Essential Role for Schizosaccharomyces pombe pik1 in Septation

Abstract

Background

Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4 ^G107S.

Principal Findings

Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25°C. At 36°C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1 ^D709A was kinase-dead, but bound Cdc4. Pik1 ^R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1 ^D709A,R838A was innocuous, pik1 ^R838A was almost innocuous, and pik1 ^D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4 ^G107S . Thus, D709 is essential for kinase activity and septation.

Conclusions

Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.