From the Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, California 94143-2240
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ABSTRACT |
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SUMO1was discovered as a small ubiquitin-related modifier that, like ubiquitin, is a polypeptide chain conjugated by an isopeptide bond to lysines in target proteins (4). SUMO conjugation, or sumoylation, is analogous to but biochemically distinct from ubiquitin conjugation (5, 6). SUMO, which is encoded by SMT3 in Saccharomyces cerevisiae, is processed by the isopeptidase Ulp1 from its precursor form (7). SUMO conjugation requires an E1 heterodimer (Uba2/Aos1), E2 protein (Ubc9), and E3 protein (either Siz1 or Siz2) to form a covalent bond with target proteins (5, 8, 9). Removal of SUMO from proteins is mediated by the cysteine proteases Ulp1 or Ulp2 with Ulp1 appearing to mediate the majority of desumoylation (3, 10). The genes encoding Smt3, Uba2, Aos1, Ubc9, and Ulp1 are required for cell growth and division (4). While ubiquitylation (conjugation of ubiquitin) often destabilizes targeted proteins, sumoylation appears to modify protein-protein interactions in some cases or acts as a ubiquitin antagonist in others (4, 11, 12). However, because a limited number of proteins modified by SUMO have been described, it is unclear whether these are the only roles for sumoylation in cells.
In budding yeast, seven proteins have been extensively characterized that are modified by SUMO. Three of these proteins are part of the essential septin complex (Cdc11, Cdc3, and Shs1), which plays a role in cytoskeletal organization and cell division. However, sumoylation of these proteins is not required for cell viability because when sumoylation is prevented by mutating the conjugated lysines, the septins retain function, and the cells continue to divide (6, 13). Proliferating cell nuclear antigen (Pol30 in budding yeast), the DNA polymerase sliding clamp, is also sumoylated (14). When the lysine that is sumoylated is mutated to an arginine, the protein is also unable to be ubiquitylated (14). Sumoylation of proliferating cell nuclear antigen antagonizes ubiquitylation and allows the cells to replicate DNA, whereas the lack of sumoylation or either monoubiquitylation or polyubiquitylation stalls replication and allows for error-free DNA repair (14). Topoisomerase II in yeast is also sumoylated, and this modification alters its activity at the centromere but is not required for cell viability (15). Both proliferating cell nuclear antigen and topoisomerase II are sumoylated in mammalian cells, suggesting that regulation by sumoylation is evolutionarily conserved (14, 16). Recently Ycs4 and Pds5 have been identified as SUMO conjugates (17, 18). Sumoylation of Ycs4 mediates its localization to rDNA and Pds5 sumoylation mitigates chromatid cohesion (17, 18); however, the lysines modified by SUMO in Pds5 and Ycs4 have yet to be identified, and it is unclear how important SUMO conjugation is for the regulation of these two proteins.
Mass spectrometry-based screens have identified additional proteins in the budding yeast proteome that are modified by SUMO (1921). In addition to reporting 22 bona fide sumoylation targets, which were confirmed by either secondary assays or identification of sumoylated peptides, these studies also identify other candidate sumoylated proteins that have yet to be confirmed by additional assays. Many are likely to be bona fide substrates, especially the SUMO substrates that were detected in multiple studies (1921). Although these screens are particularly effective in detecting relatively abundant proteins, they may miss less abundant ones. The median abundance of the 10 proteins identified in one study is in the top 5% of expressed proteins (21), and the median abundance of the 67 proteins identified in a second study is in the top 10% (20, 22).
We began our studies with an alternative approach to screen systematically for sumoylated proteins. Utilizing a collection of yeast strains epitope-tagged with the tandem affinity purification (TAP) tag (22), we crossed traits useful for the detection of sumoylation into the TAP-tagged strains, immunoprecipitated each epitope-tagged protein, and assayed for sumoylation. Our technique is systematic in that we assay individually each protein in the yeast proteome, and our expectation was that specific immunoprecipitation of each tagged protein would allow for identification of less abundant SUMO substrates. Importantly our study demonstrated the feasibility of assaying for post-translational modifications in a method complementary to other approaches.
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EXPERIMENTAL PROCEDURES |
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We transformed the resulting PCR product into a MATa strain using standard lithium acetate transformation techniques (24) and selected for canavanine resistance and leucine prototrophy. We crossed this strain (EY1254) to a MAT strain that contained a temperature-sensitive allele of ULP1 (ulp1333ts), which was generated by transformation with yIP353-ulp1-ts(333) (3). The ulp1ts allele was marked with MET15 (EY1274) by integrating a PCR product generated from the amplification of MET15 in pBSKS (EB1435) with the following oligonucleotides: 5'-GATCATTGGATATGGAATAAAGTTCAATAAACCACTTGAACGCCATCCTCATGAAAACTGT-3' and 5'-GTACAGACATCTAAATTGAGCTGGTGCTTATGCTATTCTTTACTTGTGAGAGAAAGTAGG-3'.
