CELL-CYCLE-REGULATED NUCLEAR IMPORT AND EXPORT OF CDC47, A PROTEIN ESSENTIAL FOR INITIATION OF DNA-REPLICATION IN BUDDING YEAST

The CDC47 gene was isolated by complementation of a cdc47 temperature- sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/M cm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cel l cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initi ation of DNA replication, when it is rapidly exported back into the cy toplasm. Cdc47 protein levels do not vary with the cell cycle, but exp ression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc 47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replic ation is controlled.