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Analysis of mechanism of tress-responsive and temperature-sensitive nuclear transport

应力响应和温度敏感的核运输机制分析

基本信息

批准号：
13460035
负责人：
YOSHIDA Minoru
金额：
$ 10.75万
依托单位：
RIKEN (2002)The University of Tokyo (2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The target molecule of leptomycin B (LMB), a potent antitumor agent, was genetically and biochemically identified as CRM1 , a protein reported as being required for chromosome structure control. We showed that CRM1 was a receptor for the nuclear export signal (NES) and that LMB inhibited nuclear export of proteins. It therefore became able to easily determine whether a protein of interest can be exported from the nucleus by using LMB. Mouse temperature-sensitive p53^<Val135> (tsp53) accumulates in the nucleus and acts as a wild-type at 32℃, while it is sequestered in the cytoplasm at 37℃. The cytoplasmic tsp53 relocalized into the nucleus upon inhibition of the nudear export by LMB at 37℃, whereas a mutation in a major bipartite NLS caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its NES and NLS rather than tethered to cytoplasmic structures. We showed that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C- terminal region of tsp53 at 37℃, whereas its dissociation at 32℃ allows rapid nuclear import. Using LMB, we identified a novel NES in the fission yeast transcription factor Pap1 , the function of which is abolished by oxidative stress in a manner conserved in eukaryotes. A single mutation in Crm1 (Cys-529 to Ser) caused a marked decrease in exporting Pap1 but not other typical NESs. These results suggest that the cysteine residue in Crm1 is involved in the stress-responsive nuclear export of Pap1.

一种有效的抗肿瘤剂，来普霉素B（LMB）的靶分子被遗传学和生物化学鉴定为CRM 1，一种被报道为染色体结构控制所需的蛋白质。我们发现CRM 1是核输出信号（内斯）的受体，LMB抑制蛋白质的核输出。因此，能够通过使用LMB容易地确定是否可以从细胞核输出感兴趣的蛋白质。小鼠温度敏感性p53^<Val135>（tsp 53）在32℃时聚集在细胞核中并表现为野生型，而在37℃时则被隔离在细胞质中。在37℃下，当LMB抑制核输出时，胞质tsp 53重新定位到细胞核中，而在主要的二分NLS中的突变导致组成性胞质定位，表明它通过其内斯和NLS在细胞质和细胞核之间穿梭，而不是束缚在细胞质结构中。我们发现，在37℃下，与含Hsc 70的复合物的结合阻止了NLS通过tsp 53的C-末端区域进入输入受体，而在32℃下，它的解离允许快速的核输入。使用LMB，我们确定了一个新的内斯在裂变酵母转录因子Pap 1，其功能被废除的氧化应激的方式在真核生物中保守。Crm 1的一个突变（Cys-529到Ser）导致Pap 1的输出明显减少，但不是其他典型的NES。这些结果表明，Crm 1中的半胱氨酸残基参与了Pap 1的应激反应核输出。