Role of nucleotide binding in septin-septin interactions and septin localization in Saccharomyces cerevisiae.
Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.
Nagaraj
Satish
S
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma, USA.
Rajendran
Ashok
A
Jackson
Charles E
CE
Longtine
Mark S
MS
D020816:Amino Acid Motifs; D002455:Cell Division / Q000528:radiation effects; D050296:Microbial Viability / Q000528:radiation effects; D009024:Morphogenesis / Q000528:radiation effects; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D021381:Protein Transport / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D050505:Mutant Proteins; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins