Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1
Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1
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DOI：
10.1006/excr.1998.4136
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发表时间：
1998-08-01
影响因子：
3.7
通讯作者：
Yoshida, M
中科院分区：
文献类型：
--
作者：
Kudo, N;Wolff, B;Yoshida, M
链霉素B(LMB)是链霉菌的一种代谢物，在低纳摩尔浓度下可抑制人类免疫缺陷病毒1型调节蛋白REV的核输出。最近发现LMB能抑制核输出信号受体CRM1的功能。在这里，我们展示了证据表明，在酵母和哺乳动物细胞中，LAMP直接与CRMI1结合，并且CRM1对于依赖NES的蛋白质核输出是必不可少的。与LMB的生物素化衍生物和HeLa细胞提取物的结合实验表明，CRM1是与LMB衍生物结合的主要蛋白质。将纯化的抗人CRM1抗体微量注射到哺乳动物的细胞核中，可以特异性地抑制含有NES的蛋白质的核输出，LMB也是如此。与此一致的是，当用固定化的NES和HeLa细胞提取物进行检测时，发现CRM1与NES相互作用。加入LMB或纯化的抗人CRM1抗体可破坏这种联系。在裂解酵母中也观察到了LAMP对CRM1的抑制作用。裂解酵母CRM1突变株在NES融合蛋白的核输出方面存在缺陷，但在核定位信号(NLS)融合蛋白的输入方面没有缺陷。有趣的是，一种同时含有NES和NLS的蛋白质在CRM1突变细胞或经LMB处理的细胞的细胞核中高度积累，这种蛋白质有望在细胞核和细胞质之间穿梭。这些结果有力地表明，CRM1是真核生物中LAMP的靶标，是蛋白质核输出的重要因子。(C)1998年学术出版社。
Leptomycin B (LMB) is a Streptomyces metabolite that, inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LIMB binds directly to CRMI1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LIMB was also observed in fission yeast. The fission yeast crm1 mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of the crm1 mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LIMB and is an essential factor for nuclear export of proteins in eukaryotes. (C) 1998 Academic Press.