Correspondence to: Erica S. Johnson, Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. Tel:(215) 503-4616 Fax:(215) 503-5393 E-mail:Erica.Johnson{at}mail.tju.edu.
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Abstract |
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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 G2/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.
Key Words: Smt3, SUMO, septin, cytoskeleton, protein processing
POSTTRANSLATIONAL modifications of proteins play critical roles in most cellular processes through their unique ability to cause rapid alterations in the functions of preexisting proteins, multiprotein complexes, and subcellular structures. One recently discovered modification involves the covalent attachment of the small ubiquitin-related protein SUMO to other proteins. SUMO, also known as sentrin or Ubl1 (B
, the inflammatory response regulatory protein (
SUMO is 18% identical to ubiquitin (Ub), a highly conserved 76-residue protein whose primary function is to target proteins for proteolysis, either by the 26S proteasome or by endocytosis into the lysosome or yeast vacuole (reviewed by -amino group of an internal Lys residue in the substrate. Proteins to be degraded by the proteasome are linked to a multi-Ub chain, in which successive copies of Ub are attached to an internal Lys residue of the previous Ub in the chain. Ub conjugation is carried out in three enzymatic steps, catalyzed by a Ub-activating enzyme (E1), one of a family of Ub-conjugating enzymes (E2s), and a Ub-protein ligase or recognin (E3). Ub conjugation can also be reversed by a family of deubiquitinating enzymes (Dubs) which cleave the isopeptide bond.
The SUMO and Ub conjugation pathways are entirely distinct, but share multiple similarities. In the first step of the SUMO conjugation pathway, SUMO is activated by a heterodimeric activating enzyme (E1) consisting of Aos1 and Uba2, proteins with sequence similarity to the NH2 and COOH terminus, respectively, of Ub activating enzymes (
The function of SUMO conjugation is only beginning to be understood. There are four well-characterized substrates of mammalian SUMO: RanGAP1, IB
, and two components of so-called PML nuclear bodies, PML and Sp100. In the cases of RanGAP1, Sp100, and PML, SUMO conjugation functions by altering the subcellular localization of its substrates. Attachment of SUMO targets the otherwise cytosolic RanGAP1 to the nuclear pore complex by promoting binding to Nup358/RanBP2 (
B
, where it becomes attached to the major ubiquitination site, preventing ubiquitination and thereby protecting sumoylated I
B
from proteasome-dependent proteolysis (
In S. cerevisiae, SUMO is encoded by the SMT3 gene, which was originally isolated as a high-copy suppressor of mutations in MIF2, which encodes a centromere binding protein (
The yeast bud neck is a dynamic structure that is the focus of many processes involved in the polarized growth and division of yeast cells. Its primary structural feature is a belt of 10-nm filaments whose central components are members of a family of GTP-binding coiled-coil proteins called septins (reviewed by 15 min before bud emergence. After the bud appears, the ring extends into the bud, forming a continuous hourglass-shaped structure that appears as a "double ring." During cytokinesis, the 10-nm filaments visible by electron microscopy disappear, but the septin ring, as visualized by immunofluorescence microscopy, splits in half, and the remaining septin-containing structures are disassembled during the following G1 phase.
The septin ring acts as a scaffold for the binding of other proteins (
As a first step toward understanding the physiological role of the SUMO conjugation pathway in S. cerevisiae, we were interested in identifying and characterizing substrate proteins that become modified by SUMO. We discovered that SUMO is attached to septins in a cell cycle-dependent manner and have investigated the functional consequences of impairing septin sumoylation.
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Materials and Methods |
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Media and Genetic Techniques
Standard techniques were used (-factor, 0.1 M hydroxyurea, or 15 µg/ml nocodazole (Sigma Chemical Co.).
