The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast

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 cdc42V36G 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 cdc42V36G 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.     

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae

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 bni1Delta 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 spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta 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 cla4Delta 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.     

Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p)

Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.     

SUMO modification of septin-interacting proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.     

Role of nucleotide binding in septin-septin interactions and septin localization in Saccharomyces cerevisiae

Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.     

A novel septin-associated protein, Syp1p, is required for normal cell cycle-dependent septin cytoskeleton dynamics in yeast

Septins are a family of GTP-binding proteins whose heterooligomeric complex is the basic structural element of the septin filaments found in many eukaryotic organisms. In budding yeast, septins are mainly confined at the mother–daughter junction and are required for cell morphogenesis and division. Septins undergo assembly and disassembly in accordance with the progression of the cell cycle. In this report, we identified the yeast protein Syp1p as a new regulator of septin dynamics. Syp1p colocalizes with septins throughout most of the cell cycle. Syp1p interacts with the septin subunit Cdc10p and can be precipitated by Cdc10p and Cdc12p. In the syp1Δ mutant, both formation of a complete septin ring at the incipient bud site and disassembly of the septin ring in later stages of cell division are significantly delayed. In addition, overexpression of Syp1p causes marked acceleration of septin disassembly. The fluorescence recovery after photobleaching (FRAP) assay further showed that Syp1p promotes septin turnover in different cell cycle stages. These results suggest that Syp1p is involved in the regulation of cell cycle-dependent dynamics of the septin cytoskeleton in yeast.     

Role of the septin ring in the asymmetric localization of proteins at the mother-bud neck in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, septins form a scaffold in the shape of a ring at the future budding site that rearranges into a collar at the mother-bud neck. Many proteins bind asymmetrically to the septin collar. We found that the protein Bni4-CFP was located on the exterior of the septin ring before budding and on the mother side of the collar after budding, whereas the protein kinase Kcc4-YFP was located on the interior of the septin ring before budding and moved into the bud during the formation of the septin collar. Unbudded cells treated with the actin inhibitor latrunculin-A assembled cortical caps of septins on which Bni4-CFP and Kcc4-YFP colocalized. Bni4-CFP and Kcc4-YFP also colocalized on cortical caps of septins found in strains deleted for the genes encoding the GTPase activating proteins of Cdc42 (RGA1, RGA2, and BEM3). However, Bni4-CFP and Kcc4-YFP were still partially separated in mutants (gin4, elm1, cla4, and cdc3-1) in which septin morphology was severely disrupted in other ways. These observations provide clues to the mechanisms for the asymmetric localization of septin-associated proteins.     

The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast.

The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.     

Bni5p,a septin-interacting protein,is required for normal septin function and cytokinesis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.     

Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2

The guanine nucleotide exchange factor Cdc24, the GTPase Cdc42, and the Cdc42 effectors Cla4 and Ste20, two p21-activated kinases, form a signal transduction cascade that promotes mitotic exit in yeast. We performed a genetic screen to identify components of this pathway. Two related bud cortex-associated Cdc42 effectors, Gic1 and Gic2, were obtained as factors that promoted mitotic exit independently of Ste20. The mitotic exit function of Gic1 was dependent on its activation by Cdc42 and on the release of Gic1 from the bud cortex. Gic proteins became essential for mitotic exit when activation of the mitotic exit network through Cdc5 polo kinase and the bud cortex protein Lte1 was impaired. The mitotic exit defect of cdc5-10 Deltalte1 Deltagic1 Deltagic2 cells was rescued by inactivation of the inhibiting Bfa1-Bub2 GTPase-activating protein. Moreover, Gic1 bound directly to Bub2 and prevented binding of the GTPase Tem1 to Bub2. We propose that in anaphase the Cdc42-regulated Gic proteins trigger mitotic exit by interfering with Bfa1-Bub2 GTPase-activating protein function.     

A Septin from the Filamentous Fungus A. nidulans Induces Atypical Pseudohyphae in the Budding Yeast S. cerevisiae

Background

Septins, novel cytoskeletal proteins, form rings at the bases of emerging round buds in yeasts and at the bases of emerging elongated hyphal initials in filamentous fungi.

Methodology/Principal Findings

When introduced into the yeast Saccharomyces cerevisiae, the septin AspC from the filamentous fungus Aspergillus nidulans induced highly elongated atypical pseudohyphae and spore-producing structures similar to those of hyphal fungi. AspC induced atypical pseudohyphae when S. cerevisiae pseudohyphal or haploid invasive genes were deleted, but not when the CDC10 septin gene was deleted. AspC also induced atypical pseudohyphae when S. cerevisiae genes encoding Cdc12-interacting proteins Bem4, Cla4, Gic1 and Gic2 were deleted, but not when BNI1, a Cdc12-interacting formin gene, was deleted. AspC localized to bud and pseudohypha necks, while its S. cerevisiae ortholog, Cdc12, localized only to bud necks.

Conclusions/Significance

Our results suggest that AspC competes with Cdc12 for incorporation into the yeast septin scaffold and once there alters cell shape by altering interactions with the formin Bni1. That introduction of the A. nidulans septin AspC into S. cerevisiae induces a shift from formation of buds to formation of atypical pseudohyphae suggests that septins play an important role in the morphological plasticity of fungi.     

The Cdc42p effector Gic2p is targeted for ubiquitin-dependent degradation by the SCFGrr1 complex.

Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells. In yeast, Gic1p and Gic2p are effectors of Cdc42p involved in actin polarization at bud emergence. Gic2p is expressed in a cell cycle-dependent manner and rapidly disappears shortly after bud emergence concomitant with the activation of the G1 cyclin-dependent kinase Cdc28p-Clnp. Here we have shown that the rapid disappearance of Gic2p results from ubiquitin-dependent proteolysis. Biochemical and genetic evidence demonstrates that degradation of Gic2p required the Skp1-cullin-F-box protein complex (SCF) components Cdc34p, Cdc53p, Skp1p and Grr1p, but not Cdc4p. Phosphorylation of several C-terminal sites of Gic2p served as part of the recognition signal for ubiquitination. In addition, binding of Gic2p to Cdc42p was a prerequisite for degradation, suggesting that specifically the active form of Gic2p is targeted for destruction. Finally, our data indicate that degradation of Gic2p may be part of a mechanism which restricts cytoskeletal polarization in the G1 phase of the cell cycle.     

Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.     

Identification of an amphipathic helix important for the formation of ectopic septin spirals and axial budding in yeast axial landmark protein Bud3p

Correct positioning of polarity axis in response to internal or external cues is central to cellular morphogenesis and cell fate determination. In the budding yeast Saccharomyces cerevisiae, Bud3p plays a key role in the axial bud-site selection (axial budding) process in which cells assemble the new bud next to the preceding cell division site. Bud3p is thought to act as a component of a spatial landmark. However, it is not clear how Bud3p interacts with other components of the landmark, such as the septins, to control axial budding. Here, we report that overexpression of Bud3p causes the formation of small septin rings (～1 μm in diameter) and arcs aside from previously reported spiral-like septin structures. Bud3p closely associates with the septins in vivo as Bud3p colocalizes with these aberrant septin structures and forms a complex with two septins, Cdc10p and Cdc11p. The interaction of Bud3p with the septins may involve multiple regions of Bud3p including 1-858, 850-1220, and 1221-1636 a.a. since they all target to the bud neck but exhibit different effects on septin organization when overexpressed. In addition, our study reveals that the axial budding function of Bud3p is mediated by the N-terminal region 1-858. This region shares an amphipathic helix (850-858) crucial for bud neck targeting with the middle portion 850-1103 involved in the formation of ectopic septin spirals and rings. Interestingly, the Dbl-homology domain located in 1-858 is dispensable for axial bud-site selection. Our findings suggest that multiple regions of Bud3p ensure efficient targeting of Bud3p to the bud neck in the assembly of the axial landmark and distinct domains of Bud3p are involved in axial bud-site selection and other cellular processes.     

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

Four novel suppressors of gic1 gic2 and their roles in cytokinesis and polarized cell growth in Saccharomyces cerevisiae

Gic1 and Gic2 are two Cdc42/Rac interactive binding (CRIB) domain-containing effectors of Cdc42-GTPase that promote polarized cell growth in S. cerevisiae. To identify novel genes that functionally interact with Gic1 and Gic2, we screened for high-copy suppressors of a gic1 gic2 temperature-sensitive strain. We identified two pairs of structurally related genes, SKG6-TOS2 and VHS2-MLF3. These genes have been implicated in polarized cell growth, but their functions have not previously been characterized. We found that overproduction of Skg6 and Tos2 in wild-type cells causes aberrant localization of Cdc3 septin and actin structures as well as defective recruitment of Hof1 and impaired formation of the septum at the mother-bud neck. These data suggest a negative regulatory function for Skg6 and Tos2 in cytokinesis. Consistent with this model, deletion of SKG6 suppresses the growth defects associated with loss of HOF1, a positive regulator of cytokinesis. Our analysis of the second pair of gic1 gic2 suppressors, VHS2 and MLF3, suggests that they regulate polarization of the actin cytoskeleton and cell growth and function in a pathway distinct from and parallel to GIC1 and GIC2.     

Regulation of Gic2 localization and function by phosphatidylinositol 4,5-bisphosphate during the establishment of cell polarity in budding yeast

Establishment of cell polarity is important for a wide range of biological processes, from asymmetric cell growth in budding yeast to neurite formation in neurons. In the yeast Saccharomyces cerevisiae, the small GTPase Cdc42 controls polarized actin organization and exocytosis toward the bud. Gic2, a Cdc42 effector, is targeted to the bud tip and plays an important role in early bud formation. The GTP-bound Cdc42 interacts with Gic2 through the Cdc42/Rac interactive binding domain located at the N terminus of Gic2 and activates Gic2 during bud emergence. Here we identify a polybasic region in Gic2 adjacent to the Cdc42/Rac interactive binding domain that directly interacts with phosphatidylinositol 4,5-bisphosphate in the plasma membrane. We demonstrate that this interaction is necessary for the polarized localization of Gic2 to the bud tip and is important for the function of Gic2 in cell polarization. We propose that phosphatidylinositol 4,5-bisphosphate and Cdc42 act in concert to regulate polarized localization and function of Gic2 during polarized cell growth in the budding yeast.     

A monitor for bud emergence in the yeast morphogenesis checkpoint

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.     

Shs1 plays separable roles in septin organization and cytokinesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) form the septin ring at the bud neck during vegetative growth. We show here that disruption of SHS1 caused cold-sensitive growth in the W303 background, with cells arrested in chains, indicative of a cytokinesis defect. Surprisingly, the other four septins appeared to form an apparently normal septin ring in shs1Delta cells grown under the restrictive condition. We found that Myo1 and Iqg1, two components of the actomyosin contractile ring, and Cyk3, a component of the septum formation, were either delocalized or mislocalized in shs1Delta cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10Delta and cdc11Delta cells, respectively, suggesting that Shs1 is involved in septin organization, exerting different effects on septin-ring assembly, depending on the composition of the septin subunits. Furthermore, we constructed an shs1-100c allele that lacks the coding sequence for the C-terminal 32 amino acids. This allele still displayed the genetic interactions with the septin mutants, but did not show cytokinesis defects as described above, suggesting that the roles of Shs1 in septin organization and cytokinesis are separable.