Role of Nucleotide binding in Yeast Septins.
Ashok Rajendran (1), Satish Nagaraj (1), Alina Vrabioiu (2), Christine Field (2), Mark longtine (1)
(1) Biochemistry & Molecular Biol., Oklahoma State University, 246 NRC, Stillwater, OK, 74078, United States of America;
(2) Department of Cell Biology, Harvard Medical School, #107, Louis Pasteur Avenue, Boston, MA 02115.
S. cerevisiae expresses 5 septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p and Shs1p) during mitotic growth. Septins interact with each other, colocalize to the cortex at the mother-bud neck, and are required for the localization and function of proteins involved in cytokinesis, bud-site selection and chitin deposition. Septin nucleotide binding and/or hydrolysis may regulate the interaction of septins with septins or non-septin proteins. We assayed Cdc11p mutations predicted to disrupt nucleotide binding. In contrast to cells lacking Cdc11p, at 23˚C cdc11 P-loop mutant cells are fully viable and show efficient completion of septin-dependent processes suggesting Cdc11p nucleotide binding is not required for the function of septin-interacting proteins. Cdc11p P-loop mutations show a range of minimal restrictive temperatures for viability, which correlates with the predicted defects in nucleotide binding and the extent of defects in septin localization. 2-hybrid and in vitro assays show Cdc11p interacts directly with Cdc12p in a nucleotide-binding-dependent manner. Compared to complexes from cells expressing wild-type Cdc11p, complexes from mutant cells contain substoichiometric amounts of cdc11p and have defects in filament formation. Mutant cdc11p localize inefficiently to the neck compared to wild-type Cdc11p. Thus, Cdc11p nucleotide binding promotes septin-septin interactions, septin filament formation, and septin localization.
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