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RCC1, a conserved chromosomal protein with a seven-bladed propeller is a GDP/GTP nucleotide exchange factor for RanGTPase that mediates various cellular events. We isolated 16 temperature-sensitive (ts) mutants of S. pombe RCC1-homolog, pim1+, by error-prone PCR. Five pim1ts mutants had a single mutation. The obtained pim1ts mutations and previously reported mutations were localized on similar sites in seven RCC1 repeats. Those mutations resulted in a reduced binding of Pim1 with Spi1. All pim1ts mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1ts examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, the septum was formed being uncoupled with nuclear division and abnormally widened, thus resulting in chromosomal DNA mis-segregation and the accumulation of enucleated cells. As a result, a defect of RanGEF/Pim1 abolished the orchestration of sequential mitotic events, spindle formation, septation and cytokinesis that are essential to produce two identical daughter cells.