Plasmid Constructions
The pRS315-derived (1,000 bp of the 5' flanking sequence of CDC3 and the CDC3 coding region fused to a COOH-terminal influenza virus hemagglutinin epitope tag (HA) sequence encoding GYPYDVPDYAAFL (
94-HA lacked the coding region for the NH2-terminal 94 residues of Cdc3, so that the expressed protein began MSQING. p315-PGAL-CDC3-HA expressed full-length Cdc3-HA from PGAL10. Plasmids expressing variants of Cdc3 with TEV protease cleavage sites or Lys to Arg mutations were produced by oligonucleotide-directed site-specific mutagenesis of p315-CDC3-HA using the Mutagene kit (BioRad) according to the manufacturer's instructions. Single stranded p315-CDC3-HA was produced using helper phage R408 (Promega Corp.). Partial protein sequences encoded by TEV site-containing Cdc3 constructs, including the altered segment are as follow: pCDC3-TEV-K2, RQH21ENLYFQGSD26VQI; pCDC3-TEV-K3, DGV39 ENLYFQGSQ48NDD; pCDC3-TEV-K4, GLG69ENLYFQGSQ76SEK; pCDC3-TEV-K6, IRRQ95ENLYFQGSI96NGY. Introduced sequence is underlined. Superscripts indicate positions of amino acid residues in the unaltered sequence. TEV protease cleaves between Gln and Gly in the introduced sequence. Correctly mutagenized plasmids were identified by digesting with BamHI, whose cleavage site encodes the Gly-Ser in the introduced sequence. Plasmids containing Lys to Arg mutations in CDC3 were identified by cleaving with the following restriction enzymes: K4R, PstI; K11R, BamHI; K30R, AatII; K63R, BsiWI; K415R, BamHI; K443R, MscI. All oligonucleotide sequences and construction details are available on request.
Yeast Strains
S. cerevisiae strains used are listed in Table 1. Strains in which the genomic copies of genes bear epitope tags, deletions, Lys to Arg mutations, or previously isolated mutant alleles, were produced by transforming yeast with the products of assembly PCR reactions, made as follows, which were integrated into the chromosome by homologous recombination. Three PCR products that overlapped by 1720-bp were used as the templates in a second round PCR reaction using outside primers to produce a product containing the 5' flanking sequence and/or the coding sequence of the gene of interest, with or without a tag or a mutation, followed by a selective marker and then the 3' flanking sequence. Transformants were selected for the appropriate selective marker and were screened further by immunoblotting with an mAb against the HA epitope, by amplifying the gene of interest from chromosomal DNA and digesting with an appropriate restriction enzyme diagnostic for a point mutation, or by screening for the desired temperature sensitive (ts) phenotype. Strains containing multiple altered genes were derived from the single mutants by mating and tetrad dissection. Chromosomal DNA derived from MY254 (a generous gift of M. Yuste and F. Cross, Rockefeller University, NY, NY) and STX339-1C (Yeast Genetic Stock Center) was used as source of the cdc15-2 and cdc12-1 alleles, respectively. In constructing EJY309, the SHS1 coding sequence was amplified in two overlapping pieces, one containing the K426R mutation and the other containing the K437R mutation with a BstBI restriction site included in the overlapping region between the two mutations. Cdc12 with a COOH-terminal green fluorescent protein (GFP) tag was constructed by inserting the CDC12 coding sequence into pYX242-GFP (
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Antibodies
A rabbit polyclonal antibody was raised against NH2 terminally His6-tagged SUMO(G98) (Cocalico Biologicals) and was affinity purified on a His6-FLAG-SUMO(G98) affinity column (100-kD band unrelated to Cdc3. The affinity-purified antibody also contained a small amount of antibody against this other protein. Other antibodies used were the 16B12 mAb against the HA epitope (in Fig 1 b, 3, 4 b, 5 b, and 6; Berkeley Antibody Co.), the 3F10 mAb against the HA epitope (in Fig 2 a; Boehringer Mannheim Corp.), the B5-1-2 mAb against tubulin (a generous gift of Mike Rout, Rockefeller University, NY, NY), and a rabbit polyclonal antibody against Cdc11 (Santa Cruz Biotechnology).
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Immunoblot Analyses of Whole Yeast Lysates and Immunoprecipitates
Yeast whole cell lysates were prepared as described (1 mg protein) was added to 1.5 ml RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing 2 µg/ml of each of the protease inhibitors antipain, aprotinin, chymostatin, leupeptin, and pepstatin, plus 50 mM N-ethylmaleimide (Sigma Chemical Co.). 10 µl of anti-HASepharose (Berkeley Antibody Co.) was added, followed by incubation at 4°C for 2 h with rotation. Beads were washed three times with RIPA buffer plus 0.1% SDS, resuspended in Laemmli loading buffer lacking reducing agent, and incubated at 65°C for 20 min. The supernatant was removed, supplemented to 100 mM DTT, and analyzed by SDS-PAGE and immunoblotting as described.
For the TEV protease cleavage experiment, washed HASepharose beads bearing Cdc3-HA variants were washed once with 50 mM Tris, pH 8.0, 0.5 mM EDTA, and 1% Triton X-100, and incubated with 20 U TEV protease (Life Technologies) for 4 h at 25°C in 20 µl of the same buffer. HA-Sepharose beads were pelleted by centrifugation, and the supernatant "unbound" fraction was removed. One 20 µl wash of the beads with RIPA buffer and 0.1% SDS was added to the unbound fraction, and the beads were washed three times with 1 ml RIPA buffer and 0.1% SDS and prepared for SDS-PAGE as described above to yield the "bound" fraction.
