Septin Ring Assembly Requires Concerted Action of Polarisome Components, a PAK Kinase Cla4p, and the Actin Cytoskeleton inSaccharomyces cerevisiae

Abstract

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...

Some assembly required: yeast septins provide the instruction manual

“…Phosphorylation by two protein kinases, Cla4 [28,[66][67][68] and Gin4 [39,48,69], plays a prominent role in initiating and/or stabilizing filament assembly during collar formation during emergence of the bud. Cla4 is a clear-cut ortholog of mammalian p21-activated protein kinases (PAKs), and the closest mammalian relative of Gin4 is the neuron-specific, AMPK-related, kinase-family member, BRSK1.…”

Yeast and fungal morphogenesis from an evolutionary perspective

“…Cla4 is also the major kinase that phosphorylates the GEF Cdc24, although the exact role for this phosphorylation is still controversial [74,101]. Cla4 plays a critical role in the assembly of the septin ring at the bud neck [102]. Whereas Cla4's function is more important during vegetative growth, Ste20 is essential during pheromone signalling, stress response and pseudohyphal growth [97,103].…”

The Cdc42 Effectors Ste20, Cla4, and Skm1 Down-Regulate the Expression of Genes Involved in Sterol Uptake by a Mitogen-activated Protein Kinase-independent Pathway

“…Probably the best characterized function of Cla4 is the assembly of the septin ring, which plays a fundamental role in cytokinesis and cell compartmentalization (Weiss et al, 2000;Schmidt et al, 2003;Kadota et al, 2004;Versele and Thorner, 2004). In addition, Cla4 regulates mitotic entry and exit (Hö fken and Schiebel, 2002;Seshan et al, 2002;Sakchaisri et al, 2004).…”