| Budget Amount *help |
¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 2000: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1999: ¥9,000,000 (Direct Cost: ¥9,000,000)
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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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