A Novel Protein, Psp1, Essential for Cell Cycle Progression ofSchizosaccharomyces 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 G 1 /S and G 2 /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 G 1 /S and G 2 /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.

Involvement of Moc1 in Sexual Development and Survival ofSchizosaccharomyces pombe

“…As it was previously reported that Psp1 is phosphorylated during the stationary phase, 21) we independently reexamined the phosphorylation of Moc1. Samples from the strain that expressed the moc1-HA gene on the plasmid were taken in different growth stages (Fig.…”

“…Thus, in fact, Moc1 is phosphorylated during the stationary phase, as previously reported. 21) Because it was reported that the site of phosphorylation is S333, we mutated this S333 residue to alanine and aspartate residues to test the effect of the mutation on its localization, expecting that the localization of Moc1 might be changed by mutation of S333A or S333D. Prior to observation, we first verified that the band shift of Moc1 was abolished in the S333A mutation, indicating that phosphorylation of Moc1 occurs in S333 (Fig.…”

“…This indicates the existence of regulation of Moc1 function by phosphorylation. Since the phosphorylation of S333 is thought to be conducted by Cdc2, 21) regulation of Moc1 by Cdc2 during meiosis is to be expected.…”

“…19) Here we found that Moc1 in S. pombe plays important roles in survival and sexual development in addition to the previous findings that indicate the involvement of moc1/sds23/psp1 in mitosis. 20,21) The Moc1 and its orthologous proteins contain a common domain, named CBS (cystathioninebeta-synthase), which is predicted to have a multiple trafficking function for protein-protein interaction and metabolic regulations found in proteins like AMP activating kinase [http://smart.embl-heidelberg.de]. But it is not yet clear how Moc1 works in sexual development through interaction with yet unidentified proteins.…”

“…19) The moc1 gene was found to be identical with sds23, which was isolated as multi-copy suppressor for mutations in PP1 and the 20S cyclosome/APC, 20) and also identical with psp1, which was obtained as a suppressor of temperature sensitive mutant defective in cell cycle progression. 21) Both sds23 and psp1 were isolated through screening in mitotically aberrant mutants, 20,21) but moc1 was isolated as an inducer of sexual development in S. pombe.…”