Essential Role for Schizosaccharomyces pombe pik1 in Septation

Abstract

Background Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S.Principal FindingsGene 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. 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.ConclusionsPik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.

Cooperation Between the Septins and the Actomyosin Ring and Role of a Cell-Integrity Pathway During Cell Division in Fission Yeast

“…Another hypothesis is that cdc4-s16 also compromises the function of some other Cdc4-binding partner that is part of a pathway that normally complements the pathway involving the septins. In this regard, it should be noted that there is good evidence that both S. pombe Cdc4 and its S. cerevisiae counterpart, Mlc1, interact with partners other than the type II myosin heavy chains and that these other partners are also important for cytokinesis (Stevens and Davis 1998;Boyne et al 2000;Shannon and Li 2000;Desautels et al 2001;D'Souza et al 2001;Motegi et al 2001;Win et al 2001;Lord and Pollard 2004;Luo et al 2004;Mulvihill et al 2006;Park et al 2009). The other partners include type V myosin heavy chains (S. pombe Myo51 and perhaps Myo52; S. cerevisiae Myo2), which are thought to be involved in the delivery of new plasma-membrane and cell-wall materials to the division site or (for Myo51) in some unknown function within the AMR; IQGAPs (S. pombe Rng2 and S. cerevisiae Iqg1), which may also be involved in plasma-membrane and cell-wall formation in addition to their roles in AMR function; and a phosphatidylinositol 4-kinase (S. pombe Pik1).…”

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

“…8A). Previous studies showed that overexpression of wildtype Pik1p (but not mutant Pik1-R838Ap) inhibits cell growth (Park et al, 2009), indicating that light chain binding influences Pik1p function. We also saw growth inhibition when Pik1p was overexpressed from the medium-strength nmt1-41 promoter (Fig.…”

“…We employed a pik1-R838A mutant because this point mutation targets the IQ motif and abolishes Pik1p-Cdc4p (Park et al, 2009) and Pik1p-Cam2p (Fig. 7C) interactions without compromising the essential role of Pik1p in cell growth.…”

“…Cdc4p interacts with IQGAP (D'Souza et al, 2001), a conserved protein containing six IQ motifs and a Ras GTPase-activating protein (GAP) homology domain that is essential for contractile ring assembly and cytokinesis (Eng et al, 1998). Cdc4p also interacts with a type-III phosphatidylinositol (PI) 4-kinase (Pik1p) via a single IQ motif located at the C-terminus of the kinase Park et al, 2009). Pik1p is essential for septum formation and cell division (Park et al, 2009).…”

“…Cdc4p also interacts with a type-III phosphatidylinositol (PI) 4-kinase (Pik1p) via a single IQ motif located at the C-terminus of the kinase Park et al, 2009). Pik1p is essential for septum formation and cell division (Park et al, 2009).…”