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 shs1 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 shs1 cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10 and cdc11 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.

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IN Saccharomyces cerevisiae a filamentous structure consisting of "neck filaments" was observed by electron microscopy at the bud neck, which is the cleavage site during cytokinesis (BYERS and GOETSCH 1976a). A temperature-sensitive mutation in any of CDC3, CDC10, CDC11, and CDC12 genes causes a defect in cytokinesis and disappearance of the neck filaments at the nonpermissive temperature (BYERS and GOETSCH 1976b). The amino acid sequences of the gene products deduced from the nucleotide sequences of these genes are similar to each other, and these proteins are collectively designated septins (HAARER and PRINGLE 1987; FORD and PRINGLE 1991; KIM et al. 1991; SANDERS and FIELD 1994). Two other septins, Spr3 and Spr28, are spore specific, and a seventh septin, Shs1/Sep7, was identified on the basis of sequence homology (OZSARAC et al. 1995; DE VIRGILIO et al. 1996; CARROLL et al. 1998; MINO et al. 1998).

Except for Spr3 and Spr28, septins express during vegetative growth and are localized to the bud neck during the budding cycle (HAARER and PRINGLE 1987; FORD and PRINGLE 1991; KIM et al. 1991). Approximately 15 min before bud emergence, septins are recruited to the presumptive bud site (KIM et al. 1991), which depends on activated Rho-type GTPase Cdc42 and its effectors Gic1 and Gic2 (IWASE et al. 2006). After recruitment at the incipient bud site, septins are reorganized and assembled as a higher-ordered ring structure at the mother-bud neck, which requires Cdc42, its GTPase-activating factors (Bem3, Rga1, and Rga2), Bni5, Nap1, Bni1, Cla4, Gin4, and Elm1 (CVRCKOVA et al. 1995; RICHMAN et al. 1999; BOUQUIN et al. 2000; LONGTINE et al. 2000; WEISS et al. 2000; GLADFELTER et al. 2001a, 2002, 2004; LEE et al. 2002; CAVISTON et al. 2003; GOEHRING et al. 2003; KADOTA et al. 2004; VERSELE and THORNER 2004).

Many proteins of budding yeast are known to localize to the bud neck in a septin-dependent manner (BI 2001; GLADFELTER et al. 2001b). Those are proteins functioning in septin recruitment and ring assembly (see above), cytokinesis (see below), bud-site selection (CHANT et al. 1995; SANDERS and HERSKOWITZ 1996; KANG et al. 2004), cell cycle control (FRENZ et al. 2000; YOSHIDA and TOH-E 2001), and mitotic signaling network (CARROLL et al. 1998; LONGTINE et al. 1998, 2000; BARRAL et al. 1999; LEE et al. 2002; IWASE and TOH-E 2004). Septin serves as a scaffold for these proteins to localize and maintain at the bud neck. In S. cerevisiae, assembly of the actomyosin-based contractile ring occurs in two temporally distinct stages (LIPPINCOTT and LI 1998; BI et al. 1998). Early in the cell cycle, type II myosin heavy chain Myo1 assembles at the incipient bud site. This occurs shortly after the appearance of the septin ring, in a septin-dependent and F-actin-independent manner (LIPPINCOTT and LI 1998; BI et al. 1998). Hof1 localizes to the bud neck shortly after bud emergence and is associated with the actomyosin ring during cytokinesis (VALLEN et al. 2000). In late anaphase, actin filaments are recruited to the Myo1 ring to produce an actomyosin ring (LIPPINCOTT and LI 1998; BI et al. 1998). The accumulation of actin in the cytokinetic ring was not observed in the cells depleted Iqg1, which is a member of the IQGAP family, suggesting that Iqg1 plays a role in recruitment of actin filaments to the Myo1 ring. Disruption of IQG1, but not of MYO1, is lethal in most strain backgrounds, suggesting that Iqg1 plays an additional role in cytokinesis other than actomyosin ring assembly. Iqg1 is localized to the bud neck slightly before actin ring formation (EPP and CHANT 1997; LIPPINCOTT and LI 1998). One more cytokinesis related protein, Cyk3, is involved in septum formation independent of actin ring formation, which is isolated as a multicopy suppressor of the iqg1 strain. Cyk3 is localized to the bud neck shortly after actin ring formation (KORINEK et al. 2000). Myo1, Hof1, and Cyk3 target independently to the septin ring and seem to carry out their roles in cytokinesis independently since deletion of any two causes cell lethality (VALLEN et al. 2000; KORINEK et al. 2000). However, in wild-type cells, the components of the actomyosin ring and septum formation must coordinate with each other in time and space so that cytokinesis can be carried out most efficiently (BI 2001).

As described above, septin is a dynamic entity and required for recruiting numerous proteins to the bud neck. How do septin monomers assemble to form the septin ring? How does the septin ring dissolute from the bud neck? How are bud neck proteins recruited to the right place at the right time? Mechanisms underlying septin-related phenomena remain elusive. To this end, detailed analysis of each septin gene product is inevitable. Temperature-sensitive (Ts) mutants (cdc3-1, cdc10-1, cdc11-1, and cdc12-1) have been employed for characterization of septin genes, but there is a drawback to analysis of the functions of individual septins, because all septins (or septin complexes) disappear from the bud neck together upon the shift of a septin-Ts mutant to the restrictive temperature. For this reason, until now there have been very few detailed reports about individual septin functions. Here, we analyze functions of Shs1. SHS1 has an advantage over other septin genes for conducting functional analysis because SHS1 is nonessential for cell viability. Here, we demonstrate that (i) Shs1 is not required for assembly of the septin complex in the wild-type cells; (ii) Shs1 is required for septin organization in the cdc10 cells; (iii) Shs1 inhibits the formation of the septin complex in the cdc11 cells; and (iv) Shs1 is needed for a proper function of Iqg1, Myo1, and Cyk3. Together, our results suggest that Shs1 plays multiple roles in septin organization and cytokinesis.