Isolation of SUMO-conjugated Proteins
EJY251-11b containing p315-PGAL-HFSMT3 was grown at 30°C in 4 liter of YP containing 2% raffinose and 1% galactose to an A600 of 2. Cells were harvested by centrifugation and lysed 10 min on ice in 200 ml cold 1.85 NaOH, 7.5% ß-mercaptoethanol. Protein was precipitated by addition of 200 ml 50% TCA, collected by centrifugation, and the pellet was washed with 200 ml ice-cold acetone. The pellet was resuspended in 400 ml Buffer A (6 M guanidine HCl, 100 mM sodium phosphate, 10 mM Tris/HCl, pH 8.0) and incubated at 25°C for 1 h with rotating. Lysates were clarified by centrifugation at 27,000 gmax, adjusted to pH 7.0 (measured using pH paper) with 1 M Tris base, supplemented to 20 mM imidazole, and bound in batch for 2 h to 2 ml of Ni-nitriloacetic acid (NTA) agarose (Qiagen). The Ni-NTA agarose was loaded into a column, washed with 20 ml of Buffer A, and then with 100 ml of Buffer B (8 M urea, 100 mM sodium phosphate, 10 mM Tris, pH 6.3), and then eluted with 200 mM imidazole in Buffer B. 4 ml of the eluate was added to 46 ml RIPA buffer containing 0.1% SDS, 1 mM PMSF, and 1 mM ß-mercaptoethanol, and bound in batch overnight at 4°C to 0.4 ml anti-FLAG sepharose (IBI/Kodak). The anti-FLAG Sepharose was loaded into a column, washed with 50 ml of RIPA buffer with 0.1% SDS, and then eluted with 100 mM glycine, pH 2.2, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS. The eluate was fractionated on a 520% acrylamide gel. Protein bands detected by staining with Coomassie brilliant blue were excised and identified by direct analysis of a Lys-C endoproteinase digest by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry at the Rockefeller University Protein/DNA Technology Center (NY, NY) as described (
Fluorescence Microscopy
Yeast cells were prepared for indirect immunofluorescence microscopy essentially as described (
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Results |
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Septins Are Modified by SUMO during Mitosis
To identify substrates of the SUMO pathway, we constructed a strain that expressed His6- and FLAG-tagged SUMO as its only copy of the SUMO-encoding SMT3 gene. When proteins bearing both tags were purified by affinity chromatography, the resulting fraction contained a smear of high molecular weight bands (Fig 1 a), similar to the previously observed pattern of SUMO conjugates in yeast (
To confirm that Cdc3 could be modified by SUMO and to ask whether any of the other four yeast septins expressed during vegetative growth were sumoylated, we tagged the genomic copies of the CDC3, CDC10, CDC11, CDC12, and SHS1 genes with the HA epitope tag (
To test whether septin sumoylation is cell cycle dependent, cells expressing Cdc3-HA were arrested in G1 with the mating pheromone -factor, in the S phase checkpoint with hydroxyurea, in the G2/M spindle checkpoint with nocodazole, or in late anaphase with the ts cdc15-2 allele. In all cases, >90% of cells were arrested at the appropriate point in the cell cycle (data not shown). Both Cdc3-HA and Cdc11 were most heavily modified in nocodazole-arrested cells and somewhat less so in cdc15-arrested cells (Fig 2 a). Very little, if any, of either septin was modified in
-factor- or hydroxyurea-arrested cells, although the results were partially obscured by cross-reacting bands.
To define the stages when septins are sumoylated in more detail, SUMO was visualized by double label immunofluorescence microscopy. The protein detected in addition to SUMO was either Cdc3-HA (Fig 3) or tubulin (Fig 2 b), whose localization was used to determine the stage of the cell cycle occupied by individual cells. SUMO localized to the nucleus at all points in the cell cycle (Fig 3, a, e, j, and o; data not shown), but more strikingly it localized to a ring at the bud neck only during mitosis. Specifically, a ring of SUMO appeared at the bud neck of large budded cells just before initiation of anaphase spindle elongation and nuclear division (Fig 2 b and 3 e). This SUMO ring persisted through anaphase (Fig 2 b and 3 j) and it disappeared abruptly at cytokinesis, virtually simultaneously with septin ring separation (Fig 3o and Fig p) and the beginning of spindle breakdown (Fig 2 b). Surprisingly, the SUMO ring did not completely colocalize with the septins. SUMO coincided only with the mother cell side of the septin "double ring," appearing on the side next to the undivided nucleus or on the side of the larger cell in mitotic cells (Fig 3e, Fig i, Fig j, and Fig n). Thus, SUMO conjugation to the septins was asymmetrical with respect to the motherbud axis.
