Loss of RanGEF/Pim1 activity abolishes the orchestration of Ran-mediated mitotic cellular events in S. pombe

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 temperaturesensitive (ts) mutants of S. pombe RCC1 -homolog, pim1⁺, by error-prone PCR. Five pim1^ts mutants had a single mutation. The obtained pim1 ts 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 pim1^ts mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1^ts examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, theseptum 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.