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Previous Article | Next Article 
Molecular and Cellular Biology, October 2002, p. 6906-6920, Vol. 22, No. 19
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.19.6906-6920.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Mark S. Longtine,4 and Kyung S. Lee1*
Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892,1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115,2 Department of Biology and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 27599,3 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 740784
Received 14 March 2002/ Returned for modification 23 April 2002/ Accepted 1 July 2002
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ABSTRACT |
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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 sep7 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. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Septins are a family of proteins that were identified first in yeast and subsequently in various other fungi and animals (for review, see references 20, 32, and 39). Septin family members possess at least 26% amino acid sequence identity. All of the known septins possess an N-terminal P-loop and other sequences conserved in the GTPase superfamily of nucleotide-binding proteins (8). In addition, at or near their C termini, most septins possess a predicted coiled-coil domain (for review, see reference 39) that may be important in protein-protein interactions.
In yeast, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p (or Shs1p) septins all localize to the presumptive bud site before bud emergence and remain at the mother-bud neck until after cytokinesis (11, 21, 28, 33, 45; M. S. Longtine, unpublished observations). Recent studies have indicated that the onset of contraction of the actomyosin ring results in division of the septins between the mother cell and the daughter cells (36, 38). Following cell separation, the septins disappear from the old cleavage site. At a restrictive temperature, temperature-sensitive mutations in CDC3, CDC10, CDC11, or CDC12 result in severe defects in cytokinesis and cell morphogenesis, yielding elongated, connected cells with multiple nuclei (1, 29), whereas loss of SEP7 function leads to milder bud elongation and cytokinetic defects (11, 45). Cdc3p, Cdc10p, Cdc11p, and Cdc12p copurify and form filaments in vitro (22), and they appear to be major structural components of the filaments observed at the neck by electron microscopy (9, 10, 22), although the details of protein arrangement in the filaments are not yet clear (22, 40).
The septins are thought to function as a scaffold for the localized assembly of various proteins at the mother-bud neck (20, 26, 40), including proteins important for cytokinesis. In a septin-dependent manner, the sole yeast type II myosin, Myo1p, assembles into a ring at the incipient budding site early in the cell cycle, whereas F actin is recruited to the myosin ring only late in the cell cycle, just before spindle disassembly and actomyosin ring contraction (5, 36, 37). Hof1p (or Cyk2p) also forms a septin-dependent ring at the mother-bud neck and appears to play an important role in modulating the stability of the actomyosin ring during contraction (36) and/or in septum formation (55). In addition to roles in cytokinesis, the yeast septins appear to be critical for diverse cellular functions, including the localization of chitin deposition (13), bud site selection (12), mother-daughter cell compartmentalization (3, 54), pheromone-induced morphogenesis (25), and the coordination of mitotic entry with morphogenesis (4, 11, 16, 42, 47).
To identify genes that are important for septin function, we sought high-copy suppressors of the cdc12-6 growth defect. We describe here the identification and analysis of a novel gene, BNI5. Our data suggest that Bni5p is important for providing stability to the neck-localized septins, probably through direct interactions with Cdc11p and perhaps also the other septins.
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MATERIALS AND METHODS |
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::KanMX6), HOF1/CYK2 (hof1::His3MX6), CDC10 (cdc10::KanMX6), CDC11 (cdc11::KanMX6), and SEP7/SHS1 (sep7::His3MX6) open reading frames (ORFs) were generated by the one-step gene disruption method (41). A strain expressing a BNI5-GFP2 fusion protein under endogenous BNI5 promoter control (KLY1737) was generated by C-terminally tagging the chromosomal copy of BNI5 in KLY1546 with a GFP2::KanMX6 fragment obtained by PCR with pSK1558 (a derivative of pFA6a-KanMX6 [41] that contains one additional copy of GFPS65T between the PacI and BssHII sites) as a template. Strain KLY1737 did not exhibit any detectable defects (data not shown), suggesting that Bni5p-GFP2 is functional. Strain KLY1718 was generated by introducing a plasmid containing GAL1 promoter-controlled GST-CDC11 into strain KLY1546 before disrupting the CDC11 ORF as described above. This strain grew well in galactose medium, but poorly in glucose medium (data not shown). To carry out localization and functional analyses of various forms of Cdc11p, both wild-type and mutant CDC11 alleles were C-terminally tagged with a PCR fragment containing three hemagglutinin (HA) epitopes (HA3), cloned into the URA3-based integration vector pRS306 (48), and then integrated into KLY1718 at the URA3 locus by digesting the plasmids with Sse8387I. Strain KLY1718 integrated with pRS306 itself was used as a control. To determine the localization efficiency of YFP Bni5p or YFP-Cdc10p fusions in wild-type and cdc11 mutant backgrounds, plasmid pRS314 containing either ADH1 promoter-controlled YFP-BNI5 (pKL1900) or CDC10 promoter-controlled YFP-CDC10 (pKL1901) was transformed into the KLY1718-derived strains. After repressing expression of GAL1-GST-CDC11 by growth in glucose-containing medium for various lengths of time, the localizations of these proteins were examined. YFP-Bni5p is functional, because expression of ADH1-YFP-BNI5 suppressed the cdc11-6 or cdc12-6 growth defect (data not shown). To generate strain SKY1601, the CDC3 locus in strain KLY1546 was C-terminally tagged with a PCR fragment containing nine myc epitopes (myc9) bridged with a TEV protease (Life Technologies, Rockville, Md.) cleavage site. Either full-length CDC11-HA3 or truncated cdc11-HA3 (amino acids 1 to 385) cloned in pRS306 was then integrated into SKY1601 at the URA3 locus (as described above) to generate strains SKY1824 and SKY1825, respectively.
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Growth conditions and media.
Yeast cell culture and transformations were carried out by standard methods (46). Yeast extract-peptone (YEP)-glucose, YEP-galactose, and synthetic media were used as appropriate. For cell cycle synchronization, MATa cells were arrested with 5 µg of 
mating pheromone (Sigma, St. Louis, Mo.) per ml for 3 h at 30°C and then released into fresh growth medium. To select against cells containing URA3 plasmids, cells were streaked onto synthetic minimal medium (SDM) supplemented with 1 g of 5-fluoroorotic acid (5-FOA) per liter (6).
Two-hybrid assays. Quantitative ß-galactosidase assays were performed essentially as described previously (2) with reporter plasmid pSH18-34. The assays shown in Fig. 6 used diploid strains obtained by mating isolates of strains EGY48 and EGY194 that had been individually transformed with a pEG202-NLS-based plasmid or with a pJG4-5-based plasmid (as described above); the assays shown in Table 5 used isolates of strain EGY48 that had been cotransformed with a pEG202-based plasmid and with a pJG4-5-based plasmid.
Immunoprecipitation and immunoblotting. Cell lysates were prepared in TED buffer, composed of 40 mM Tris-Cl (pH 7.5), 0.25 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride] (Pefabloc; Boehringer Mannheim, Indianapolis, Ind.), 10 µg of pepstatin A per ml (Sigma), 10 µg of leupeptin per ml (Sigma), and 10 µg of aprotinin per ml (Sigma), with an equal volume of glass beads (Sigma) as described previously (50). Immunoprecipitation was carried out as described previously (51). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (2). Western blot analyses of total lysates were carried out with anti-GFP (Clontech, Palo Alto, Calif.), anti-HA.11 (Babco, Richmond, Calif.), anti-LexA (Santa Cruz Biotechnologies, Santa Cruz, Calif.), anti-T7 (Novagen), anti-GST (Clontech), anti-myc (Santa Cruz Biotechnologies), anti-Cdc28 (a gift of R. Deshaies, California Institute of Technology, Pasadena, Calif.), and anti-Clb2 (a gift of D. Morgan, University of California, San Francisco, Calif.) antibodies as described previously (51), using the ECL enhanced chemiluminescence detection system (Pierce, Rockford, Ill.).
Preparation of recombinant proteins and in vitro protein-protein interaction studies. Recombinant T7-Bni5p-His6, GST, GST-Cdc3p, GST-Cdc10p, GST-Cdc11p, and GST-Cdc12p fusion proteins were expressed from plasmids pKL1995, pGEX-KG, pGEX-4T/CDC3, pGEX-4T/CDC10, pKL1993, pGEX-4T/CDC11, and pGEX-4T/CDC12 in Escherichia coli. T7-Bni5p-His6 was partially purified with the use of a Ni-nitrilotriacetic acid column (Qiagen, Valencia, Calif.) according to the manufacturer's protocol, and GST or GST-septins were purified by using glutathione-Sepharose beads (Sigma). In addition, T7-Bni5p-His6 was synthesized in vitro by using the T7-coupled rabbit reticulocyte system (Promega, Madison, Wis.). To investigate the interaction between Bni5p and the septins, in vitro-translated, 35S-labeled T7-Bni5p-His6 was added to either bead-bound GST-septins or bead-bound GST as a control, and then the mixture was incubated in a binding buffer (1x phosphate-buffered saline containing 0.5% NP-40) for 1 h at 4°C. The resin was then washed five times with the binding buffer. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and then analyzed by autoradiogram after SDS-PAGE. To further investigate the interaction between Bni5p and Cdc11p, T7-Bni5p-His6 partially purified from E. coli was added to either bead-bound GST-Cdc11p or bead-bound GST, and then the mixture was incubated in a binding buffer as described above. Bound Bni5p was separated by SDS-PAGE and detected by immunoblotting with anti-T7 (Novagen) or anti-GST (Clontech) antibodies.
Cell staining and immunofluorescence microscopy. To visualize plasma membranes, cells were stained with DiI (Molecular Probes, Eugene, Oreg.) as described previously (36). To determine whether septa were formed between the cell bodies, chitin was stained as described previously (36) with Calcofluor (Fluorescent Brightener 28; Sigma), and 100-nm-interval serial sections were obtained with either a Bio-Rad MRC 1024 confocal scan head mounted on a Nikon Optiphot microscope with a x60 planapochromat lens or a Leica TCS spectrophotometer confocal microscope. Indirect immunofluorescence was performed as described previously (34). Briefly, cells were fixed with 3.7% formaldehyde, and Cdc11p was visualized with an anti-Cdc11p antibody (Santa Cruz Biotechnologies) and a rhodamine-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody. Localization of GFP- or YFP-fused proteins was examined after fixing cells as described above. Similar results were obtained with unfixed cells. DNA was stained with 4',6'-diamidino-2-phenylindole (DAPI).
Time-lapse imaging. Cells were grown overnight in selective medium and placed on agarose pads as described previously (56). Living cells were imaged at room temperature on an Eclipse E600 microscope with differential interference contrast (DIC) optics and a Nikon 100/1.40 oil immersion objective. Images of Bni5p-GFP and Tub1p-GFP were collected through a neutral density filter with a value of 8 every 1 min with 0.8 s of exposure to fluorescent light by using a cooled RTE/CCD 782Y Interline camera (Princeton Instruments, Trenton, N.J.). The shutter was controlled automatically with a D122 shutter driver (UniBlitz, Rochester, N.Y.) and WinView 1.6.2 software (Princeton Instruments).
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RESULTS |
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100,000 transformants of strain M-1907 for growth at 32°C, recovering 30 plasmids that permitted growth at 32°C but not at 37°C. Sequencing of the ends of the inserts in these plasmids identified seven different genomic regions. Three plasmids that showed very good suppression had a region of overlap spanning nucleotides 323823 to 321482 of chromosome XIV. This region contains a single complete ORF of 1,347 bp (YNL166C), suggesting that overexpression of YNL166C was responsible for the observed suppression. This evidence and other data discussed below led us to rename YNL166C as "BNI5" (for Bud Neck Involved 5).
Next, we asked if overexpression of BNI5 could suppress the temperature-sensitive growth defects of other septin mutants. Indeed, overexpression of BNI5 either from a high-copy plasmid or under control of the GAL1 promoter suppressed the cdc10-1, cdc11-6, and sep7 mutants, in addition to the cdc12-6 mutant (Fig. 1 and Table 3). In contrast, no suppression of a cdc3-6 mutant was detected at several temperatures examined (Table 3). It is noteworthy that overexpression of BNI5 resulted in better suppression of the septin mutants tested than did overexpression of GIN4 (M. Longtine, unpublished data), which was previously shown to have a positive role in septin organization (40). These data suggest that Bni5p has a positive role in septin function.
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30% of KLY1831 cells were connected as short chains and/or displayed cell bodies that were more elongated than those of the wild type (Fig. 2A). In almost all cases, cells with normal morphology appeared also to have normal septin organization, as judged by the YFP-Cdc10p fluorescence signals. In contrast, most of the morphologically abnormal cells either lacked neck-localized YFP-Cdc10p (Fig. 2A, arrows) or displayed aberrantly organized YFP-Cdc10p structures (Fig. 2A, barbed arrows). In some cases, the aberrant septin structures appeared as a set of bars parallel to the mother-bud axis. Immunostaining with an anti-Cdc11p antibody revealed that the aberrant structures also contained Cdc11p (Fig. 2B) and thus, presumably, the other septins as well. Similar septin bars have been observed in cells carrying mutations in GIN4, CLA4, or NAP1, genes whose products appear to be involved in septin function and organization (40, 42).
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cells were grown at 37°C, sonicated, and stained with DiI to reveal the plasma membrane. In this assay, approximately 50% (n = 90) of internal mother-bud necks of connected cells with three or more cell bodies appeared to have a shared cytoplasm (Fig. 2C). bni5 cells were also stained with Calcofluor to assay for primary septum formation in the internal necks of cells with three or more cell bodies. Serial optical sectioning with a confocal microscope revealed discontinuous chitin deposition in 90% of such necks (n = 110) (Fig. 2D), indicating defective primary septum formation. Together, these observations suggest that Bni5p plays a role in cytokinesis. Because the septins are essential for cytokinesis and septin structures are frequently abnormal in bni5 mutant cells, it is likely that the observed cytokinesis defects in bni5 strains are related to the abnormal septin organization.
Exacerbation by bni5 of septin and hof1 mutant defects.
To further examine the interaction of Bni5p and the septins, a BNI5 deletion was introduced into cdc3-6, cdc10-1, cdc11-6, cdc12-6, and sep7 mutants. When cultured at temperatures that are normally semipermissive for these septin mutations, all double-mutant cells exhibited severely elongated buds and an apparent exacerbation of the cytokinesis defects (Fig. 3A). Compared to bni5 and septin single-mutant strains, bni5 cdc10-1, bni5 cdc11-6, bni5 cdc12-6, and bni5 sep7 double-mutant strains, but not a bni5 cdc3-6 double-mutant strain, also exhibited enhanced growth defects at elevated temperatures (Fig. 3C and data not shown). We also compared the localization of YFP-Cdc10p in cdc12-6 and bni5 cdc12-6 mutants. In the cdc12-6 strain, YFP-Cdc10p appeared to localize normally at 23°C, and approximately 80% of the cells possessed neck-localized YFP-Cdc10p after 3 h at 30°C (Fig. 3B and D). In contrast, in the bni5 cdc12-6 double-mutant strain, YFP-Cdc10p was often aberrantly localized at 23°C and was largely absent from the necks after 3 h at 30°C (Fig. 3B and D). Together, these observations suggest that Bni5p contributes to the maintenance or stability of neck-localized septins.
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was synthetically lethal with a hof1 mutation (Fig. 3E). Tetrad analysis of a diploid strain heterozygous for the bni5 and hof1 mutations also showed that bni5 hof1 double-mutant spores were incapable of forming colonies at 23°C. Microscopic analysis of the double-mutant segregants (Fig. 3F) revealed that they produced several interconnected cell bodies before cessation of growth, which appeared to result from cell lysis. In contrast, a bni5 myo1 double-mutant strain did not exhibit any detectable synthetic defect (data not shown), suggesting that Bni5p and Myo1p may function in the same pathway.
Cell cycle-dependent expression and localization of Bni5p.
To investigate the localization of Bni5p, the chromosomal BNI5 gene was tagged at its C-terminal end with two tandem copies of GFP sequences. The tagged Bni5p appeared to be fully functional (see Materials and Methods). Microscopic observation of asynchronously growing cells revealed a band of Bni5p-GFP at the mother-bud neck in most budded cells (Fig. 4A). However, Bni5p was not detectably localized in most unbudded cells or in some cells with nascent buds, as well as in some large-budded cells (Fig. 4A). Immunostaining with an anti-Cdc11p antibody revealed that the Bni5p-GFP band corresponded approximately to that of the septins (Fig. 4B). Although the Bni5p band often appeared to occupy only a portion of the region defined by the septin staining (Fig. 4B, arrows), this appearance might just result from the greater strength of the Cdc11p signal relative to that of the Bni5p-GFP signal. Interestingly, Bni5p-GFP localization was not detected in some unbudded or nascent-budded cells, or in some large-budded cells, even when the septins were clearly present (Fig. 4B, barbed arrows). These data suggest that Bni5p arrives at the bud site approximately coincident with bud emergence (and thus 10 to 15 min later than the septins [21, 33]) and dissociates from the septin scaffold before cytokinesis.
To explore further the timing of Bni5p localization and to investigate whether the changes in localization reflect changes in Bni5p abundance, we examined Bni5p-GFP levels and localization in a synchronous culture. Bni5p-GFP levels were low in the -factor-arrested cells and for 20 min after release (Fig. 4C). They then increased abruptly between 20 and 40 min and peaked at 50 to 70 min, approximately coincident with the G2/M peak in Clb2p levels (Fig. 4C). These observations are consistent with microarray data indicating that BNI5 mRNA is enriched in the S phase (52; Saccharomyces Genome Database, Stanford University, Calif.). The percentage of cells with detectably localized Bni5p-GFP also increased abruptly between 20 and 40 min after release and paralleled (but perhaps lagged slightly behind) the appearance of buds (Fig. 4D). Between 70 and 90 min after release, the number of cells with detectably localized Bni5p-GFP first fell abruptly, even though the number of budded cells remained high, and then began to increase again, presumably as new buds were formed in the next cell cycle (Fig. 4D). These data support the inferences about the timing of Bni5p localization and delocalization that were made from the observations on asynchronous cells, and they also suggest that the changes in Bni5p localization may reflect, at least in part, changes in Bni5p abundance during the cell cycle.
To further explore the timing of Bni5p delocalization from the neck, we made time-lapse observations of living cells expressing both Bni5p-GFP and Tub1p-GFP. As shown in Fig. 5, the band of Bni5p disappeared abruptly, without any detectable change in diameter, 2 to 4 min before spindle disassembly. Interestingly, Bni5p localization to the neck was still apparent in the cdc5-1 and cdc15-2 mutants, which are defective in exit from mitosis, after 3.5 h at a restrictive temperature (data not shown). Since the spindle disassembles at the onset of cytokinesis (36), these observations indicate that Bni5p delocalizes from the neck after mitotic exit has been triggered, but before the onset of cytokinesis.
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mutant conditionally expressing GAL-GST-CDC11 (KLY1718) was further transformed either with plasmid pKL1900 (expressing YFP-BNI5 under ADH1 promoter control) or with plasmid pKL1901 (expressing YFP-CDC10 under endogenous CDC10 promoter control), and the localization of YFP-Bni5p or YFP-Cdc10p was determined upon depletion of GST-Cdc11p by a shift to glucose-containing medium. The YFP-Bni5p rings completely disappeared from the bud necks within 1 h of the shift (Fig. 6A), whereas the YFP-Cdc10p signal was stable for at least several hours under the same conditions (data not shown). In contrast, YFP-Bni5p localized normally to the neck in cdc10 cells at 23°C (Fig. 6A), as well as in hof1 (strain RLY292) and myo1 (strain RLY332) cells (data not shown). In a sep7 mutant (strain KLY3937 containing plasmid pKL1900), the localization of YFP-Bni5p was severely impaired at temperatures from 23 to 37°C. However, at 23°C, approximately 10% of the population still exhibited normal-looking YFP-Bni5p rings (data not shown). Taken together, these observations suggest that Bni5p localization depends on the septins and perhaps particularly on Cdc11p. In addition, because the neck filaments are disorganized in a cdc10 background (22), these observations suggest that Bni5p, like Bud4p, Cdc3p, and Cdc11p (17, 22), does not depend on intact neck-filament structures for its normal localization.
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To investigate which domain(s) of Cdc11p may be important for the interaction with Bni5p, and hence for Bni5p localization, we integrated into strain KLY1718 (cdc11 with a GAL1-GST-CDC11 plasmid) various HA-tagged Cdc11p constructs under normal CDC11 promoter control (Fig. 6B and C). These strains were then transformed with a plasmid expressing either ADH1-YFP-BNI5 or YFP-CDC10. Cells were fixed after depleting GST-Cdc11p by growth in 2% glucose for 1 h, and the percentages of budded cells with neck-localized YFP-Bni5p or YFP-Cdc10p were determined. In strains expressing either full-length Cdc11p1-415 or Cdc11p1-385, YFP-Bni5p was detectable at approximately 90% of the necks in budded cells (Fig. 6D). This result is striking because Cdc11p1-385 lacks a canonical leucine zipper motif (Leu384-X6-Leu391-X6-Leu398-X6-Leu405) that comprises about half of the 57-amino-acid coiled-coil domain predicted by the Coils program (44). In contrast, YFP-Bni5p localization was greatly diminished or abolished in the strains expressing Cdc11p31-385 or Cdc11p1-200 (Fig. 6D), even though these proteins were expressed at levels comparable to those of the others (Fig. 6C). Under the same conditions, YFP-Cdc10p localization at the bud neck was consistently detected in all the strains (Fig. 6D). These data suggest that the N-terminal region (amino acids 1 to 385) of Cdc11p is sufficient for Bni5p localization and that the C-terminal coiled-coil domain is not involved in this event.
Apparently normal function of Cdc11p lacking the coiled-coil domain.
The neck localization of Bni5p in the Cdc11p1-385 cells suggested that Cdc11p1-385 itself also localizes to the neck. Indeed, immunostaining revealed that Cdc11p1-385 localized to the neck as well as did the full-length protein, whereas the other mutant Cdc11p proteins did not (Fig. 6D and E). We then examined the abilities of the Cdc11p truncation mutants to complement the cdc11 growth defect. Consistent with the localization data, Cdc11p1-385 appeared to complement the growth defect as well as did the full-length protein, while the other truncated proteins did not (Fig. 6F). Finally, we examined whether Cdc11p1-385 could form a complex with other septin proteins. In strains expressing a myc-tagged Cdc3p and either HA-tagged full-length Cdc11p or HA-tagged Cdc11p1-385, immunoprecipitation with anti-HA antibodies was equally effective in coprecipitating Cdc3p-myc (Fig. 6G). This observation is consistent with the results of a matrix of two-hybrid tests involving the full-length and partial septin proteins (Table 5). In particular, although no interactions were observed with the C-terminal domain of Cdc11p, the N-terminal domain appeared to be capable of interacting with itself, with the N-terminal domain of Cdc12p, and with Cdc3p and Cdc10p. With regard to possible models of septin assembly (see Discussion), it was also of interest in these experiments that no homotypic interactions were observed with the Cdc3p constructs (although these did interact with other septins) and that the C-terminal domain of Cdc12p appeared to interact well not only with itself but also with the C-terminal domain of Cdc3p (Table 5). Taken together, these data suggest that the predicted coiled-coil domain of Cdc11p is not required for interaction with other septins, for neck localization, or for other vital aspects of Cdc11p function, and that the assembly of septin complexes involves complex interactions involving both the N- and C-terminal portions of the proteins.
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DISCUSSION |
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and a temperature-sensitive septin mutation display a phenotype that is more severe than those of the single mutants. Fourth, bni5 is synthetically lethal with deletion of HOF1/CYK2, a gene whose product appears to interact with the septins and other proteins involved in cytokinesis (31, 36, 55). Finally, both two-hybrid data and GST pull-down experiments suggest that Bni5p interacts directly with Cdc11p, a suggestion supported by the observations that Bni5p is present at the neck in a cdc10 or a sep7 strain but disappears rapidly from the neck when Cdc11p is depleted in a viable cdc11 strain. The GST pull-down experiments also suggest that Bni5p may interact directly, but more weakly, with the other septins. Although many proteins have now been shown to localize to the mother-bud neck in a septin-dependent manner (26), suggesting a scaffold function for the septin array, there are very few cases in which strong evidence exists for a specific and/or direct interaction with a particular septin. For example, although the available evidence does suggest that Bni4p interacts specifically with Cdc10p (13) and that Gin4p interacts specifically with Cdc3p (40), these putative interactions have not yet been observed with the isolated proteins. Thus, the results with Bni5p are important in suggesting that at least some proteins are recruited to the neck by direct interaction with the septins and that interaction with particular septins (e.g., Cdc11p in the case of Bni5p) may be largely responsible for the recruitment in particular cases.
Possible roles of Bni5p.