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Identification of the SUMO Attachment Sites in Cdc3
To determine the function of septin sumoylation, we wanted to generate yeast mutants in which septins would not be modified. The most direct way to eliminate septin sumoylation specifically was to identify the Lys residues that serve as SUMO attachment sites and to replace them with Arg residues, which cannot be modified, thereby eliminating septin sumoylation without disturbing conjugation to other substrates.
Our initial approach to identifying SUMO attachment sites on Cdc3 was to affinity-purify SUMO-Cdc3 conjugates from strains expressing Cdc3-HA and His6- and FLAG-tagged SUMO(G98) or SUMO(A98). These preparations varied only in the identity of the COOH-terminal residue of SUMO, which is the residue involved in the isopeptide bond with the substrate. This material was digested with trypsin and with endoproteinase Lys C and analyzed by MALDI-TOF mass spectrometry with the goal of identifying species varying by the 14 mass unit difference between the COOH-terminal Gly and Ala in the two preparations. One such species was identified, with a molecular weight of 2,327 D, which was consistent with SUMO attachment at Lys11 of Cdc3 (data not shown).
Examination of the sequence surrounding Lys11 revealed that three other Lys residues in the NH2-terminal domain of Cdc3 were embedded in similar sequence motifs having the consensus (IVL)KXE, which appeared to be a potential sumoylation site consensus sequence. Since there are multiple SUMO-Cdc3 conjugate bands (Fig 1), such a consensus sequence might be used in either of two different ways. SUMO might be attached at one or the other of these Lys residues as a chain containing SUMOSUMO linkages, analogous to the multi-Ub chain. Alternatively, multiple single copies of SUMO might be attached with one SUMO moiety per Lys residue. To distinguish between these possibilities, we designed a series of constructs containing cleavage sites for the TEV protease at different sites along the NH2-terminal domain of Cdc3, either after the first two Lys residues (TEV-K2), after the third (TEV-K3), after the fourth (TEV-K4), or after the sixth (TEV-K6; see Fig 4 a). These constructs also contained a COOH-terminal HA tag, which was used to immunoprecipitate the Cdc3 variants from lysates of nocodazole-arrested yeast. Immunoprecipitated proteins were cleaved with TEV protease while still bound to the beads and separated into an unbound fraction containing the fragment NH2-terminal to the cleavage site, and a bound fraction containing the COOH-terminal cleavage product. This experiment showed that for each additional Lys residue included NH2-terminal to the TEV site, an additional sumoylated species appeared on the NH2-terminal product and disappeared from the COOH-terminal product. There was one major NH2-terminal SUMO-containing species from TEV-K2, two from TEV-K3, and three from TEV-K4 and TEV-K6 (Fig 4 e), and there were two major SUMO-containing COOH-terminal cleavage products from TEV-K2, one from TEV-K3, and none from TEV-K4 or TEV-K6 (Fig 4 d, cleavage products are indicated; lanes 2, 3, and 5 contained uncleaved species, see Fig 4 b). These results were generally consistent with a model where single copies of SUMO are attached to Cdc3 at three to four major sites, at Lys-4 and/or Lys-11, at Lys-30, and at Lys-63. A separate experiment in which these Lys residues were altered by site-directed mutagenesis showed that both Lys-4 and Lys-11 can serve as sumoylation sites (data not shown).
