| Résumé | Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S. Principal Findings: Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-offunction allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25ºC. At 36ºC, 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. Pik1D709A was kinase-dead, but bound Cdc4. Pik1R838A 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, pik1D709A,R838A was innocuous, pik1R838A was almost innocuous, and pik1D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4G107S. Thus, D709 is essential for kinase activity and septation. |
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