CUP1pHA3-SMT3(GG) was generated by amplifying SMT3(GG) (processed Smt3 ends with two C-terminal glycines) with oligonucleotides that generated PacI and AscI sites, and the digested product was ligated into pRS426 (3, 25). To replace the native SMT3 locus, we used the following oligonucleotides to amplify the new SMT3 allele from pRS426-CUP1p-HA3-SMT3(GG) plasmid (EB1445) and replace the genomic locus, marking the locus with URA3: 5'-CCGTACGGCGGGGCACTTTTGAAACGTTTTTGTGCATCCTCGCGCGTTTCGGTGATGACG-3' and 5'-CCAGGGTTTCCGAAGATAAAGTGGCATCTTTCCATTTACCGGCCGCTCTAGAACTAGTGG-3'.
We sequenced the integrated ORF to confirm that no additional mutations were introduced. The final MAT strain (EY1295) was grown to logarithmic phase, mixed with the TAP-tagged strains in 96-well plates (Nalge Nunc International, Rochester, NY) and allowed to grow overnight at 25 °C. To select for diploids, the strains were pinned onto SD (synthetic medium with dextrose) lacking histidine (His), uracil (Ura), and methionine (Met) utilizing a 96-pinning tool (V & P Scientific, San Diego, CA) and allowed to grow for 2 days at 30 °C. The strains were then pinned into liquid SD His Ura medium, grown overnight at room temperature, pinned onto solid sporulation medium, and allowed to sporulate for at least 7 days. Meiosis was monitored by light microscopy, and the resulting tetrads were pinned onto SD His Ura Met medium also lacking leucine (Leu) and arginine (Arg) and supplemented with 50 µg/ml canavanine and grown at room temperature for 4 days to select for appropriate haploid strains. The resulting colonies were pinned into 96-well plates with liquid SD His Ura Met Leu Arg +canavanine medium and allowed to grow for 2 days at room temperature. Recipes for all media preparations are available upon request. Cells were diluted 1:1000 and allowed to grow to stationary phase in SD His Ura Met Leu. We screened the resulting strains in this study.
Generation of Cell Lysates and Assay for SUMO Conjugation
Strains in 96-well plates were grown to stationary phase in SD His Ura Met Leu and diluted 1:20 into YEPD (24) + 500 µM CuSO4 in 2-ml-deep-well plates (Greiner Bio-One, Frickenhausen, Germany) and grown at 25 °C for 8 h in a HiGro Shaker (Gene Machines, Ann Arbor, MI). Between 2 and 10 ml of cells were grown, harvested, and lysed as described previously (26) except that the lysis buffer consisted of 25 mM Tris-Cl, pH 8, 5 mM EDTA, 0.5% Triton X-100, 10 mM NaCl, 50 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 2.5 mM benzamidine, and 1 µg/ml pepstatin. After hypotonic lysis, we added NaCl to a final concentration of 250 mM and removed insoluble cell debris by centrifugation at 2000 x g for 20 min. One microgram of biotin-IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and 5 µl of buffer-equilibrated streptavidin-Sepharose (Amersham Biosciences) were added to the supernatant (26). After 20 min, we resuspended the Sepharose by pipetting the mixture. After 40 min total, we centrifuged the plates, removed the supernatant, washed the Sepharose once with fresh wash buffer (lysis buffer + 240 mM NaCl), and transferred the Sepharose to 96-well filter-tip plates (Orochem, Westmont, IL) (26). We washed the beads three times with 0.5 ml of ice-cold wash buffer and eluted the immunoprecipitation with 20 µl of 2x SDS sample buffer at 65 °C. We loaded 13 µl of the eluate into wells of 415% Tris Criterion gels (Bio-Rad) and blotted the electrophoresed proteins to nitrocellulose as described previously (26). First we probed for hemagglutinin (HA) reactivity with 0.5 µg/ml 12CA5 antibody and anti-mouse antibody conjugated to horseradish peroxidase (Bio-Rad), then boiled the blot in 1% SDS for 2 min, rinsed away the SDS, and probed for TAP reactivity with 0.1 µg/ml Fc (Jackson Immunoresearch Laboratories, Inc.) and anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad). We used SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) to detect horseradish peroxidase. We considered proteins to be immunoprecipitated if a band of approximately the predicted size was detected with a 1-min exposure to film. We repeated the screen twice for all proteins that were scored as SUMO-interacting and considered proteins as reproducibly associated with SUMO when they were scored as SUMO-interacting in at least one additional assay.
Parameters for Network Visualization System (Osprey)
We imported the 82 SUMO-interacting targets into Osprey 1.2.0 (27), selected them, and visualized all interactions among these proteins using data from three large scale data sets (2830). We inverted the selection and removed nodes with the connection minimum filter set at 2. We grouped nodes into logical complexes; for example, all RSC subunits were grouped together.
Assay for Direct Targets of SUMO
TAP strains (ULP1wt) that were scored as reproducibly SUMO-interacting were transformed with pRS426-CUP1p-HA-His6-SMT3(GG) (EB1436). Strains with and without the plasmid were grown to midlogarithmic growth at 30 °C in 50 ml of SD Ura or SD supplemented with amino acids, respectively. We induced the CUP1 promoter with 0.5 mM CuSO4 for 2.5 h. We lysed cells in 100 mM NaH2PO4, 10 mM Tris, 8 M urea, pH 8, and 10 mM N-ethylmaleimide by bead-beating with glass beads at room temperature and cleared the extract by centrifugation at 13,000 x g for 10 min. The cleared extract was incubated with 50 µl of Co2+-iminodiacetic acid (IDA)-Sepharose (Sigma) for 30 min. We washed bound proteins in 1.5-ml tubes four times with 1 ml of lysis buffer and then eluted proteins in lysis buffer + 50 mM EDTA. We added 2x sample buffer for 15 min, and 13 µl were loaded onto Criterion gels (26).
Generation of Myc-tagged ORFs
Each candidate ORF was amplified by PCR to generate 500 bp upstream of the start codon flanked by a NotI site and a PacI site at the 3'-end of the ORF without a stop codon so that the ORF was in-frame with 13 Myc epitopes. We ligated the Myc tag and ORF into pRS313 (25, 31) and transformed the plasmids into an S288c strain containing EB1436. Denatured extracts were prepared and processed as described above except that proteins bound to the Co2+-IDA resin were only washed twice with 1 ml of lysis buffer. We detected the Myc tag on immunoblots utilizing the mouse monoclonal antibody 9E10 at a concentration of 0.2 µg/ml. We generated mutations in codons encoding lysines by oligonucleotide-mediated overlapping PCR using Vent polymerase (New England Biolabs, Beverly, MA). We sequenced the ORFs to confirm there were no other mutations. To determine whether mutated proteins were able to substitute for wild-type function, we inactivated the RPS3, TUP1, and RSC58 ORFs utilizing PCR gene replacement with the Candida glabrata LEU2 gene in a diploid strain and sporulated and dissected tetrads with the appropriate covering plasmid (32).
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RESULTS |
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Development of a High Throughput Immunoprecipitation Technique to Identify SUMO-associated Proteins
We designed a high throughput assay to immunoprecipitate TAP-tagged proteins and to determine whether the immunoprecipitated protein was conjugated to SUMO. We performed high throughput immunoprecipitations by adding biotinylated human IgG and streptavidin-Sepharose to extracts derived from the TAP-tagged strains containing ulp1ts and HA3-SMT3. IgG binds with high affinity and specificity to the TAP tag, and streptavidin binds with extremely high affinity to biotin. Importantly this protocol minimizes the cross-reactivity of IgG bands during immunoblot analysis because the biotin-streptavidin interaction is not disrupted by the elution buffer (Fig. 1C).
After immunoblot analysis to detect the TAP tag, we determined the efficiency of immunoprecipitation. Of the 4034 extracts analyzed, we were able to immunoprecipitate 2893 proteins (or 72%). We were able to immunoprecipitate 80% of the proteins that were above the median protein abundance in the proteome as determined by genome-wide immunoblot analysis (22). Importantly our success rate for immunoprecipitating and visualizing even the least abundant proteins was 46% (Fig. 1D). When we compared the ability to immunoprecipitate membrane-bound proteins relative to the proteins defined as soluble, there was no decrease in the immunoprecipitation efficiency of the membrane proteins. This may be a consequence of poor definition of membrane proteins (26) or more likely inclusion of the vesicle fraction in the immunoprecipitation because we performed a relatively low speed centrifugation to remove insoluble proteins during the preparation of cell lysate.