Genetic manipulations:

The yeast strains used in this study are listed in Table 1. Yeast cells were grown on rich medium (YPD) that consisted of 2% polypepton (Nihon Seiyaku), 1% Bacto yeast extract (Difco), 2% glucose, 0.04% adenine sulfate, and 0.005% uracil, or on synthetic complete medium (SC) that consisted of 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose, and appropriate supplements (SHERMAN et al. 1983). Drop-out medium was prepared by dropping out the indicated nutrient from SC. SC–Ura, for example, indicates SC without uracil. YPgal or SCgal contained 2% galactose instead of 2% glucose in YPD or SC medium, respectively. Sporulation medium consisted of 1% potassium acetate. Medium for a two-hybrid screening was prepared by adding 30 mM 3-amino-1,2,4-triazole (3-AT) into SC–Leu–Trp–His medium. Standard yeast genetic manipulations were described previously (SHERMAN et al. 1983). Yeast transformations were performed by the lithium acetate method (ITO et al. 1983; GIETZ and SCHIESTL 1991). Escherichia coli DH5 [supE44 lacU169 (80lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used for construction and propagation of plasmids. E. coli cells were grown in Luria–Bertani broth (LB) that consisted of 0.8% Bacto tryptone (Difco), 0.5% Bacto-yeast extract (Difco), and 0.5% NaCl. Sodium ampicillin (40 mg/liter) was supplemented to LB medium as appropriate. E. coli was grown at 37°. E. coli transformation was carried out as described by INOUE et al. (1990). Agar was added (2%) to prepare solid media. Plasmids used in this study are listed in Table 2 .

View this table: In this window In a new window   TABLE 1 Yeast strains used in this study

 

View this table: In this window In a new window   TABLE 2 Vectors and plasmids used in this study

 

DNA manipulation:

DNA preparation, restriction enzyme analysis, and agarose gel electrophoresis were carried out as described by SAMBROOK et al. (1989). Polymerase chain reaction (PCR) was performed using the Expand High Fidelity PCR system (Roche Diagnostics) according to the manufacturer's instructions. The activity of a gene cloned by the PCR method was confirmed by its ability to complement the defect of the gene of interest. Nucleotide sequences were determined by the dideoxy chain termination method (SANGER et al. 1977) using an automated DNA sequencer (ABI 370A, Applied Biosystems). The adenine nucleotide of ATG corresponding to the putative initiation codon is defined as nucleotide +1.

Yeast two-hybrid analysis:

Two-hybrid analysis was performed using strain J21 containing the LexA DBD fusion plasmid and AD fusion plasmid. The extent of two-hybrid interaction was estimated by assaying ß-galactosidase activity in each Leu⁺ Trp⁺ transformant. ß-Galactosidase activity was expressed as described (GUARENTE 1983). One unit = (1000 x OD_{420 nm})/(t x OD_{600 nm}), where t is the reaction time in minutes.

Morphological observation:

Cells were fixed with 5% formaldehyde for 30 min. After fixation, cells were washed with PBS (140 mM NaCl, 2.7 mM KCl, and 3.8 mM Na₂HPO₄) three times and suspended in PBS. The fluorescence of GFP was observed with an epifluorescence microscope [BX60 (Olympus) or IX70 (Olympus)] and photographed with an automatic camera [PM-C35DX (Olympus)] or a chilled CCD camera [SENSYS III (Nippon Roper)].

Preparation of cell lysates:

Approximately 10⁸ cells were disrupted with glass beads in lysis buffer [0.1 M Tris-HCl (pH 7.5), 0.2 M NaCl, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 5% glycerol] containing protease inhibitors [1 mM phenyl methyl sulfonyl fluoride (PMSF), 1 µg/ml antipain, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin] and diluted with the same buffer to a final concentration of 8 µg total protein per microliter. Samples were boiled with SDS–PAGE sample buffer [12.5 mM Tris-HCl (pH 6.8), 2% glycerol, 0.4% SDS, 1% ß-mercaptoethanol, 0.0002% Bromophenol Blue] for 5 min, and the resulting supernatant was subjected to immunoblot analysis.

Deletion of SHS1 causes a defect in cytokinesis but not in septin-ring assembly at the restrictive temperature:

Among septin genes (CDC3, CDC10, CDC11, CDC12, and SHS1) that are expressed in vegetative cells, SHS1 has been most poorly investigated. To determine whether or not Shs1 exerts a distinct function from other septins in septin ring assembly and/or cytokinesis, we constructed shs1 cells in the W303 background. Proper disruption of SHS1 was verified by PCR (data not shown). The shs1 cells grew slowly at 25° and did not grow at 20° (Figure 1A). At the restrictive temperature, the shs1 cells formed chains or clusters (Figure 1B). The zymolyase-treated shs1 cells were still unseparated, suggesting that they have a cytokinesis defect (data not shown). These results indicate that Shs1 plays a role in cytokinesis. To determine whether there is any functional redundancy between Shs1 and other septins, multicopy plasmid carrying each of septin genes was introduced into the shs1 strain. As shown in Figure 1C, the cold sensitivity of shs1 cells was suppressed by CDC11 on a multicopy plasmid. A higher dosage of CDC11 partially alleviated the morphological and cytokinetic defects of shs1 cells (Figure1D).

