Abstract. Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDCIO, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. BioL 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normallooking ring structures at the bases of the first zygotic buds.

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p. INTRODUCTIONBud formation in the yeast Saccharomyces cerevisiae has served as an important model for studies of eukaryotic cell polarization (Hall, 1992;Nelson, 2003). In wild-type cells, nonrandom bud sites are selected by a system that involves cortical marker proteins and a signaling pathway based on the Ras-type GTPase Rsr1p Kang et al., 2004;Pruyne et al., 2004). GTP-bound Rsr1p then promotes the localization and/or localized activation of Cdc24p, the guanine-nucleotide-exchange factor (GEF) for the Rho-type GTPase Cdc42p, and of Cdc42p itself; the localized activation of Cdc42p then causes polarization of the cytoskeletal and secretory systems, which leads to the polarized growth of the bud Kozminski et al., 2003;Pruyne et al., 2004;Shimada et al., 2004).Among the proteins recruited early to the presumptive bud site are the septins. This widely conserved family of GTP-binding, filament-forming proteins functions in cytokinesis and other processes, many of which involve the organization of specialized regions of the cell cortex (Longtine et al., 1996;Gladfelter et al., 2001b;Longtine and Bi, 2003;Hall and Russell, 2004). S. cerevisiae has seven septins, five of which (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p) are expressed in vegetative cells, where they form heterooligomeric complexes and localize interdependently to the bud site (Kim et al., 1991;Longtine et al., 1996;Frazier et al., 1998;Mortensen et al., 2002;Vrabioiu et al., 2004). About 10 min before bud emergence, the septins form a ring at the cell cortex. The bud then emerges through this ring, which concurrently reorganizes into an hourglassshaped collar that spans the mother-bud neck. This reorganization coincides with a major decrease in the exchangeability of septin subunits, presumably reflecting the formation of more stable higher-order structures at this time (Caviston et al., 2003;Dobbelaere et al., 2003;. The septin collar remains at the neck until cytokinesis, when it splits into two rings as the actomyosin ring contracts an...

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