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However, this model does not completely explain the pattern of high molecular weight SUMO conjugates on Cdc3, which consisted of seven bands between 95 and
200 kD (Fig 4 c, lanes 15). For several reasons, it is more likely that the additional bands reflect the presence of another mobility-altering factor, rather than attachment of multiple copies of SUMO at the same Lys residue. One is that cleavage at the TEV-K2 site produced only two detectable SUMO-containing COOH-terminal fragments (Fig 4 d, lane 2). This result can be explained most easily if a single mobility-altering factor lies NH2-terminal to the TEV site at position 27. Once the NH2 terminus is cleaved off, the number of bands associated with the COOH-terminal fragment would reflect the number of attached SUMO moieties, whereas NH2-terminal fragments would still be present in two forms for every SUMO moiety. This explanation is also consistent with the pattern of NH2-terminal cleavage products, where there is a minor band above each of the three major SUMO-containing bands from TEV-K4 and TEV-K6 (Fig 4 e, lanes 4 and 5). A second reason is that several of the SUMO-Cdc3 bands and corresponding NH2-terminal TEV cleavage product bands appear to be too close together to differ by a whole SUMO moiety. Free SUMO runs at
20 kD on SDS-PAGE, but two of the pairs of SUMO-Cdc3 bands appeared to differ by significantly less than 20 kD, and the first two sets of major and minor NH2-terminal cleavage products appeared to differ by 10 kD or less (Fig 4 c, lane 1, and e, lanes 4 and 5). A third reason is that the TEV-K4 variant significantly reduced the amount of the third SUMO-containing conjugate with a proportionate increase in the second band, but there was no reduction in intensity or change in position of the fourth or sixth SUMO-containing bands (Fig 4 c, lane 4, cf lanes 13, and 5). This result is most easily explained if the TEV-K4 mutation reduced the other mobility-altering factor without affecting SUMO conjugation. We do not know what this other factor is. It might be another posttranslational modification, but it is also possible that conjugates bearing the same number of SUMO moieties could have different mobilities resulting from SUMO attachment at different sites.
Mutational Analysis of Sumoylation Site Lys Residues on Septins
Inspection of the Cdc11 and Shs1 sequences for sumoylation site consensus sequences revealed that Cdc11 contained one such sequence near its COOH terminus and that Shs1 contained two such sequences in its COOH-terminal coiled-coil domain (Fig 5 a). To determine the effects of eliminating these Lys residues on septin sumoylation and on cellular function, we made yeast strains in which the genomic copies of septins were replaced with mutant versions lacking these Lys residues. Deleting the coding sequence for the 94 NH2-terminal residues of Cdc3 eliminated the vast majority of the SUMO-Cdc3 conjugates in nocodazole-arrested cells, confirming the conclusion that most SUMO is attached to this NH2-terminal domain (Fig 5 b, lanes 1 and 2). However, Cdc3-94 was still sumoylated at a very low level by one copy of SUMO (Fig 5 b, lane 2; note the band running at the position of full-length Cdc3-HA). This result was confirmed by immunoprecipitating with the anti-HA antibody and immunoblotting with an antibody against SUMO (data not shown). Mutating two more Lys residues in CDC3-
94, Lys-415 in the sequence IKQD, and Lys-443 in the sequence AKLE, had no effect on this residual sumoylation (data not shown). The only other sequence in Cdc3 that resembles the sumoylation site consensus sequence is the AKSD containing Lys-287, which is in the septin homology domain. This Lys residue is conserved in all members of the septin family in all organisms, except for Shs1 and one hypothetical open reading frame in S. pombe. Mutant cdc3 containing a Lys to Arg mutation in this position was unable to complement the lethality of the cdc3
strain, even when all the NH2-terminal sumoylation site Lys residues were present (data not shown). It is likely that this result reflects a requirement for this Lys residue in some septin function other than SUMO conjugation. When we mutated Lys-412 of Cdc11, and Lys-426 and Lys-437 of Shs1 to Arg, similar results were observed. In both cases, the vast majority of SUMO conjugates were eliminated, but a small amount of residual conjugation remained, reflecting low levels of SUMO conjugation at other sites (Fig 5 b, lanes 36; data not shown).
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As all of the strains with sumoylation site mutations in only one of the septins still contained substantial SUMO rings at the bud neck, we proceeded to construct a triple mutant in which all of Cdc3, Cdc11, and Shs1 lacked the major sumoylation sites. Analysis of a series of strains in which progressively fewer septins contained sumoylation sites demonstrated that the majority of the total SUMO in nocodazole-arrested cells was attached to Cdc3 (Fig 5 c, lane 2, cf lane 1) and that the most of the remainder was attached to Cdc11 or Shs1 (Fig 5 c, lanes 24). Thus, septins are by far the most abundant SUMO conjugates at this point in the cell cycle. However, a longer exposure of this blot revealed a large number of lower abundance substrates in the triple mutant strain (data not shown).
