Yeast Ulp1, an Smt3-specific protease, associates with nucleoporins

Yeast Smt3 is a ubiquitin-like protein similar to the mammalian SUMO-1. Cdc3, a septin component, is known to be modified by Smt3. The level of this modification was affected by Smt3-specific protease mutation ulp1-ts or overexpression of ULP1. By two-hybrid screening, we isolated 5 UIP (Ulp1 interacting protein) genes. UIP1 was identical to NUP42 encoding a component of the nuclear pore complex (NPC). Gle1, another NPC-associating component, also interacted with Ulp1 in the two-hybrid system and co-immunoprecipitation experiment. Thus Ulp1 associates with nucleoporins and may interact with septin rings in the telophase.     

Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RING-like domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1.Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.     

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.     

Comparative analysis of yeast PIAS-type SUMO ligases in vivo and in vitro

SUMO/Smt3, a ubiquitin-like modifier, is known to conjugate other proteins and modulate their functions in various processes. Recently, Ull1/Siz1 was discovered as a novel PIAS-type E3 required for septin sumoylation in yeast. We demonstrate here that the second PIAS-type Nfi1/Siz2 is also a SUMO ligase. It interacted with Smt3, SUMO/Smt3 conjugating enzyme Ubc9 and a septin component Cdc3 in the two-hybrid system. The region containing the RING-like domain of Nfi1/Siz2 bound directly to Ubc9 and Cdc3, but not to Smt3. Nfi1/Siz2 stimulated Smt3 conjugation to Cdc3 in vitro. In this in vitro system, Smt3 formed polymeric chains in the presence of higher concentrations of E1 and E2 enzymes. When the lysine(15) residue of Smt3 was substituted with arginine, Smt3 chain-polymerization was abolished. Using this polysumoylation-deficient mutant Smt3, we found that Cdc3 and Nfi1/Siz2 were modified with Smt3 at multiple sites. Finally we found that the C-terminal truncated form of Ull1/Siz1 was mis-localized in vivo, but retained its SUMO ligase activity in vitro. We discuss the regulation of these SUMO ligases in vivo and in vitro.     

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.     

Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam, which is an axin-binding protein promoting beta-catenin degradation

A novel SUMO-1/Smt3-specific isopeptidase, SMT3IP2/Axam2 (Smt3-specific isopeptidase 2), was cloned and characterized. The catalytic domains in the carboxyl-terminal region were very much similar to those of other SUMO-1/Smt3-specific proteases, but the amino-terminal part was quite different. The enzyme specifically bound to Smt3a and Smt3b but not to SUMO-1. The SMT3IP2 expressed by Escherichia coli could cleave SUMO-1, Smt3a, or Smt3b from a SUMO-1/RanGAP1, Smt3a/RanGAP1, or Smt3b/RanGAP1 conjugate, respectively, and had the activity of a carboxyl-terminal hydrolase to produce a glycine residue in the carboxyl terminus of these ubiquitin-like proteins. The sequence data indicated that the amino acid sequence of SMT3IP2 was mostly identical to that of rat Axam, which binds to Axin and promotes the degradation of beta-catenin, although its amino-terminal region was much shorter than that of Axam. Therefore, we designated this isopeptidase SMT3IP2/Axam2. When human SW480 cells were transfected with wild-type SMT3IP2/Axam2, the beta-catenin disappeared. When the cells were transfected with the SMT3IP2/Axam2 C500A mutant, which had neither isopeptidase nor carboxyl-terminal hydrolase activity, or with the 1-352 mutant, which lacked the catalytic domain of the enzyme, again the beta-catenin disappeared, indicating that the enzyme activities were not necessary for the instability of beta-catenin in this transfection assay system and that its competition with Dvl for binding to Axin may be important for the instability of beta-catenin as suggested previously for Axam (Kadoya, T., Kishida, S., Fukui, A., Hinoi, T., Michiue, T., Asashima, M., and Kikuchi, A. (2000) J. Biol. Chem. 275, 37030-37037). The involvement of its enzyme activities in the Wnt signaling pathway remains to be elucidated.     

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.     

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.     

Phosphorylation-dependent septin interaction of Bni5 is important for cytokinesis

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Delta. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.     

Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3(+) gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3Delta cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3(+) is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.     

A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase.

A novel Smt3-specific isopeptidase, SMT3IP1, was cloned using a yeast two-hybrid screen with Smt3b as bait. The clone, named SMT3IP1 (Smt3-specific isopeptidase 1), which bound to Smt3b but not SUMO-1 in the two-hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1. The catalytic domains in the C-terminal region were very similar, but the N-terminal region was quite different to other enzymes. The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved. SMT3IP1 expressed by the baculovirus-expression system had the ability to cleave SUMO-1 or Smt3b from SUMO-1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO-1 conjugate. Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO-1 in vitro. The enzyme did not cleave Nedd8 from Nedd8/cullin-1. Nor did it cleave ubiquitin from ubiquitinated p53. SMT3IP1 was localized almost exclusively at the nucleolus during interphase. The N-terminal sequence was responsible for the nucleolar localization of this enzyme. Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated.     

Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.     

Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.     

Septins,under Cla4p regulation,and the chitin ring are required for neck integrity in budding yeast

CLA4, encoding a protein kinase of the PAK type, and CDC11, encoding a septin, were isolated in a screen for synthetic lethality with CHS3, which encodes the chitin synthase III catalytic moiety. Although Ste20p shares some essential function with Cla4p, it did not show synthetic lethality with Chs3p. cla4 and cdc11 mutants exhibited similar morphological and septin localization defects, including aberrant and ectopic septa. Myo1p, which requires septins for localization, formed abnormally wide rings in cla4 mutants. In cultures started with unbudded cells, an inhibitor of Chs3p activity, nikkomycin Z, aggravated the abnormalities of cla4 and cdc11 mutants and gave rise to enlarged necks at the mother-bud junction, leading to cell death. It is concluded that Cla4p is required for the correct localization and/or assembly of the septin ring and that both the septin ring and the Chs3p-requiring chitin ring at the mother-bud neck cooperate in maintaining the neck constricted throughout the cell cycle, a vital function in budding yeast.     

Septin pairs, a complex choreography

Septins form a filamentous collar at the mother-bud neck in budding yeast. In cytokinesis, this collar splits into two rings and the septin complexes undergo a dramatic reorientation. Using fluorescence polarization microscopy, DeMay et al. (2011. J. Cell Biol. doi:10.1083/jcb.201012143) now demonstrate that septin complexes assemble as paired filaments in vivo and reveal new insights into septin organization during cytokinesis.     

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.     

The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II

In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.     

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.