View larger version (70K): In this window In a new window Download PPT slide   FIGURE 1.— Phenotype of an shs1 disruptant. (A) Disruption of SHS1. One of the alleles of SHS1 in W303a/ was replaced with the shs1::HIS3 gene (Masa1). The resulting heterozygous diploid cells were sporulated and dissected. One wild-type and two disruptant segregants were chosen among the segregants and streaked across YPD plates, each of which was incubated at 20° for 4 days, or at 25°, 30°, and 37° for 2 days. (B) Cell morphology of shs1 cells. The shs1 cells (Masa2) and wild-type cells (W303a) were cultivated at 30° to midlogarithmic phase and then shifted to 20°. The cultures were incubated for another 6 hr. Bar, 10 µm. (C) Suppression of shs1 cells by a different septin gene on a multicopy vector. The indicated plasmid (CDC3, pM-9; CDC10, pM-10; CDC11, pM-11; CDC12, pM-12; SHS1, pM-13) was introduced into the shs1 cells (Masa2). One representative was selected from each transformation experiment and streaked on two SC–Ura plates, one of which was incubated at 20° for 4 days (left) and the other at 30° for 2 days (right). (D) Cell morphology of shs1 cells carrying CDC11 on a multicopy vector. The indicated plasmid (SHS1, pM-13; CDC11, pM-11; vector, pYO326) was introduced into the shs1 cells (Masa2). Cells were cultivated at 30° to midlogarithmic phase and then shifted to 20°. The cultures were incubated for another 6 hr. Bar, 10 µm.

 
Localization of any of Cdc3, Cdc10, Cdc11, and Cdc12 at the bud neck is dependent on the presence of the other wild-type septins at 37° (HAARER and PRINGLE 1987; FORD and PRINGLE 1991; KIM et al. 1991). To examine whether localization of Shs1 depends on other septins, we constructed a series of septin mutant strains carrying the SHS1-GFP fusion gene and the GFP signal was observed. Shs1-GFP localized to the bud neck in each of the septin mutants at a permissive temperature (25°) but not at a restrictive temperature (37°). In the wild-type strain, Shs1-GFP is localized to the bud neck irrespective of temperature (Figure 2A). Thus, the localization of Shs1 at the bud neck needs a normal set of other septins.

View larger version (78K): In this window In a new window Download PPT slide   FIGURE 2.— Localization of septin-GFPs in a septin mutant. (A) Localization of Shs1-GFP. The chromosomal SHS1 gene of each of the septin mutants was replaced with the SHS1-GFP gene. Cells of cdc3-1 SHS1-GFP (Masa11, a and a'), cdc10-1 SHS1-GFP (Masa12, b and b'), cdc11-1 SHS1-GFP (Masa13, c and c'), cdc12-1 SHS1-GFP (Masa14, d and d'), and SHS1-GFP (Masa6, e and e') were grown at 25° to midlogarithmic phase and a part of each culture was shifted to 37°. After 4 hr of incubation at 37°, cells were harvested, fixed with 5% formaldehyde, and GFP was observed. Bar, 10 µm. (B) Localization of septin-GFPs in the shs1 strain. Septin-GFP fusion genes were separately introduced into the shs1 cells (Masa2). The shs1 cells expressing one of septin-GFP fusion protein were cultivated at 30° to midlogarithmic phase and then shifted to 20°. Cells were harvested at 6 hr after the shift, and GFP was observed. Bar, 10 µm. (a and a') Cdc3-GFP (pM-1); (b and b') Cdc10-GFP (pM-2); (c and c') Cdc11-GFP (pM-3); (d and d') Cdc12-GFP (pM-4); (e and e') Masa6.

 
To examine whether localization of other septins depends on Shs1, shs1 cells carrying each of the septin-GFP fusions were cultured to midlogarithmic phase at 30° and shifted to the restrictive temperature (20°). After 6 hr of incubation, chained cells were observed in all the cultures except that of the shs1 cells expressing Shs1-GFP. In all cases except shs1 cells containing SHS1-GFP, the GFP signals were found exclusively at the bud neck of the outer most cells (Figure 2B). These results suggest that other septins in shs1 cells have undergone through the normal cell cycle-regulated disassembly and assembly process, i.e., septins that are disassembled at the bud neck after cytokinesis in the previous cell cycle are reassembled into ring structures at the next budding site. These results also indicate that any of the septins, Cdc3, Cdc10, Cdc11, and Cdc12 can be incorporated into the septin ring without Shs1.

Shs1 is required for contractile ring assembly and septum formation:

As described above, shs1 cells are defective in cytokinesis in spite of the presence of the septin complexes at the bud neck. To determine whether the contractile ring was formed or not in shs1 cells, we tried to observe the localization of Iqg1 in shs1 cells. To this end, we attempted to construct an shs1 iqg1::IQG-GFP strain. The shs1 cells were crossed to the strain whose genomic IQG1 was replaced with IQG1-GFP. Surprisingly, we failed to obtain shs1 cells containing iqg1::IQG1-GFP, indicating that IQG1-GFP did not function fully and that shs1 and iqg1::IQG1-GFP were synthetically lethal (Figure 3A). Consistent with this observation is our finding that shs1 and iqg1-1 also showed synthetic lethality (data not shown). In contrast, cdc10 iqg1::IQG1-GFP cells were viable (Figure 3B), even though cdc10 cells displayed more severe growth and morphological defects than shs1 cells (Figure 4, A and B). Additionally, double mutants of cdc3-1 iqg1::IQG1-GFP, cdc10-1 iqg1::IQG1-GFP, cdc11-1 iqg1::IQG1-GFP, and cdc12-1 iqg1::IQG1-GFP were found to be viable (data not shown), suggesting that the synthetic lethality observed between shs1 and IQG1-GFP or iqg1-1 is due to an SHS1-specific function, not a general function of the septin genes.

