The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Abstract

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42 V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42 V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth. INTRODUCTIONThe septin family of proteins was first recognized in yeast and now appears to be ubiquitous in fungi and animals, although not in plants (Longtine et al., 1996;Field and Kellogg, 1999;Trimble, 1999;Nguyen et al., 2000;Momany et al., 2001;Macara et al., 2002). Typical septins have a variable N-terminal region, a conserved core that includes the elements of a GTP-binding site, and a variable C-terminal region that (in all but a few cases) includes a predicted coiled-coil domain. In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al., 1996;Frazier et al., 1998;Hsu et al...

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

“…The idea that there may be multiple types of septin structures (rings and gauzes) is consistent with the observation that exchange of septin subunits changes dramatically during the cell cycle (Caviston et al, 2003;Dobbelaere et al, 2003). Both rings ( Figure 7C) and gauzes (Figure 8) consist of filaments organized into a mesh-like structure.…”

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

“…The septin dependence of Bnr1p provides a likely explanation for the synthetic lethality between defects in Bni1p function and septins (Longtine et al, 1996), which recapitulate the lethality of the simultaneous loss of Bni1p-and Bnr1p-dependent cable assembly ( Figure 5A). Septindependent recruitment of Bnr1p also might explain the delay in Bnr1p recruitment to the bud neck until after bud emergence, because during this transition the septins undergo significant changes in their organization (reviewed in Gladfelter et al, 2001; recent results in Caviston et al, 2003;Dobbelaere et al, 2003).…”

Some assembly required: yeast septins provide the instruction manual

“…In S. cerevisiae in G1, an apparent cap, or patch, of septins in which the subunits are highly mobile as judged by fluorescence recovery after photobleaching (FRAP) experiments [47,48] accumulates in the juxta-membrane region immediately subtending the incipient bud site. Septins seem to be more concentrated at the edge of this patch than at its center.…”

“…In the next cell cycle, the initial recruitment of the septin patch to the incipient bud site and its transition into a rimmed disk depends on function of the small, Rho-family GTPase, Cdc42 (Reviewed in [61]). It has been reported that the effects of Cdc42 require the conversion of Cdc42-GTP to Cdc42-GDP [64], via the action of either the cognate Cdc42-GAPs (Rga1, Rga2 and Bem3) [47] or, as yet, unknown effectors [65].…”