Next we probed the immunoblot to determine whether each TAP-tagged protein was sumoylated (Fig. 1C). Utilizing the 12CA5 mouse monoclonal antibody, we determined whether there was significant HA reactivity in the immunoprecipitate. We observed three different results. 1) There was no HA reactivity, suggesting that the protein is not sumoylated. 2) There was HA reactivity that corresponds to a protein migrating at the same size as the TAP-tagged protein. We observed this HA reactivity with the most abundant, efficiently isolated TAP-tagged proteins, and this reactivity is likely a consequence of 12CA5 cross-reacting with the TAP tag. We did not consider these candidate sumoylated proteins. 3) There was HA reactivity corresponding to a size larger than the major TAP-reactive band. We considered the proteins in category 3 candidate sumoylated proteins. For many sumoylated proteins, we observed multiple HA-reactive bands migrating slower than the major TAP-reactive band, suggesting these proteins may be multiply sumoylated. Because often only a small fraction of a given protein is sumoylated, when we probed for the TAP tag there was little or no TAP tag reactivity overlapping with the HA-reactive band.
Identification of Direct Targets of Sumoylation
Eighty-two proteins reproducibly associated with HA reactivity (category 3 from above) in our immunoprecipitation assay, suggesting an association with SUMO. Because our assay is performed under non-denaturing conditions, the HA reactivity can be a consequence of an associated protein that is sumoylated. Therefore, we utilized a secondary denaturing screen with a six histidine (His6) tag on SUMO to determine which candidates were directly conjugated to SUMO. To determine direct SUMO conjugates, we transformed the 82 TAP-tagged strains that scored positive in our primary screen with a plasmid expressing His6-Smt3 under the control of the CUP1 promoter. Utilizing the original TAP-tagged strain (with ULP1wt) allowed us to eliminate the possibility that the protein was only sumoylated in the presence of the ulp1ts allele. The TAP-tagged strains containing His6-Smt3 were lysed in 8 M urea to disrupt non-covalent protein-protein interactions, and SUMO and its conjugates were purified on immobilized Co2+-IDA resin, eluted, and immunoblotted with antibodies that recognize the TAP tag. By comparing strains with and without His6-Smt3, we identified proteins that were covalently modified by SUMO given that SUMO modification leads to slower migrating forms of the protein (Fig. 2). We demonstrated that 13 proteins were directly conjugated to SUMO. Eight proteins were previously demonstrated to be sumoylated: Aos1, Ubc9, Shs1, Cdc11, Sod1, Rsc8, Rsc58, and Tup1 (6, 1921); and five were new direct sumoylation targets: Ysh1, Nut1, Rps3, Taf8, and Gsy2. These new targets are involved in transcription, ribosome function, mRNA processing, and cell survival during stress conditions (3337).
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Complexes That Are Sumoylated
We performed our primary screen under non-denaturing conditions; therefore, we expected to identify SUMO-interacting proteins that, while not directly conjugated to SUMO, are associated in complexes with sumoylated proteins. To determine whether the proteins we identified were complexed with each other, we utilized a visualization program called Osprey (27) to display protein-protein interactions (Fig. 3). We chose stringent parameters, allowing there to be no more than one degree of freedom from the identified sumoylation targets, and only examined published protein-protein interaction data bases (2830). Of the 82 proteins that interact with SUMO in our assay, 47 interact with proteins that we have demonstrated to be directly modified by SUMO. We identified four complexes associated with SUMO that are essential for growth: the TFIID, mediator, septin, and RSC complexes. By repeated immunoprecipitation of multisubunit complexes, the non-denaturing immunoprecipitation minimizes two limitations of the TAP-tagged yeast collection: not all proteins are C-terminally tagged, and not all proteins are able to be immunoprecipitated.
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Confirmation of Proper Identification of Target Proteins
Although systematic strain collections are powerful tools, individual results must be confirmed in an independent manner. To confirm that the direct sumoylation targets that we identified are the annotated proteins we expect, we amplified nine target ORFs from a wild-type genome using PCR and cloned the ORF into a plasmid that C-terminally tagged the ORF with Myc epitopes. We chose these nine ORFs because they had not been identified as direct SUMO targets when we began these studies. We transformed the plasmids encoding the Myc-tagged proteins into a strain that contained a plasmid expressing His6-Smt3. Exponentially grown cells were harvested and lysed under denaturing conditions. We enriched for sumoylated proteins on Co2+-IDA resin and immunoblotted for Myc reactivity. In the His6-Smt3-enriched fraction, we looked for the presence of Myc-reactive bands that both migrated slower than the major Myc-reactive band present in crude extract and were enriched. By tagging the ORF with an epitope other than the TAP tag, we eliminated the possibility of an antibody binding to the TAP tag nonspecifically. Of the nine sumoylated proteins, we confirmed that eight are indeed modified by SUMO (Fig. 4A ). However, Gsy2-Myc sumoylation was not detected in this assay. This could be caused by the changed epitope tag or more likely by the inconsistent determination of Gsy2-TAP as a direct SUMO target. Given that Gsy2 activity varies depending upon environmental conditions (35), it is possible that robust sumoylation of Gsy2 is dependent on growth conditions.