To minimize any non-SUMOrelated effects from deleting the entire Cdc3 NH2-terminal domain, we constructed a different cdc3 sumoylation site mutant in which all four attachment site Lys residues were replaced with Arg. This mutant's effects on Cdc3 sumoylation were indistinguishable from those of the deletion mutant (data not shown), and subsequent experiments were done using this cdc3 allele. We analyzed the sumoylation site triple mutant (cdc3-R4,11,30,63 cdc11-R412 shs1-R426,437) for phenotypes and found that in most respects it was very similar to the parental wild-type strain. Its growth rate was virtually indistinguishable from that of the wild-type (data not shown), with a doubling time of 95 min, versus 94 min for wild-type, a statistically insignificant difference. This strain was not hypersensitive to either high or low temperatures, to 1 M sorbitol, to DNA damaging agents (UV light or methyl methane sulfonate), to the microtubule depolymerizing drug benomyl, or to the cell wall perturbing agent Calcofluor white (data not shown). It mated efficiently and sporulated with the same efficiency as wild-type to produce viable segregants (data not shown). Haploid cells correctly positioned their bud sites axially, and most diploid cells positioned their bud sites bipolarly, although both this strain and the parental wild-type diploid strain had a significant frequency of cells with random bud sites (data not shown). The actin cytoskeleton, as visualized by rhodamine-phalloidin staining, also appeared normal in the triple mutant (data not shown).
We also analyzed the triple mutant by double-label immunofluorescence microscopy with antibodies against SUMO and against the HA tag on the septins. The triple mutant displayed a dramatic reduction in SUMO staining at the bud neck (Fig 6, ah), although a faint ring could be seen in a few cells (data not shown), and other cells contained a bulge of SUMO staining at the bud neck near the nuclear envelope that did not appear to colocalize completely with the mother cell half of the septin ring (Fig 6 e). This slight SUMO localization to the bud neck probably resulted from the residual sumoylation of the septins, although some other bud neck protein may also be sumoylated at a very low level.
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The striking difference that we noted between the triple mutant strain and wild-type was that in the mutant, at least one extra septin ring could be observed in virtually all budded cells (Fig 6d, Fig g, and Fig h). This is in contrast to the wild-type strain, in which budded cells never contained septin rings other than at the neck of the growing bud (Fig 6 b). The presence of these extra septin rings was a synthetic phenotype of the triple mutant, as none of the single mutants or the cdc11-R412 shs1-R426,437 double mutant had this property (data not shown). To test the hypothesis that the extra septin rings in the triple mutant were remnants of septin rings from previous cells divisions, we compared the localization of the extra septin rings with that of the bud scars. Every time an S. cerevisiae cell divides, the division site on the mother cell is marked by a chitin-containing bud scar, stainable with the fluorescent dye Calcofluor white, which persists through the lifetime of the cell. Although they sometimes had many bud scars, wild-type budded cells never contained more than the one septin ring at the base of the growing bud (Fig 6i and Fig j). In the mutant, however, a septin ring colocalized with virtually every bud scar (Fig 6k and Fig l), indicating that the extra septin rings are likely to be undisassembled septin rings from previous bud sites. Unlike mother cells, daughter cells do not have Calcofluor-staining structures marking the division site. Examination of budded cells lacking bud scars revealed that they always contained exactly one extra septin ring, indicating that daughter cells are also defective in septin ring disassembly (Fig 6m and Fig n).
There are two simple reasons why the triple sumoylation site mutant might be defective in septin ring disassembly. One is that attachment of SUMO to the septin ring promotes disassembly. The other is that the sumoylation-site Lys residues also have some other function, and that this other function promotes septin ring disassembly. For example, the purpose of these Lys residues could be to serve as ubiquitination sites, or perhaps acetylation or methylation sites. One way to distinguish between these possibilities might be to determine whether mutants in the SUMO conjugation pathway also have septin ring disassembly defects. Examination of Cdc11 localization in ubc9 (Fig 7 a) and uba2 (data not shown) ts mutants showed that no extra septin rings were visible in cells from either strain, either when grown at the permissive temperature or after transfer to the restrictive temperature. This result may suggest that SUMO conjugation, per se, does not promote septin ring disassembly. Alternatively, it may indicate that, although SUMO conjugation may promote septin ring disassembly, as long as these ts mutants retain enough SUMO conjugating activity to divide, they also retain sufficient SUMO conjugation to disassemble their septin rings.
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In contrast to a previously published report (
To test whether septins are ubiquitinated or degraded during cytokinesis, we synchronized a culture of cdc15-2 cells by incubating them at 37°C, which arrests them in late anaphase, and releasing them at 25°C to allow them to complete cytokinesis (Fig 8). Between 30 and 50 min after release, all the SUMO-conjugated forms of Cdc3 and Cdc11 disappeared from these cells. However, there was no significant reduction in the steady-state level of either Cdc3 or Cdc11 at this point, which is consistent with the observation by immunofluorescence microscopy that septin rings do not disappear suddenly at cytokinesis. Also, in several different experiments, we never observed high molecular weight ubiquitinated Cdc3 species. However, we did sometimes observe minor bands that might be degradation products of Cdc11 (Fig 8 c) and of Cdc3-HA (data not shown). We conclude that, at most, a small fraction of Cdc3 and Cdc11 is degraded immediately at cytokinesis.
