Analysis of the functions of RCC1 gene involved in chromosomal condensation

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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.