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/Mcm 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 cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 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 replication is controlled.
Dalton
S
S
Roche Institute of Molecular Biology, Nutley, NJ 07110, USA.
Whitbread
L
L
D000595:Amino Acid Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016193:G1 Phase; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D064168:Minichromosome Maintenance Complex Component 7; D008938:Mitosis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7