2000 Fiscal Year Final Research Report Summary
ANALYSIS OF NUCLEAR EXPORT MECHANISM AND IDENTIFICATION OF NOVEL PROTEINS EXPORTED FROM THE NUCLEUS USING LEPTOMYCIN
| Project/Area Number |
11460037
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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 | THE UNIVERSITY OF TOKYO |
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Principal Investigator |
YOSHIDA Minoru GRADUATE SCHOOL OF ACTICULTURE AND LIFE SCIENCES, THE UNIVERSITY OF TOKYO ASSOCIATE PROFESSOR, 大学院・農学生命科学研究科, 助教授 (80191617)
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| Project Period (FY) |
1999 – 2000
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| Keywords | leptomycin / CRMI / nuclear export signal / oxidative stress / transcriptional regulation / fission yeast |
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| Research Abstract |
The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal (NES) of proteins. However, the mechanism by which LMB inhibits CRM1 still remaints unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene named crml-Kl conferred LMB resistance on wild-type S.pombe and Crml-Kl no longer bound biotinylated LMB.^1H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its αβ-unsaturated δ-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation. Using LMB, we found a novel NES in Pap1, which was sensitive to oxidative stress. Pap1 was localized normally in the cytoplasm but was accumulated in the nucleus when Crm1 was inactivated by a temperature-sensitive mutation or by treatment with leptomycin B, a specific export inhibitor. Deletion and mutational analyses identified several important amino acids in a 19-amino acid region as a nuclear export signal (NES). Strikingly, unlike classical NESs such as the HIV Rev NES, the Pap1 NES lost the function upon treatment with oxidants such as diethyl maleate (DEM). The oxidative stress response is conserved through evolution, as GFP-flused proteins bearing the Pap1 NES expressed in mammalian cells responded to DEM.
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[Publications] Hoshino, H., Kobayash, A., Yoshida, M., Kudo, N., Oyake, T., Motohashi, H., Hayashi, N., Yamamoto, M., and Igarashi, K.: "Oxidative stress abolishes leptoomycin B-sensitive nuclear export of transcription repressor Bach2 that counteracts activation of Maf recognition element."J.Biol.Chem.. 275. 15370-15376 (2000)
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[Publications] Ki, S.W., Ishigami, K., Kitahara, T., Kasahara, K., Yoshida, M., and Horinouchi, S.: "Radicicol binds and inhibits mammalian ATP citrate lyase."J.Biol.Chem.. 275. 39231-39236 (2000)
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[Publications] Verdel, A., Curtet, S., Brocard, M.-P., Rousseaux, S., Lemercier, C., Yoshida, M., and Khochbin, S.: "Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm."Curr.Biol.. 10. 747-749 (2000)
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[Publications] Callanan, M., Kudo, N., Gout, S., Brocard, M.-P., Yoshida, M., Dimitrov, S., and Khochbin, S.: "Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis."J.Cell Sci.. 113. 451-459 (2000)
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「研究成果報告書概要(欧文)」より
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