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We also tested whether the sumoylation site triple mutant interacted genetically with either SUMO conjugation pathway mutants or septin mutants. A quadruple mutant also containing a chromosomally integrated version of the uba2 ts10 allele grew at a range of rates similar to those seen with the uba2 ts10 strain alone (data not shown), which grew very poorly and was heterogeneous. Thus, the attachment site mutations did not strongly exacerbate or suppress the phenotypes of the uba2 ts10 mutant. However, when we crossed the sumoylation site triple mutant to a cdc12-1 mutant, we obtained the surprising result that the cdc3-R4,11,30,63 mutation alone was synthetically lethal with the cdc12-1 mutation. cdc12-1 single mutants grow well with near normal morphology at 25°C, but lose septin rings and develop the distinctive phenotypes of septin mutants at 37°C (see introduction). At 25°C, cdc3-R4,11,30,63 cdc12-1 cells overexpressing wild-type CDC3-HA exhibited near normal morphology (Fig 9 a). When CDC3-HA expression was repressed, the double mutant developed the phenotypes of septin mutants, even at 25°C, producing branched, elongated cells and failing to undergo cytokinesis (Fig 9 b). This effect was not general for all septin mutants, as the sumoylation site mutations did not exacerbate the phenotypes of the cdc10-1 mutant (data not shown).
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Discussion |
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We have identified the septins Cdc3, Cdc11, and Shs1 as the three SUMO conjugation pathway substrates that account for the most abundant sumoylated species during yeast mitosis. Septin sumoylation was highly regulated, occurring only during mitosis and only on the mother cell side of the bud neck. We identified the major SUMO attachment site Lys residues in these septins and found that they are surrounded by a short consensus sequence that has also been observed around the mammalian SUMO attachment sites (
While this manuscript was in preparation, another paper was published reporting that Cdc3 is a SUMO substrate (-factor-, hydroxyurea- and nocodazole-arrested cells; that the SUMO-Cdc3-GFP conjugate persists through cell lysis under native conditions (in our hands, Cdc3-HA is rapidly desumoylated under these conditions; data not shown); and that Cdc3-GFP disappears from the bud neck upon transfer of ubc9 ts cells to the nonpermissive temperature. The first four of these results also contradict other published reports about Cdc3, where Cdc3 runs on SDS-PAGE as a single band of the expected size for the unmodified protein (
Our results indicate that the G2/M cell cycle arrest phenotypes of the SUMO conjugation pathway mutants probably do not result from a reduction in septin sumoylation. The sumoylation site triple mutant, which eliminated the vast majority of SUMO conjugation to the septins, grew at the same rate as wild-type and did not exhibit any cell cycle arrest phenotype. Furthermore, a uba2 ts mutation did not exacerbate the phenotype of the triple mutant, indicating that SUMO conjugation to other sites on the septins or to some other substrate is not compensating for the loss of the major septin sumoylation sites. It is still possible that the minor septin sumoylation sites play a distinct essential role for which the major sites cannot substitute. However, we believe it is more likely that the SUMO substrates involved in the essential and cell cycle-related roles of the SUMO pathway participate in an entirely different cellular function, possibly chromosome segregation, as SMT3, the SUMO-encoding gene, was identified as a high copy suppressor of a mutant impaired in chromosome segregation (
Septin sumoylation might be controlled by the cell cycle machinery through regulation of SUMO attachment, SUMO removal, or both. It is difficult to determine the fate of sumoylated septins at cytokinesis because of the small fraction modified, but it is most likely that the septins are desumoylated by a SUMO-specific isopeptidase such as Ulp1 or the similar protein Ulp2/Smt4 (
Several issues depend on the structure of the septin-containing neck ring, which has not been described in detail. Purified septins form linear filaments in vitro, and the present model for the structure of the ring holds that it consists of parallel septin filaments running through the bud neck parallel to the mother-bud axis (
The data on septin-SUMO conjugation is less consistent with the model where various regions are preestablished, because it is possible for the daughter side to be sumoylated.