View larger version (118K): In this window In a new window Download PPT slide   FIGURE 3.— The interactions between the septins and cytokinesis genes. (A) The double mutant between shs1 and iqg1 was lethal. Tetrad analysis of diploid cells from a cross between shs1 cells (Masa2) and iqg1::IQG1-GFP cells (Masa98) was carried out. Tetrads were dissected and grown on YPD plate at 25° for 7 days. Genotypes of the segregants are indicated on the right. +, SHS1 or IQG1; –, shs1 or iqg1::IQG1-GFP. (B) The double mutant between cdc10 and iqg1 was viable. Tetrad analysis of diploid cells from a cross between cdc10 cells (Masa22) and iqg1::IQG1-GFP cells (Masa98) was carried out. Tetrads were dissected and grown on YPD plate at 25° for 9 days. Genotypes of the segregants are indicated on the right. +, CDC10 or IQG1; –, cdc10 or iqg1::IQG1-GFP. (C) The localization of Iqg1-GFP in an shs1 disruptant. The IQG1-GFP fusion gene (IQG1pUG35) was introduced into the shs1 cells (Masa2) and wild-type cells (W303a). Cells were cultivated at 30° to midlogarithmic phase and then shifted to 20°. Cells were harvested at 6 hr after the shift, and GFP was observed. Arrows indicate Iqg1-GFPs, and arrowheads indicate mislocalized Iqg1-GFPs. Bar, 10 µm. (a, a', b, and b') Wild type at 20°; (c, c', d, and d') shs1 at 20°; (e, e', f, and f') wild type at 30°; (g, g', h, and h') shs1 at 30°. (D) The localization of Myo1-GFP in an shs1 disruptant. The MYO1-GFP fusion gene was integrated into shs1 cells (Masa2) and wild-type cells (W303a). Cells were cultivated and observed as described in C. Arrows indicate Myo1-GFPs, and an arrowhead indicates mislocalized Myo1-GFP. Bar, 10 µm. (a, a', b, and b') Wild type (Masa105) at 20°; (c, c', d, and d') shs1 (Masa104) at 20°; (e, e', f, and f') wild type (Masa105) at 30°; (g, g', h, and h') shs1 (Masa104) at 30°. (E) The localization of Cyk3-8xGFP in an shs1 disruptant. The CYK3-8xGFP cells (YAT3200) were crossed with the shs1 cells (Masa2), and the resulting diploid cells were sporulated and dissected. shs1 cells containing CYK3-8xGFP and YAT3200 cells were cultivated and observed as described in C. Arrows indicate Cyk3-8xGFPs, and an arrowhead indicates mislocalized Cyk3-8xGFP. Bar, 10 µm. (a, a', b, and b') Wild type (YAT3200) at 20°; (c, c', d, and d') shs1 (Masa2175) at 20°; (e, e', f, and f') wild type (YAT3200) at 30°; (g, g', h, and h') shs1 (Masa2175) at 30°.

 

View larger version (51K): In this window In a new window Download PPT slide   FIGURE 4.— SHS1 was required for septin assembly in the cdc10 cells. (A) The shs1 cdc10 cells were lethal. The cdc10 cells (Masa24) were crossed with the shs1 cells (Masa2), and the resulting diploid cells (Masa31) containing GAL1-CDC10 plasmid (pM-92) were sporulated and dissected. A set of spore clones derived from one tetra-type ascus was streaked on YPD plates at 25° for 7 days and on YPGal plate at 25° for 4 days. shs1 cdc10, Masa90; shs1, Masa106; cdc10, Masa102; wild type, Masa107. (B) The morphology of the shs1 cdc10 cells. The set of spore clones derived from one tetra-type ascus, which was used in (A) was grown to midlogarithmic phase at 25° in SCgal-Trp medium, and then harvested, washed, and resuspended into SC-Trp medium, where the expression of CDC10 was shut off, at 25°. After 8 hr of incubation, cells were harvested and observed. Bar, 10 µm. (a) cdc10 shs1 (Masa90); (b) shs1 (Masa106); (c) cdc10 (Masa102); (d) wild type (Masa107). (C) Septin-GFP was not localized at the bud neck in the cdc10 shs1 cells. The set of spore clones derived from one tetra-type ascus that was used in A was employed in this experiment. CDC12-GFP plasmid (pM-4) was introduced into each strain and grown to midlogarithmic phase at 25° in SCgal–Ura medium and then harvested and washed. Cells were resuspended into SC–Ura medium to shut off GAL1-CDC10 expression at 25°C. After 8 hr of incubation, cells were harvested and subjected to GFP observation. Bar, 5 µm. (a and a') cdc10 shs1 (Masa90[pM-4]); (b and b') shs1 (Masa106[pM-4]); (c and c') cdc10 (Masa102[pM-4]); (d and d') wild type (Masa107[pM-4]).