Except for the possible coiled-coil domains, the sequence of Bni5p displays no known motifs or other clues to Bni5p function. Nonetheless, the bni5 mutant phenotype and the timing and pattern of Bni5p localization allow some speculations about possible functions of this protein. Because Bni5p localizes to the neck only after the septins are already there, it cannot be involved in their initial recruitment or assembly. However, the partial loss of septin localization and aberrant septin rings that are observed in the bni5 mutant suggest that Bni5p, like Gin4p, Cla4p, Elm1p, and Nap1p (7, 40, 42), contributes to the organization and/or stability of the septin array. Although the precise nature of the role of Bni5p is not yet clear, one interesting possibility is suggested by a comparison of previous electron and light microscopic observations. Byers and Goetsch observed that the apparent septin filaments were not fully evident by electron microscopy until the bud had emerged and that they seemed to disappear before cytokinesis (9, 10). However, studies using immunofluorescence and GFP-tagged proteins have shown that the septin proteins arrive at the presumptive bud site 10 to 15 min before bud emergence and typically remain at the division site for some time after cytokinesis and cell separation are complete (21, 33, 36). Thus, the localization of Bni5p to the neck at about the time of bud emergence and its abrupt disappearance just before cytokinesis suggest that it might be involved in the assembly of the higher-order septin structure that gives the appearance of filaments in the electron microscope. Because it does not appear that this higher-order structure is necessary for most aspects of septin function (22, 40), the nonlethality of the bni5 mutation is consistent with this hypothesis.
Another interesting possibility is suggested by the recent evidence that activation of the GTPase Tem1p triggers splitting of the septin array, an event that immediately precedes, and may be a prerequisite for, contraction of the actomyosin ring during cytokinesis (38). Thus, the timing of Bni5p delocalization suggests that it might be a Tem1p target, the dissociation of which from the septins is necessary for the splitting of the septin array. However, Tem1p presumably has at least one other relevant target, because most cells of a bni5 mutant appear to have a continuous septin array and to complete cytokinesis normally.
In elucidating the role(s) of Bni5p, it will also be necessary to account for the apparent lack of Bni5p homologues in other organisms. Particularly striking is the lack of an unequivocal homologue in Candida albicans, which also reproduces by budding and has a septin family whose sequences and assembly seem very similar to those in S. cerevisiae (15, 23, 57). However, it remains possible that improvements in the annotation of the C. albicans sequence will resolve this apparent paradox.
Roles of the predicted coiled-coil domains in septin assembly and function.
Most of the known septins contain predicted coiled-coil domains at or near their C termini (39), and one simple model for septin assembly suggested that these domains might function in the formation of septin homodimers that were subunits of the heteromeric complexes (19). This model is difficult to reconcile with the dimensions of septin complexes isolated from cdc10 and cdc11 strains (22). In addition, we have shown here that Cdc11p1-385 (which lacks the leucine zipper motif that comprises about half of the predicted coiled-coil domain) is able to associate with Cdc3p and localize apparently normally to the mother-bud neck. Moreover, in two-hybrid tests, a fragment of Cdc11p lacking the entire predicted coiled-coil domain was able to self-associate, whereas a fragment of 74 amino acids that included the entire 57-amino-acid predicted coiled-coil domain was not. Indeed, taken as a whole, the two-hybrid data (see also reference 14) suggest that the assembly of septin complexes involves a complicated set of both homomeric and heteromeric interactions that involve both the N-terminal and C-terminal domains. Higher-resolution structural data will presumably be necessary to clarify the details of septin complex assembly.
The septin coiled-coil domains may also be involved in interactions with the many proteins that are recruited to the neck in a septin-dependent manner (26). In this regard, however, it is striking that Cdc11p1-385 was able not only to recruit Bni5p to the neck but also to rescue fully the growth defect of a cdc11 mutant. In addition, the two-hybrid data suggest that the coiled-coil domain of Cdc12p may be involved both in homomeric interactions and in interactions with the coiled-coil domain of Cdc3p. Additional structure-function studies of the various septins and the septin-interacting proteins will be necessary to clarify these issues.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants NIH GM59964 (R.L.) and NIH GM31006 (J.R.P.).
P.R.L., S.S., and H.-S.R. contributed equally to this work.
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Present address: Department of Medical Biochemistry, University of Geneva, Geneva, Switzerland.
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REFERENCES |
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