Essential Role for Schizosaccharomyces pombe pik1 in Septation 英文参考文献.docVIP

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Essential Role for Schizosaccharomyces pombe pik1 in Septation 英文参考文献.doc

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Essential Role for Schizosaccharomyces pombe pik1 in Septation 英文参考文献

EssentialRoleforSchizosaccharomycespombepik1in Septation Jae-SookPark1.,SarahK.Steinbach1.,MichelDesautels2,SeanM.Hemmingsen1,3* 1Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 2Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 3Plant Biotechnology Institute, National Research Council Canada, Saskatoon, Saskatchewan,Canada Abstract Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S . PrincipalFindings:Genedeletionrevealedthatpik1isessential.Incellswithpik1deleted,ectopicexpressionofaloss-of- functionallele, created by fusion toatemperature-sensitive dihydrofolate reductase,allowed normal cellproliferation at 25uC.At36uC,cellsarrestedwithabnormallythick,misplacedorsupernumerarysepta,indicatingadefectlateinseptation. InadditiontobeingGolgiassociated,ectopicallyexpressedGFP-taggedPik1wasobservedatthemedialcellplanelatein cytokinesis.Newalleles,createdbysite-directedmutagenesis,wereexpressedectopically.LipidkinaseandCdc4-binding activityassayswereperformed.Pik1D709Awaskinase-dead,butboundCdc4.Pik1R838AdidnotbindCdc4,butwasanactive kinase.GenomicintegrationofthesesubstitutionsinS.pombeandcomplementationstudiesinSaccharomycescerevisiae pik1-101cellsrevealedthatD709isessentialinbothcaseswhileR838isdispensable.InS.pombe,ectopicexpressionofpik1 wasdominantlylethal;while,pik1D709A,R838A wasinnocuous,pik1R838Awasalmostinnocuous,andpik1D709Aproducedpartial lethalityandseptationdefects.Thepik1ectopic expressionlethalphenotype wassuppressed in cdc4G107S.Thus,D709is essentialforkinaseactivityandseptation. Conclusions:Pik1kinaseactivityisrequiredforseptation.ThePik1R838residueisrequiredforimportantprotein-protein interactions,possiblywithCdc4. Citation:ParkJ-S,SteinbachSK,DesautelsM,HemmingsenSM(2009)EssentialRoleforSchizosaccharomycespombepik1inSeptation.PLoSONE4(7):e