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We mutated at least one conjugation site in three other proteins: Rps3, Rsc58, and Tup1 (Fig. 4B). Because RPS3 is an essential gene, we examined whether removal of the majority of sumoylation causes lethality. We find that sumoylation of Rps3K212R is dramatically decreased and that this mutant protein is able to replace the wild-type function of Rps3 during growth in rich medium (data not shown). It is possible that residual sumoylation is sufficient for Rps3 function or that sumoylation of Rps3 is not an essential modification during standard laboratory growth conditions. Interestingly lysine 212 has also been identified as a target of ubiquitylation (39), allowing for the possibility that sumoylation acts as a ubiquitin antagonist. Similarly Tup1K269R is defective in sumoylation. To determine whether sumoylation regulates Tup1 activity, we assayed the TUP1K269R allele for the ability to complement a tup1 strain (40). The mutant protein complemented the flocculation phenotype of the tup1
, and microarray analysis demonstrated that fewer than 10% of Tup1-dependent genes changed expression more than 2-fold between the wild-type and Tup1K269R proteins (data not shown). The fact that the TUP1K269R mutant does not have a dramatic phenotype could be explained by redundant sumoylation of Ssn6, a binding partner of Tup1 that has recently been demonstrated to be sumoylated (20), or by residual sumoylation of Tup1, which we observed in some experiments. Finally Rsc58K322R,K328R is significantly less modified by SUMO than wild-type Rsc58, and this mutant protein allows the cells to grow in standard growth medium in the absence of the wild-type protein. Because we identified Rsc8 as a direct SUMO conjugate as well and others have demonstrated that Rsc2 is modified by SUMO (19), other RSC subunits may need to be mutated before the role of SUMO conjugation in RSC function becomes clear. Thus, it is likely that multiple sumoylation site mutants, either in single proteins or in complexes, will have to be generated to determine the role of SUMO in the regulation of these proteins.
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DISCUSSION |
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We identified new sumoylated proteins that are involved in important processes within the cell. Concurrent mass spectrometry studies have confirmed that Sod1, Rsc8, Rsc58, and Tup1 are sumoylated (1921), supporting the utility and complementary nature of our screen. We identified Nut1, Ysh1, and Taf8 as directly sumoylated proteins, and these proteins are present in the cell at less than 2500 copies/cell, which is below the median protein abundance (22). Furthermore the new direct SUMO conjugates that we identified were nuclearly localized (43), which is consistent with the primarily nuclear localization of Smt3, Aos1, Uba2, and Ubc9 (6, 43). The mass spectrometry-based approaches and our approach have identified some of the same substrates, e.g. Sod1 and Tup1, but each study has unique candidates as well (1921). While it is likely that the identified substrates that overlap in all of the studies are bona fide SUMO conjugates, further research is required to validate the unique candidates in each study.
Many of the recently identified targets of sumoylation have demonstrated roles in transcription (3436, 44, 45). Important mammalian transcription factors that control cell proliferation are sumoylated (11), and our results along with those of others suggest that the regulation of transcription by sumoylation is an evolutionarily conserved process (19). Furthermore the loss of activity of two of the substrates we identified, Rps3 and Sod1, contributes to the human diseases xeroderma pigmentosum class D (46) and familial amyotrophic lateral sclerosis (47), respectively, and understanding how sumoylation regulates the function of these proteins in budding yeast may provide insights into these diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, December 13, 2004, DOI 10.1074/mcp.M400166-MCP200
1 The abbreviations used are: SUMO, small ubiquitin-related modifier; HA, hemagglutinin; IDA, iminodiacetic acid; ORF, open reading frame; SD, synthetic yeast medium with 2% dextrose; TAP, tandem affinity purification; E1, SUMO-activating enzyme; E2, SUMO carrier protein; E3, SUMO-protein isopeptide ligase; Ura, uracil.
* k was supported by National Institutes of Health Postdoctoral Research Fellowship GM20762 and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Howard Hughes Medical Inst., University of California, 600 16th St., Genentech Hall, Rm. GH-S472D, San Francisco, CA 94143-2240. Tel.: 415-476-2212; Fax: 415-514-2073;E-mail: oshea{at}biochem.ucsf.edu.
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REFERENCES |
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