ANALYSIS OF NUCLEAR EXPORT MECHANISM AND IDENTIFICATION OF NOVEL PROTEINS EXPORTED FROM THE NUCLEUS USING LEPTOMYCIN

核输出抑制剂来普霉素B（LMB）的细胞靶点已被鉴定为CRM 1（exportin 1），其为蛋白质的核输出信号（内斯）的进化保守受体。然而，LMB抑制CRM 1的机制仍然不清楚。在对LMB表现出极高抗性的粟酒裂殖酵母突变体中，CRM 1在Cys-529处被Ser取代。命名为crml-Kl的突变基因赋予野生型粟酒裂殖酵母LMB抗性，并且Crml-Kl不再结合生物素化的LMB。1H NMR分析表明，LMB通过迈克尔型加成结合N-乙酰基-L-半胱氨酸甲酯，这与LMB通过其αβ-不饱和δ-内酯共价结合Cys-529的巯基的想法一致。当HeLa细胞与生物素化的LMB一起培养时，唯一共价结合的细胞蛋白质是CRM 1。这些结果表明，单个半胱氨酸残基决定了LMB的敏感性，并被LMB选择性烷基化，导致CRM 1失活。利用LMB，我们发现了一个新的内斯在Pap 1，这是敏感的氧化应激。Pap 1通常定位于细胞质中，但当Crm 1被温度敏感突变或用一种特异性输出抑制剂来霉素B处理而失活时，Pap 1在细胞核中积累。缺失和突变分析确定了19个氨基酸区域中的几个重要氨基酸作为核输出信号（内斯）。引人注目的是，与经典的NES如HIV Rev内斯不同，Pap 1内斯在用氧化剂如马来酸二乙酯（DEM）处理后失去了功能。氧化应激反应在进化过程中是保守的，因为哺乳动物细胞中表达的携带Pap 1内斯的GFP融合蛋白对DEM有反应。

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.