Another question is why such a small fraction of each of the septins is modified. If the septin polypeptides that are sumoylated are functionally and structurally equivalent to the ones that are not, it would be less likely that SUMO is blocking Lys residues against attachment of some other modification, since these Lys residues would still be available on the vast majority of potential substrates. However, the various septin polypeptides may not be equivalent, either because all positions in the septin lattice may not be equivalent structurally or because of the presence of other posttranslational modifications. We did see variant forms of SUMO-Cdc3 conjugates, which may bear another posttranslational modification. There did not appear to be proportional amounts of a corresponding variant of unsumoylated Cdc3, suggesting that the other modification may target the same subpopulation or that one modification is a prerequisite for the other.
The triple sumoylation site mutant is the first mutant isolated with a defect in septin ring disassembly. We have mentioned two models for how the triple mutant might affect disassembly of the septin ring. One is that SUMO itself promotes septin ring disassembly, and the other is that the sumoylation-site Lys residues play some other role in disassembly, possibly by serving as ubiquitination sites. In the second model, SUMO might play a regulatory role by preventing the Lys residues from taking part in the other function. Alternatively, the other function might be completely independent from SUMO.
Any model for SUMO involvement in septin ring disassembly has to explain two sets of seemingly contradictory results. One is that SUMO is attached only to the mother cell side of the bud neck, but the septin rings fail to be disassembled in both the mother cell and in the daughter cell (Fig 6). The other is that SUMOseptin conjugates disappear suddenly at cytokinesis, but disassembly of the septin rings does not take place until G1 of the next cycle. The simplest way to explain the symmetry of the phenotype is if the SUMO-related event takes place in the center of the septin ring. During cytokinesis, an actomyosin contractile ring forms in the center of the septin ring (
Either model can also explain the timing of SUMO removal and septin ring disassembly. If SUMO itself promotes disassembly, SUMO might be involved in an initial event, either the SUMO-dependent association with the septin ring of another protein that is later involved in disassembly, or direct SUMO participation in a preliminary disassembly event, whose effects only become apparent later in the cell cycle. Using this model, it is easier to explain the fact that only a small fraction of each of the septins is modified, because a small amount of modified septin might be sufficient to attract some other protein to the bud neck. On the other hand, the model where SUMO prevents disassembly by inhibiting some other process is more consistent with the timing of desumoylation, as septin ring disassembly actually starts after SUMO is removed. Removal of SUMO might serve as an initiation signal, allowing access to other proteins that trigger the septin rearrangements that take place during and after cytokinesis. However, as mentioned above, the main problem with this model is the extremely low percentage of septin polypeptides that are actually modified. This seems like an inefficient way to block an interaction or modification, since >95% of all sites still would be available. Another key question related to this model is whether any fraction of the septins is ubiquitinated and degraded at this point in the cell cycle. We were unable to detect high molecular weight Ub conjugates or any dramatic reductions in the steady-state levels of Cdc3 or Cdc11, but it is possible that a small fraction of the septins may be degraded during cytokinesis.
Another important question is whether septin sumoylation plays other roles in initiation of anaphase, during anaphase, or in cytokinesis. It is still possible that septin sumoylation participates in a process carried out by two or more partially redundant pathways, or that it is part of a checkpoint monitoring a process that we have not perturbed in any of our experiments. Further analysis of genes that interact with the septin sumoylation site mutant should lead to a clearer picture of the role of septin sumoylation in yeast growth.
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Footnotes |
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1 We are calling the product of the Saccharomyces cerevisiae SMT3 gene SUMO to bring the yeast terminology for this modification in line with the mammalian terminology.
2 Abbreviations used in this paper: GFP, green fluorescence protein; HA, influenza virus hemagglutinin epitope tag; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; NTA, nitriloacetic acid; RanGAP1, Ran GTPase-activating protein; ts, temperature sensitive; Ub, ubiquitin.
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Acknowledgements |
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We thank John Pringle, Mark Longtine, Maria Yuste, Fred Cross, Mike Rout, Jonathan Rosenblum, Jürgen Dohmen, and Stefan Jentsch for strains, plasmids, and antibodies; and members of the Rockefeller University Protein/DNA Technology Center for DNA sequencing and especially Farzin Gharahdaghi for MALDI-TOF analysis. We also thank John Pringle and Ray Deshaies for helpful discussions, and Lucy Pemberton, Markus Albertini, and especially, Nabeel Yaseen and Jonathan Rosenblum, for critical reading of the manuscript.
This work was supported by the Howard Hughes Medical Institute (G. Blobel).
Submitted: 15 September 1999
Revised: 11 October 1999
Accepted: 20 October 1999
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References |
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