Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4.
Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.
Dept. of Molecular and Cell Biology, University of California, Berkeley, Room 16, Barker Hall, Berkeley, CA 94720-3202, USA.
Matthias
M
Versele
Jeremy
J
Thorner
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D048429:Cell Size; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors
C099504:CDC12 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C061178:cdc10 protein, S pombe; D006160:Guanosine Triphosphate; C095212:CLA4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C512892:CDC10 protein, S cerevisiae; D020558:GTP Phosphohydrolases