Analysis of the functions of RCC1 gene involved in chromosomal condensation

具有rcc1基因突变的tsBn2细胞在S期同步化后，温度漂移到39.5cmC，导致过早复制的染色体凝聚。RCC1编码核小G蛋白RAN的鸟核苷酸交换因子。我们试图分离与RCC1相互作用的因子，以研究RCC1控制染色体凝集的机制。通过双杂交筛选，获得了5个RanBP1和RanBP2基因的cDNA。依赖于RAN的存在，RanBP1与RCC1形成复合体，并在体外抑制RCC1从RAN释放鸟嘌呤核苷酸。另一方面，通过间接免疫荧光观察，我们发现RanBP1在体内定位于胞浆。RanBP2编码358 kDa的核孔蛋白，N端有亮氨酸富集区，C端有亲环素同源区，中间有8次重复的锌指结构。通过免疫荧光和免疫电子显微镜观察，我们发现该蛋白定位于核孔的胞浆细丝。抗RanBP2的多克隆抗体抑制了核蛋白的输入，表明这是核运输系统所必需的。

The tsBN2 cell, which has a mutation in the RCC1 gene, causes condensation of prematurely replicated chromosomes by the temperature-shift to 39.5ﾟC after synchronization at S phase. RCC1 encodes a guanine nucleotide exchanging factor for the nuclear small G protein, Ran. We tried to isolate the factors interacting with RCC1 to investigate the mechanisms of RCC1 function to control the chromosomal condensation. By 2-hybrid screening, 5 cDNAs from RanBP1 and RanBP2 genes were obtained. RanBP1 formed complex with RCC1 depending upon presence of Ran and inhibited release of the guanine nucleotide from Ran by RCC1 in vitro. On the other hand, we found that RanBP1 localized cytosole in vivo by indirect immunofluorescence observation. RanBP2 encoded the 358 kDa nucleopore-protein, and it had the leucine-rich region in N-terminus, the region homologous to cyclophilin in C-terminus, and 8 times repeated Zn-finger in the middle region. We found that this protein localized at cytosolic filaments of the nucleopores by immunofluorescence and immuno-electron microscope observations. The polyclonal antibody against RanBP2 inhibited protein import of the nuclear protein, indicating that this was essential for the nuclear transport system.