 
To explore the interaction between SHS1 and IQG1 further, we constructed an shs1[IQG1-GFP] strain and an SHS1[IQG1-GFP] strain and compared GFP signals in these strains. We found that Iqg1-GFP in the shs1 background was much less localized to the bud neck than that in the SHS1 background and some shs1 cells displayed mislocalized Iqg1-GFP near the bud neck as a dot at 20° and 30° (Figure 3C). Because Iqg1 plays a role in recruiting actin filaments to the Myo1 ring (EPP and CHANT 1997; LIPPINCOTT and LI 1998), it is conceivable that the localization of Myo1 would also depend on Shs1. As expected, Myo1-GFP was delocalized or mislocalized from the bud neck like Iqg1-GFP in most shs1 cells at 20° and 30° (Figure 3D) whereas Myo1-GFP was localized to the bud neck of the wild-type cells under the same conditions. These results clearly indicate that Shs1 is required for the actomyosin contractile ring formation at the bud neck. Furthermore, we examined whether the septum formation would depend on Shs1, because shs1 and a mutant of the actomyosin ring component iqg1 showed synthetic-lethal growth. A component of septum formation machinery, Cyk3-8xGFP, which is localized at the bud neck in the wild-type cells, was also delocalized or mislocalized from the bud neck in most of shs1 cells at 20° and 30° (Figure 3E). These results indicate that Shs1 is also required for septum formation.

Shs1 is required for septin organization in cdc10 cells:

Shs1 is not required for the septin-ring assembly in otherwise wild-type backgrounds (Figure 2B), but Shs1 plays a role in septin organization when the function of one of other septin genes is compromised. Masa31 cells (shs1/SHS1 cdc10/CDC10) containing plasmid pM-92 (CDC10 driven by the GAL1 promoter) were sporulated and subjected to tetrad analysis, and the shs1 cdc10 cells containing pM-92 were obtained on a YPGal plate. These cells did not grow when CDC10 driven by the GAL1 promoter was shut off on a glucose plate (YPD), indicating that shs1 and cdc10 are synthetically lethal (Figure 4A). The shs1 cdc10 cells showed severe morphological and cytokinetic defects (Figure 4B, a). To analyze septin ring formation in the shs1 cdc10 cells, we observed localization of a septin using Cdc12-GFP. In most of shs1 cdc10 cells, Cdc12-GFP was not observed at the bud neck (Figure 4C, a). These results suggest that the lethality of the shs1 cdc10 cells is likely caused by the absence of the septin complexes at the bud neck and that Shs1 is required for septin organization at the bud neck in cdc10 cells.

Shs1 inhibits septin organization in cdc11 cells:

Next, we performed genetic crosses between the shs1 strain and one of other septin mutants to examine functional interactions between them. Data shown in Figure 5A indicate that the double mutants of cdc3-1 shs1, cdc10-1 shs1, and cdc12-1 shs1 were lethal, but the cdc11-1 shs1 cells were viable. Since cdc11 in the W303 background is lethal but in the YPH background is temperature-sensitive, we carried out the following experiments with a cdc11 mutant of the YPH background. cdc11 cells that are viable at 25° were crossed with shs1 cells with the YPH background, and a set of four spore clones in a tetratype ascus was recovered at 25°. cdc11 segregants were not able to grow at 30°, whereas cdc11 shs1 double disruptants grew at 30° (Figure 5B). These results indicate that the disruption of the SHS1 gene allows the cdc11 cells to grow. We also obtained same results in other 18/18 of cdc11 shs1 segregants (data not shown). This conclusion is supported by the following experiments. In cdc11 cells, nearly all Cdc12-GFP was delocalized from the bud neck (Figure 5C, e'–g'), and some weak GFP signals were localized to the bud cortex (arrows in Figure 5C, g') at the restrictive temperature. In contrast, Cdc12-GFP was concentrated to the bud-neck region in most cdc11 shs1 cells at the same temperature, although Cdc12-GFP did not make a tight ring structure (Figure 5C, a'–c'). Furthermore, overproduction of SHS1 did not cause growth defect in the wild-type cells, but inhibited growth of the cdc11 cells (Figure 5D). The morphology of the cdc11 cells containing overproduction of SHS1 was more elongated than that of the cdc11 cells containing vector plasmid, although overproduction of SHS1 also caused the morphological defect in the wild-type cells (Figure 5E). These results indicate that Shs1 inhibits assembly of the septin complex in the cdc11 cells. Additionally, it should be emphasized that, Cdc12-GFP was localized to all the bud necks in the chained cells of the cdc11 shs1 strain (Figure 5C, a'–c') and the cdc11 strain at a permissive temperature (data not shown). This observation is in a clear contrast to the fact that septins are delocalized after cytokinesis and move to the next budding site in the wild-type cells. The results shown in Figure 5C indicate that Cdc11 is essential for the disassembly of the septin ring after cytokinesis.

