Septin-Dependent Assembly of a Cell Cycle-Regulatory Module inSaccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G 2 /M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G 2 delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).

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

“…Further study of two cdc42 effector-loop mutants and of the roles of Bem3p, Rga1p, and Rga2p, which are three GTPase-activating proteins (GAPs) for Cdc42p (Zheng et al, 1993;Stevenson et al, 1995;Johnson, 1999;Smith et al, 2002), has suggested that Cdc42p GTPase cycling is involved in septin-ring formation (Gladfelter et al, 2002). In particular, it was suggested that Cdc42p, like the assembly GTPase EF-Tu in protein synthesis, participates in septin-ring formation by shuttling septin complexes to the assembling ring structure (Gladfelter et al, 2002), a view that contrasts with the more common view of activated (i.e., GTP-bound) Cdc42p activating effectors in signaling pathways in a Ras-like manner (Johnson, 1999).In addition to Cdc42p, its GEF, and its GAPs, several other proteins including Bni5p, Nap1p, and the protein kinases Cla4p, Gin4p, and Elm1p are also known to be involved in formation of the normal septin ring (Cvrcková et al, 1995;Longtine et al, 1998aLongtine et al, , 2000Sreenivasan and Kellogg, 1999;Bouquin et al, 2000;Weiss et al, 2000;Lee et al, 2002). Cla4p may function in the same pathway as Cdc42p, because it is a Cdc42p effector (Cvrcková et al, 1995;Weiss et al, 2000), although it is not clear whether the interaction between Cla4p and Cdc42p is required for its role in septin organization.…”

“…Mutation of the PAK-kinase gene CLA4 produces significant defects in septin organization and cell morphology (Cvrcková et al, 1995;Richman et al, 1999;Longtine et al, 2000;Weiss et al, 2000; Figure 5A). However, a bem3⌬ rga1⌬ cla4⌬ triple mutant had defects in both cell morphology ( Figure 5A) and septin organization ( Figure 5B) that were much more severe than those of either the cla4⌬ single mutant or the bem3⌬ rga1⌬ double mutant (cf.…”

“…The cell-morphology and septin-organization defects in the bem3⌬ rga1⌬ rga2⌬ and bem3⌬ rga1⌬ gin4⌬ triple mutants were only partially suppressed (indeed, hardly at all in the case of the bem3⌬ rga1⌬ gin4⌬ strain) by deletion of SWE1 (unpublished data). As Swe1p is an essential component of the morphogenesis checkpoint that causes a G2 delay in response to morphogenetic defects (Lew and Reed, 1995;Barral et al, 1999;Shulewitz et al, 1999;Longtine et al, 2000), the phenotypes of the triple mutants must not result primarily from activation of this checkpoint. …”

Some assembly required: yeast septins provide the instruction manual

“…One set of proteins that is recruited to the septin collar in budding yeast includes the protein kinases Hsl1 and Swe1 (the S. cerevisiae homolog of Wee1 [87]) and a protein-arginine methyltransferase called Hsl7 [84][85][86] (Figure I). In response to perturbation of septin filament (or actin filament) organization, Swe1 is stabilized and, hence, its ability to phosphorylate a conserved tyrosine residue in the cyclin B-bound form of the Cdc28 cyclindependent kinase persists.…”

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

“…Septins are recruited to the nascent bud site in a Cdc42p-dependent manner, and then remain at that location as a ring around the neck of the emerging and growing bud, and finally divide into two rings at cell separation ( Figure S2A and B; for review, see Field and Kellogg, 1999;Gladfelter et al, 2001;Faty et al, 2002;Longtine and Bi, 2003). However, the septins play a variety of roles, promoting the proper activation of the B-cyclins for cell cycle progression (Barral et al, 1999;McMillan et al, 1999;Shulewitz et al, 1999;Longtine et al, 2000;Hanrahan and Snyder, 2003), maintaining a diffusion barrier between the bud and mother cortices , and recruiting cytokinetic (Bi et al, 1998;Lippincott and Li, 1998) and cell wall-synthesizing machinery (DeMarini et al, 1997) to the bud neck. To determine whether septins influence actin cable organization, we examined Tpm1p distribution in yeast with a temperaturesensitive CDC10 septin (cdc10-1) (Hartwell, 1971).…”