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

leptomycin B （LMB）是一种有效的抗肿瘤药物，其靶分子被遗传学和生物化学鉴定为CRM1，这是一种被报道为控制染色体结构所需的蛋白质。我们发现CRM1是核输出信号（NES）的受体，而LMB抑制蛋白的核输出。因此，使用LMB可以很容易地确定感兴趣的蛋白质是否可以从细胞核中输出。小鼠温度敏感型p53^< val135> (tsp53)在32℃时聚集在细胞核中，作为野生型，而在37℃时则被隔离在细胞质中。37℃LMB抑制核输出后，胞质tsp53重新定位到细胞核中，而主要的双部NLS突变引起了组成性的胞质定位，表明它通过其NES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上。我们发现，与含hsc70复合物的结合阻止了NLS在37℃时通过tsp53的C端区域进入输入受体，而在32℃时它的解离允许快速的核输入。利用LMB，我们在分裂酵母转录因子Pap1中发现了一个新的NES，其功能被氧化应激以一种在真核生物中保守的方式取消。Crm1的单个突变（Cys-529到Ser）导致输出Pap1的显著减少，但其他典型的NESs没有明显减少。这些结果表明，Crm1中的半胱氨酸残基参与了Pap1的应激性核输出。

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.