View larger version (132K): In this window In a new window Download PPT slide   FIGURE 5.— Interactions between SHS1 and CDC11. (A) Phenotypes of the double mutants. The shs1::HIS3 cells (Masa2) containing SHS1 on multicopy plasmid (pM-13) were crossed with each of the septin mutants (cdc3-1, YAT1775; cdc10-1, YAT1769; cdc11-1, YAT1773; cdc12-1, YAT1765). A double mutant containing pM-13 from each cross was selected and streaked across FOA plate (+FOA) and SC–Ura plate (–FOA), which were incubated at 25° for 4 days. cdc3-1 shs1, Masa86; cdc10-1 shs1, Masa87; cdc11-1 shs1, Masa88; cdc12-1 shs1, Masa89; shs1, Masa103. (B) cdc11 was suppressed by shs1. The cdc11 cells (Masa27) were crossed with the shs1 cells (Masa4), and the resulting diploid cells were sporulated and dissected. A set of spore clones derived from one tetra-type ascus was streaked on two YPD plates, one of which was incubated at 30° and the other at 25° for 2 days. (C) Cdc12-GFP localization in the cdc11 shs1 and cdc11 cells. Plasmid carrying the CDC12-GFP fusion gene (pM-4) was introduced into a set of tetrad segregants described in B. Each transformant was grown to midlogarithmic phase at 25° and then shifted to 30°. After 4 hr of incubation, cells were harvested and subjected to GFP observation. Arrows indicates cortical Cdc12-GFPs. Bar, 10 µm. (a, a', b, b', c, and c') cdc11 shs1; (d and d') shs1; (e, e', f, f', g, and g') cdc11; (h and h') wild type. (D) Growth inhibition of cdc11 cells by overproduction of Shs1. GAL1-GFP-SHS1 plasmid (pM-91) and vector plasmid (pGAL-GFP) were separately introduced into the cdc11 cells (Masa27) and wild-type cells (YPH499). Two transformants from each transformation experiment were streaked on SC–Ura plate and SGal–Ura plate at 25° for 4 days. (E) The morphology of the cdc11 cells overproducing Shs1. A set of transformants used in D was grown to midlogarithmic phase at 25° in SCgal–URA medium for 15 hr and then observed. Bar, 10 µm. (a) cdc11 (Masa27[pM-91]); (b) cdc11 (Masa27[pGAL-GFP]); (c) wild type (YPH499[pM-91]); (d) wild type (YPH499[pGAL-GFP]).

 

Isolation and characterization of the SHS1-100c mutant:

To determine physical interactions between septins, we evaluated two-hybrid interactions between Shs1 and other septins. Shs1 interacted strongly with Cdc12 and very weakly with Cdc11, Cdc3, and Cdc10 (Table 3). On the basis of these results, we generated a new shs1 mutant that is defective in interaction with Cdc12. First, we constructed deletion derivatives of SHS1, which were subjected for examining interaction with CDC12 by the two-hybrid assay. Interestingly, any region of Shs1 so far tested did not interact with Cdc12 except for 1–354 amino acids of Shs1 as shown by FARKASOVSKY et al. (2005) (Figure 6A). The lack of interaction between Cdc12 and derivatives of Shs1 was not due to a lack of production of derivative proteins, as these derivatives were produced at a level similar to those of full length Shs1 (Figure 6B, data not shown, except polypeptide 1–519 and polypeptide 312–551). During the course of this experiment, we generated an shs1 mutant missing 99 bp encoding the C-terminal 32 amino acid (aa) residues of Shs1, designated the shs1-100c mutant (+1 aa to +519 aa). This mutant exhibited no phenotypic change in growth irrespective of temperature (Figure 6C). Additionally, the Shs1-100c-GFP fusion was localized to the bud neck like the wild-type Shs1-GFP fusion, and it did not cause a morphological change to cells whose SHS1 had been replaced by shs1-100c-GFP (Figure 6D). However, shs1-100c, like shs1, was found to be synthetically lethal with cdc10 (Figure 7A), and Cdc12-GFP was delocalized from the bud neck in cdc10 shs1-100c cells (our unpublished data). On the other hand, shs1-100c partially suppressed temperature-sensitivity of cdc11 cells (Figure 7B), as does shs1, and Cdc12-GFP was concentrated to the bud-neck region in most of cdc11 shs1-100c cells at 30° like cdc11 shs1 cells (data not shown). These results indicate that the shs1-100c allows the cdc11 cells to grow. We also found the same results in the other 17/17 of cdc11 shs1-100c segregants (data not shown). In contrast to shs1, the shs1-100c iqg1::IQG1-GFP and shs1-100c iqg1-1 strains were viable (Figure 7C and data not shown about shs1-100c iqg1-1 strain). As reported by TONG et al. (2004), we confirmed that shs1 cyk3 was lethal and found that shs1-100c cyk3 was viable (our unpublished data). Furthermore, Iqg1-GFP, Myo1-GFP, and Cyk3-8xGFP were localized normally to the bud neck in shs1-100c strain (Figure 7D). These results suggest that the C-terminal 32 residues of Shs1 play an important role in septin organization but not in the Shs1-dependent localization of cytokinesis proteins Iqg1, Myo1, and Cyk3.

View this table: In this window In a new window   TABLE 3 Two-hybrid interactions between Shs1 and other septins

 

View larger version (50K): In this window In a new window Download PPT slide   FIGURE 6.— Phenotypes of the shs1-100c allele. (A) Two-hybrid interaction between Cdc12 and Shs1 truncated versions. Cells of J21 containing DBD-Cdc12 (pM-19) were transformed with AD-Shs1 (pM-93 (+1 aa to +90 aa), pM-94 (+91 aa to +318 aa), r26-5 (+312 aa to +551 aa), pM-95 (+1 aa to +253 aa), pM-2001 (+1 aa to +354 aa), pM-96 (+48 aa to +433 aa), pM-98 or pM109 (+1 aa to +519 aa), or pM-25 or pM-26 (+1 aa to +551 aa). The ß-galactosidase activity of each transformant was measured. Shs1 derivatives that gave a higher ß-galactosidase activity than a vector control were indicated as +, and those that gave a lower activity than a vector control were indicated as –. Each transformant was streaked on SC-Trp-Leu-His plate. +, growth; –, no growth. (B) Western blot analysis of AD-Shs1. Cell lysate prepared from the J21 cells carrying pM-25 (+1 aa to +551 aa), pM-109 (+1 aa to +519 aa), r26-5 (+312 aa to +551 aa), or pACT (vector plasmid) was subjected to electrophoresis on a 7.5% SDS–polyacrylamide gel followed by Western blot analysis using anti-GAL4 AD antibody (CLONTECH). Cdc28 was detected with anti-PSTAIRE antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as an internal reference. (C) Growth characteristics of the shs1-100c cells. shs1 cells (Masa2), shs1-100c cells (Masa91), and wild-type cells (W303a) were streaked across two YPD plates, each one of which was incubated at 30° for 3 days and the other at 18° for 4 days. (D) Localization of shs1-100c-GFP. SHS1–GFP (pM-101), shs1-100c-GFP (pM-102), and vector plasmids (pTS910CU) were separately introduced into the shs1 cells (Masa2). Cells were cultivated at 30° to midlogarithmic phase and then shifted to 20°. Cells were harvested at 6 hr after the shift, and GFP was observed. Bar, 5 µm. (a and a') Shs1–GFP; (b and b') shs1-100c-GFP; (c and c') vector.

