Characterization of functional domains of Saccharomyces cerevisiae septins

Abstract

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-septins. We assayed septin mutations predicted to disrupt nucleotide binding. In contrast to cells lacking a particular septin, at 23�C septin nucleotide-binding mutant cells are fully viable and show efficient completion of septin-dependent processes suggesting septin nucleotide binding is not required for the function of septin-interacting proteins. Septin nucleotide-binding mutations show temperature-sensitive viability, which correlates with the predicted defects in nucleotide binding and with the extent of defects in septin localization. Two-hybrid and in vitro assays show septins interact directly with each other in a nucleotide-binding-dependent manner. Thus, septin nucleotide binding promotes septin-septin interactions and septin localization. To identify the particular regions in septins involved in protein-protein interactions, I used alanine-scanning mutagenesis to mutate charged residues (presumed to be on the surface of the protein) in Cdc11p. I describe the effects of these mutations on cell viability, cell division, cell morphogenesis and on septin-septin interactions.