_2-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 Yap1 NES.
On the other hand, several genes responsive for glutathione metabolism are known to be up-regulated by Yap1. Glyoxalase I detoxifies methylglyoxal in the presence of glutathione, although expression of its gene (GLO1) is not regulated by Yap1. In the present study, we demonstrate that Yap1 is constitutively concentrated in the nucleus and activates expression of its target gene in a glo1Δ strain. Expression of Yap1 target gene in the glo1Δ mutant is further accelerated if cells are exposed to oxidative stress. Interestingly, the constitutive activation of Yap1 in the glo1Δ mutant is observed under anaerobic conditions. Steady state levels of methylglyoxal in the glo1Δ mutant are approximately 2-times higher than those in wild type. Expression of the gloA gene coding for glyoxalase I of Escherichia coli in the glo1Δ mutant restores intracellular methylglyoxal levels and consequently suppresses the constitutive activation of Yap1. Methylglyoxal is known to modify arginine and lysine residues of protein. Taken together, we propose a possibility that Yap1 is activated by methylglyoxal which is independent from oxidative modification. Less
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[Publications] Bae Gyo Jung, Kyun Oh Lee, Seung Sik Lee, Yong Hun Chi, Ho Hee Jang, Soon Suk Kang, Kyunghee Lee, Dongbin Lim, Sung Chul Yoon, Dae Jin Yoon, Yashiharu Inoue, Moo Je Cho, Sang Yeol Lee: "A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase"Journal of Biological Chemistry. 277(15). 12572-12578 (2002)
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