 

View larger version (75K): In this window In a new window Download PPT slide   FIGURE 7.— Phenotypes of the shs1-100c allele. (A) Genetic interaction with CDC10. Tetrad analysis of diploid cells from a cross between cdc10 cells (Masa24) and shs1-100c (Masa91) was carried out. Tetrads were dissected and grown on YPD plate at 25° for 4 days. Genotypes of the segregants are indicated in the right. +, CDC10 or SHS1. –, cdc10 or shs1-100c. (B) Genetic interaction with CDC11. The cdc11 cells (Masa27) were crossed with the shs1-100c cells (Masa93), and the resulting diploid cells were sporulated and dissected. A set of spore clones derived from one tetra-type ascus was streaked on two YPD plates, one of which was incubated at 30° and the other at 25° for 3 days. (C) Genetic interaction with IQG1. Tetrad analysis of diploid cells from a cross between iqg1::IQG1-GFP cells (Masa98) and shs1-100c (Masa91) was carried out. Tetrads were dissected and grown on YPD plate at 25° for 7 days. Genotypes of the segregants are indicated on the right. +, IQG1 or SHS1. –, iqg1::IQG1-GFP or shs1-100c. (D) The localization of Iqg1-GFP, Myo1-GFP, Cyk3-8xGFP in shs1-100c. The shs1-100c cells (Masa91) containing the IQG1-GFP fusion plasmid (IQG1pUG35), MYO1-GFP integrated shs1-100c cells (Masa2154), and CYK3-8xGFP integrated shs1-100c cells (Masa2187) were cultivated and observed as described in Figure 3, C–E. Bar, 5 µm. (a and a') Iqg1-GFP; (b and b') Myo1-GFP; (c and c') Cyk3-8xGFP.

 

Shs1 acts differently in cytokinesis from other septins:

Like other septins, Shs1-GFP was delocalized in a temperature-sensitive mutant of cdc3, cdc10, cdc11, or cdc12. In contrast, Cdc3-, Cdc10-, Cdc11-, and Cdc12-GFP were all assembled into a ring structure at the bud neck of shs1 cells at the restrictive temperature. Under such a condition, shs1 cells displayed a severe defect in cytokinesis and cell separation and were not able to grow in spite of presence of the septin ring. This surprising finding indicates that Shs1 plays a specific role in cytokinesis differently from other septins. This conclusion is supported by our observations that shs1 and IQG1-GFP, and iqg1-1, were synthetically lethal (Figure 3A; our unpublished data) and that Iqg1, Myo1, and Cyk3 were delocalized or mislocalized from the bud neck in shs1 cells (Figure 3, C–E). Additionally, shs1 cyk3 and shs1 hof1 are synthetically lethal (TONG et al. 2004; our unpublished data). These findings strongly suggest that Shs1 plays supportive roles in cytokinesis by localizing cytokinetic factors to the bud neck.

Cytokinesis in S. cerevisiae is carried out by the coordinated actions of the actomyosin-based contractile ring and septum formation (BI 2001; FINGER 2005). Proteins involved in cytokinesis, such as Myo1, Iqg1, and Hof1, target to the bud neck in a septin-dependent manner (BI et al. 1998; LIPPINCOTT and LI 1998; VALLEN et al. 2000; BI 2001; GLADFELTER et al. 2001b), but it is not clear how individual septins contribute to this targeting process. Here, we showed that Iqg1 and Myo1 were delocalized or mislocalized from the bud neck in shs1 cells in spite of the presence of other septins at the bud neck. Because Iqg1 is required for the recruitment of actin to the Myo1 ring (EPP and CHANT 1997; LIPPINCOTT and LI 1998), our finding indicates that Shs1 is required for the assembly of the actomyosin contractile ring at the bud neck. However, in most strain backgrounds, the loss of the actomyosin contractile ring is not sufficient to cause cell death, although myo1 null cells form short chains of cells (BI et al. 1998), suggesting that cytokinesis could be carried out by septum formation without the contractile ring, albeit less efficiently (BI 2001). We also observed that Cyk3, a protein that is thought to be involved in septum formation (KORINEK et al. 2000; BI 2001), failed to localize in shs1 cells, suggesting that Shs1 is also required for septum formation. Thus, the severe defects of cytokinesis of shs1 are likely to be caused by defects in both the ac-tomyosin contractile ring and the septum formation. In contrast to shs1 cells, iqg1 mutants did not show synthetic lethality with cdc10 or other septin mutants. Furthermore, Myo1 localized to the bud neck in cdc10 cells at the permissive temperature, even though the growth and morphological defects of cdc10 cells are much more pronounced than in the isogenic shs1 cells that are cultured under the same conditions (J. LUO and E. BI, unpublished data). Together, our results suggest that Shs1, but not other septins, plays a direct role in orchestrating cytokinetic factors in cytokinesis.

