Kudo N, Khochbin S, Nishi K, Kitano K, Yanagida M, Yoshida M, Horinouchi S
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku Tokyo 113.
J Biol Chem. 1997 Nov 21;272(47):29742-51. doi: 10.1074/jbc.272.47.29742.
Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity. cDNA encoding a human homolog of Crm1 was cloned. Human CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S. pombe Crm1. hCRM1 weakly complemented the cold-sensitive mutation of S. pombe crm1-809, as did S. pombe crm1+. Overproduction of hCRM1 under the control of a series of nmt1 promoters suppressed cell proliferation in wild-type S. pombe in an expression level-dependent manner. A similar inhibitory effect was also observed for crm1+. Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei. Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa. Closely similar phenotypes between hCRM1- and S. pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S. pombe crm1+. Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle. Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm. A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal. All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.
粟酒裂殖酵母的Crm1是一种对增殖和染色体区域维持至关重要的核蛋白，它可能是雷帕霉素B的作用靶点，雷帕霉素B是一种具有细胞周期阻滞活性的抗真菌和抗肿瘤抗生素。编码Crm1人类同源物的cDNA被克隆出来。人类CRM1（hCRM1）由1071个氨基酸组成，其序列与粟酒裂殖酵母Crm1有52%的同源性。hCRM1对粟酒裂殖酵母crm1 - 809的冷敏感突变有微弱的互补作用，粟酒裂殖酵母crm1 +也有此作用。在一系列nmt1启动子的控制下过量表达hCRM1以表达水平依赖的方式抑制野生型粟酒裂殖酵母的细胞增殖。对crm1 +也观察到类似的抑制作用。过量表达hCRM1或粟酒裂殖酵母Crm1的细胞明显比未诱导的细胞大，并且含有紧密和碎片化的细胞核。此外，荧光增白剂染色表明，这些细胞中的大多数每个细胞形成两个隔膜，并在隔膜周围积累大量几丁质或其相关多糖。hCRM1诱导细胞和粟酒裂殖酵母Crm1诱导细胞之间非常相似的表型表明，克隆的cDNA编码粟酒裂殖酵母crm1 +的功能同源物。对从同步化的哺乳动物细胞中分离的RNA进行Northern印迹分析表明，哺乳动物CRM1的表达在G1晚期开始，并在G2/M期达到峰值，尽管其蛋白质水平在细胞周期中没有变化。在NIH3T3细胞中瞬时表达与绿色荧光蛋白（GFP）融合的hCRM1表明，hCRM1优先定位于核膜，在核质和细胞质中也可检测到。粟酒裂殖酵母crm1突变导致含有核输出信号的GFP融合蛋白的核输入，但含有核定位信号的GFP融合蛋白的分布没有变化。所有这些数据表明，CRM1是一个新的细胞周期调控基因，对依赖核输出信号的蛋白质核输出至关重要。
