A novel protein, Psp1, essential for cell cycle progression of Schizosaccharomyces pombe is phosphorylated by Cdc2-Cdc13 upon entry into G0-like stationary phase of cell growth

Abstract

A novel gene, psp1+, which functionally complements a temperature- sensitive mutant defective in cell cycle progression both in G1/S and G2/M has been isolated from the genomic and cDNA libraries of Schizosaccharomyces pombe. Disruption of this gene is lethal for cell growth at 30 °C indicating that it is an essential gene for vegetative cell growth. Western analysis of the protein by polyclonal antibody made from glutathione S-transferase-Psp1 fusion protein indicated that the Psp1 protein exists in two different molecular weight forms depending on the growth state of the cell. In vitro experiments with a phosphatase showed that this difference is due to phosphorylation. The dephosphorylated form of the protein is dominant in actively growing cells whereas the phosphorylated form becomes the major species when cells enter the stationary phase. The Cdc2-Cdc13 complex is shown to phosphorylate the GST-Psp1 fusion protein in vitro, and site- directed mutagenesis and phosphoamino acid analysis indicated that the serine residue at position 333 in the carboxyl-terminal region is required for phosphorylation. In situ fluorescein isothiocyanate-conjugated antibody staining showed that this protein tends to be localized to both ends of the cell upon entry into the stationary phase of cell growth. However, overexpression of the novel protein Psp1 in actively growing cells inhibits cell growth causing accumulation of DNA (4n or 8n). Thus we speculate that Psp1 can function at both G1/S and G2/M phases complementing the defect of the new mutant we have isolated. It is likely that Psp1 is required both for proper DNA replication and for the process of mitosis.