Antagonistic roles of Shs1 in septin organization are visualized in cdc10 and cdc11 cells:

Septin mutants were originally isolated as temperature-sensitive cdc mutants (HARTWELL et al. 1970). Later, it was found that the requirement of individual septins for cell viability vary depending on the septin genes and the strain backgrounds. In the genetic background of W303, CDC10 is dispensable whereas CDC11 is dispensable in the genetic background of YPH, and either CDC10 or CDC11 is dispensable in the strains used by FRAZIER et al. (1998). The fact that shs1 cdc11 cells are viable suggests that three septins, Cdc3, Cdc10, and Cdc12 are sufficient for the minimal essential function of the septin complex. The fact that the cold-sensitive growth phenotype of the shs1 cells was suppressed by multicopy CDC11 (Figure 1C) implies that Shs1 and Cdc11 have a partially redundant function. However, our observations that Shs1 overproduction did not suppress temperature sensitivity of the cdc11-1 mutant (our unpublished data) and that overproduction of Shs1 was toxic to the cdc11 cells (Figure 5D) indicate that Shs1 and Cdc11 are not functionally interchangeable. These seemingly conflicting genetic observations can be explained by assuming that Shs1, but not Cdc11, is able to interact with a polarity or bud-cortex localized protein, such as Spa2 (MINO et al. 1998). This assumption is supported by the septin localization data presented in Figure 5C. With this assumption, one could imagine that in cdc11 cells, Shs1 channels most of the septin complex to the bud cortex (see arrows in cdc11 cells in Figure 5C, g') and by extrapolation, overexpression of Shs1 is toxic to cdc11 cells, presumably due to the more severe sequestering of the septin complex from the bud neck to the bud cortex. In contrast, overexpression of Cdc11 suppresses the growth defect of shs1 cells presumably by promoting septin complex formation at the bud neck. This simple assumption also explains why shs1 suppresses the growth defects of cdc11, because other existing septins in the shs1 cdc11 cells are now able to localize to the bud neck and promote cell survival. In contrast to the suppression of cdc11 cells by shs1, cdc10 and shs1 are synthetically lethal. These data suggest that Shs1 and Cdc10 play complementary roles in septin complex and/or filament formation. Cdc3, Cdc11, Cdc12, and Shs1 have both P-loop and coiled-coil domains in their sequences, although Cdc10 has a P-loop domain but not a coiled-coil domain. Septins form a conserved protein family, but they are not so similar (28.1–39.4%) to each other, suggesting that each septin may have a unique or differentiated function. To fully understand the phenotypic differences between shs1 cdc10 strain and shs1 cdc11 strain, it is essential to perform analysis of subunit composition of the septin complex produced in these double mutants. Moreover, MORTENSEN et al. (2002) reported that Shs1 interacts with Gin4 kinase independently of Cdc11 in cla4 and nap1 cells. Cdc3 is phosphorylated in vivo, which plays a role in septin ring disassembly (TANG and REED 2002). Three septins, Cdc3, Cdc11, and Shs1, are sumolylated during mitosis, but Cdc10 and Cdc12 are not (JOHNSON and BLOBEL 1999; TAKAHASHI et al. 1999). Thus, each septin receives specific or specific combinations of modifications. These different modifications may also contribute to phenotypic differences.

The roles of Shs1 in cytokinesis and septin organization are separable:

Genetic analysis of the shs1-100c mutant provided us new insights into the function of Shs1. Although the shs1-100c cells behave like wild-type cells, we found that the shs1-100c cdc10 and shs1-100c cdc11 double mutants displayed the same growth phenotypes as shown by the shs1 cdc10 and shs1 cdc11 double mutants, respectively. These results indicate that Shs1 lacking the C-terminal 32 amino acids does not interact with a certain component, most likely Cdc12, to cause the phenotypes shown by the double mutants. On the contrary, we found that shs1-100c iqg1::IQG1-GFP, shs1-100c iqg1-1, and shs1-100c cyk3 cells were viable (Figure 7C and our unpublished data). Moreover, localizations of Iqg1, Myo1, and Cyk3 were normal in shs1-100c (Figure 7D). As summarized in Figure 8, these results suggest that Shs1 lacking the C-terminal 32 amino acids cannot function properly in septin organization, as manifested by its genetic interactions with cdc10 and cdc11, but this mutant version of Shs1 is still competent for its role in the assembly of the actomyosin ring and septum formation. Thus, Shs1 appears to play separable roles in septin organization and cytokinesis.

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 8.— A model for the roles of Shs1 in septin organization and cytokinesis. Shs1 exerts different effects on septin organization depending on the subunit compositions of the septin complexes. Shs1 promotes and inhibits septin ring assembly in cdc10 and cdc11 cells, respectively. Shs1 lacking the C-terminal 32 amino acids can carry out its function in cytokinesis normally, including the recruitment of the components involved in the actomyosin contractile ring assembly such as Myo1 and Iqg1 as well as those involved in septum formation such as Cyk3. In contrast, the last 32 amino acids of Shs1 are required for its role in septin organization (see text for details).

 

The authors would like to thank Y. Kikuchi, Y. Matsui, T. Sasaki, J. Takeuchi, T. Michimoto, and S. Yoshida for strains, plasmids, and helpful discussions. M.I. was a recipient of a grant from Japan Society for the Promotion of Science for a predoctoral fellow. Research on cytokinesis in the Bi lab is supported by the American Cancer Society grant RSG-02-039-01-CSM.

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Communicating editor: M. D. ROSE

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