Park Jae-Sook, Steinbach Sarah K, Desautels Michel, Hemmingsen Sean M
Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
PLoS One. 2009 Jul 9;4(7):e6179. doi: 10.1371/journal.pone.0006179.
Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4(G107S).
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 degrees C. At 36 degrees 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.
Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.
粟酒裂殖酵母pik1编码一种磷脂酰肌醇4-激酶，据报道可与Cdc4结合，但不与Cdc4(G107S)结合。
基因缺失表明pik1是必需的。在缺失pik1的细胞中，与温度敏感的二氢叶酸还原酶融合产生的功能丧失等位基因的异位表达，使得细胞在25℃时能够正常增殖。在36℃时，细胞因隔膜异常增厚、位置错误或数量过多而停滞，表明在隔膜形成后期存在缺陷。除了与高尔基体相关外，在胞质分裂后期还在内侧细胞平面观察到异位表达的绿色荧光蛋白标记的Pik1。通过定点诱变产生的新等位基因被异位表达。进行了脂质激酶和Cdc4结合活性测定。Pik1(D709A)激酶失活，但能结合Cdc4。Pik1(R838A)不与Cdc4结合，但却是一种活性激酶。这些取代在粟酒裂殖酵母中的基因组整合以及在酿酒酵母pik1-101细胞中的互补研究表明，在这两种情况下D709都是必需的，而R838是可有可无的。在粟酒裂殖酵母中，pik1的异位表达具有显性致死性；而pik1(D709A,R838A)无害，pik1(R838A)几乎无害，pik1(D709A)产生部分致死性和隔膜缺陷。在cdc4(G107S)中，pik1异位表达致死表型受到抑制。因此，D7基团对激酶活性和隔膜形成至关重要。
隔膜形成需要Pik1激酶活性。Pik1的R838残基对于重要的蛋白质-蛋白质相互作用是必需的，可能是与Cdc4相互作用。
