A NOVEL PROTEIN, PSP1, ESSENTIAL FOR CELL-CYCLE PROGRESSION OF SCHIZOSACCHAROMYCES-POMBE IS PHOSPHORYLATED BY CDC2-CDC13 UPON ENTRY INTO G(0)-LIKE STATIONARY-PHASE OF CELL-GROWTH

A novel gene, psp1(+), which functionally complements a temperature-se nsitive mutant defective in cell cycle progression both in G(1)/S and G(2)/M has been isolated from the genomic and cDNA libraries of Schizo saccharomyces pombe, Disruption of this gene is lethal for cell growth at 30 degrees C indicating that it is an essential gene for vegetativ e 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 depen ding on the growth state of the cell, In vitro experiments with a phos phatase showed that this difference is due to phosphorylation. The dep hosphorylated form of the protein is dominant in actively growing cell s whereas the phosphorylated form becomes the major species when cells enter the stationary phase, The Cdc2-Cdc13 complex is shown to phosph orylate the GST-Psp1 fusion protein in vitro, and site-directed mutage nesis and phosphoamino acid analysis indicated that the serine residue at position 333 in the carboxyl-terminal region is required for phosp horylation. In situ fluorescein isothiocyanate-conjugated antibody sta ining showed that this protein tends to be localized to both ends of t he cell upon entry into the stationary phase of cell growth, However, overexpression of the novel protein Psp1 in actively growing cells inh ibits cell growth causing accumulation of DNA (4n or 8n), Thus we spec ulate that Psp1 can function at both G(1)/S and G(2)/M phases compleme nting 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.