Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1⌬ cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2⌬ cla4-75-td, bud6⌬ cla4-75-td, and pea2⌬ cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4⌬ mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process. INTRODUCTIONThe development of both unicellular and multicellular organisms requires cells to respond to intracellular and extracellular cues that direct growth and division. These signals regulate polarized cell growth, maintenance of cell shape, cell motility, and cytokinesis (Drubin and Nelson, 1996). Polarized cell growth is a complex process that requires the directed organization of the actin cytoskeleton and the coordinated function of many polarity proteins and signal transduction cascades. Cells of the yeast Saccharomyces cerevisiae grow by budding, a process in which a rigid cell wall is locally expanded as a result of polarized secretion (Pruyne and Bretscher, 2000a). Before bud emergence, cells polarize the actin cytoskeleton toward the future bud site and assemble a septin ring at that site.The septins are a conserved family of filament-forming proteins that play important roles in a variety of cell functions in fungal and animal cells (Longtine et al., 1996;Trimble, 1999;Gladfelter et al., 2001;Longtine and Bi, 2003).Typical septins have a variable N-terminal region, a conserved core that includes the element of a GTP-binding site, and a variable C-terminal region. Septins were first identified as temperature-sensitive cdc mutants in S. cerevisiae (Hartwell, 1971). Septins are assembled into a ring before bud formation and remain as a collar subjacent to the plasma membrane at the mother-bud neck for most of the cell cycle. The septin ring is assembled by the copolymerization of the septins Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p into filaments (Frazier et al., 1998;Field and Kellogg, 1999). Purified septins from yeast form filaments of 7-9-nm diameter and of v...

A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1DN ). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1D. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PFY1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82 were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1D sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1DN phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.

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