Essential Role for Schizosaccharomyces Pombe Pik1 in Septation

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2009 Jul 10

PMID 19587793

Citations 14

Authors

Jae-Sook Park

Sarah K Steinbach

Michel Desautels

Sean M Hemmingsen

Affiliations

Soon will be listed here.

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 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.

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

Citing Articles

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References

Humbel B, Konomi M, Takagi T, Kamasawa N, Ishijima S, Osumi M . In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe. Yeast. 2001; 18(5):433-44. DOI: 10.1002/yea.694. View

Eng K, Naqvi N, Wong K, Balasubramanian M . Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr Biol. 1998; 8(11):611-21. DOI: 10.1016/s0960-9822(98)70248-9. View

Gietz R, Sugino A . New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988; 74(2):527-34. DOI: 10.1016/0378-1119(88)90185-0. View

Guertin D, Trautmann S, McCollum D . Cytokinesis in eukaryotes. Microbiol Mol Biol Rev. 2002; 66(2):155-78. PMC: 120788. DOI: 10.1128/MMBR.66.2.155-178.2002. View

Balla A, Balla T . Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol. 2006; 16(7):351-61. DOI: 10.1016/j.tcb.2006.05.003. View

Desautels M, Den Haese J, Slupsky C, McIntosh L, Hemmingsen S . Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe, interacts with a phosphatidylinositol 4-kinase. J Biol Chem. 2000; 276(8):5932-42. DOI: 10.1074/jbc.M008715200. View

Demmel L, Beck M, Klose C, Schlaitz A, Gloor Y, Hsu P . Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth. Mol Biol Cell. 2008; 19(3):1046-61. PMC: 2262966. DOI: 10.1091/mbc.e07-02-0134. View

Godi A, Pertile P, Meyers R, Marra P, Di Tullio G, Iurisci C . ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol. 1999; 1(5):280-7. DOI: 10.1038/12993. View

Han G, Audhya A, Markley D, Emr S, Carman G . The Saccharomyces cerevisiae LSB6 gene encodes phosphatidylinositol 4-kinase activity. J Biol Chem. 2002; 277(49):47709-18. DOI: 10.1074/jbc.M207996200. View

10.

Bahler J, Wu J, Longtine M, Shah N, McKenzie 3rd A, Steever A . Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 1998; 14(10):943-51. DOI: 10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y. View

11.

Mahon E, Hawrysh A, Chagpar R, Johnson L, Anderson D . A-Raf associates with and regulates platelet-derived growth factor receptor signalling. Cell Signal. 2005; 17(7):857-68. DOI: 10.1016/j.cellsig.2004.11.006. View

12.

Moreno S, Klar A, Nurse P . Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991; 194:795-823. DOI: 10.1016/0076-6879(91)94059-l. View

13.

Wu J, Pollard T . Counting cytokinesis proteins globally and locally in fission yeast. Science. 2005; 310(5746):310-4. DOI: 10.1126/science.1113230. View

14.

Balasubramanian M, McCollum D, Chang L, Wong K, Naqvi N, He X . Isolation and characterization of new fission yeast cytokinesis mutants. Genetics. 1998; 149(3):1265-75. PMC: 1460233. DOI: 10.1093/genetics/149.3.1265. View

15.

Craven R, Griffiths D, Sheldrick K, Randall R, Hagan I, Carr A . Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene. 1998; 221(1):59-68. DOI: 10.1016/s0378-1119(98)00434-x. View

16.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

17.

Schu P, Takegawa K, Fry M, Stack J, Waterfield M, Emr S . Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993; 260(5104):88-91. DOI: 10.1126/science.8385367. View

18.

Forsburg S . Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 1993; 21(12):2955-6. PMC: 309706. DOI: 10.1093/nar/21.12.2955. View

19.

Audhya A, Foti M, Emr S . Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics. Mol Biol Cell. 2000; 11(8):2673-89. PMC: 14948. DOI: 10.1091/mbc.11.8.2673. View

20.

Lord M, Pollard T . UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II. J Cell Biol. 2004; 167(2):315-25. PMC: 2172548. DOI: 10.1083/jcb.200404045. View