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• W2091602394 abstract "SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms. SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms. Many types of post-translational protein modifications alter protein function, and in some cases the modifying group is itself a protein. The prototypical example of this is ubiquitin, a small, highly conserved polypeptide that is reversibly linked to many different proteins (probably thousands) in the cell (1Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1278) Google Scholar). Polypeptides distinct from but related to ubiquitin, called ubiquitin-like proteins or Ubls, can also be ligated to proteins (2Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (362) Google Scholar, 3Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (Smt3 in the yeast Saccharomyces cerevisiae) is a divergent Ubl that has crucial roles in many organisms (4Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (569) Google Scholar, 5Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar). Vertebrates have four SUMO variants, SUMO1–SUMO4, whereas yeasts have only one. Only human SUMO1, and not the other SUMO variants, can substitute for the essential yeast protein (6Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (68) Google Scholar). Activation and conjugation reactions involving SUMO have much in common with those of ubiquitin (reviewed in Refs. 2Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (362) Google Scholar, 5Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar, and 7Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (646) Google Scholar). Attachment of substrates to Smt3/SUMO depends on a heterodimeric SUMO-activating enzyme (E1), 1The abbreviations used are: E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO-protein ligase; ORF, open reading frame; MS, mass spectrometry; GBD, Gal4 DNA-binding domain; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside.1The abbreviations used are: E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO-protein ligase; ORF, open reading frame; MS, mass spectrometry; GBD, Gal4 DNA-binding domain; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. called Uba2-Aos1 in yeast, and a SUMO-conjugating enzyme (E2), Ubc9. Both enzymes form transient thiolester bonds with the C terminus of SUMO. Substrate recognition factors (E3s) that stimulate the transfer of SUMO from E2 to substrate have also been identified. SUMO, like ubiquitin, is always synthesized in precursor form, requiring enzymatic removal of a C-terminal peptide. Specialized proteases, called Ubl-specific proteases or Ulps, are responsible for these SUMO processing reactions and for reversing the post-translational attachment of SUMO to proteins. Unlike ubiquitin ligation, many sumoylation sites in proteins match a short consensus sequence, hKXE, where h represents a hydrophobic residue, X is any residue, and lysine (K) is the site of SUMO attachment (5Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar). This consensus can be rationalized by structural data showing the direct binding of the Ubc9 E2 to the conserved elements of this sequence (8Bernier-Villamor V. Sampson D.A. Matunis M.J. Lima C.D. Cell. 2002; 108: 345-356Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 9Hochstrasser M. Mol. Cell. 2002; 9: 453-454Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). This E2-substrate interaction is unlikely to be sufficient for specific substrate discrimination in the cell, so E3 factors are expected to be important for achieving the requisite in vivo specificity. The SUMO system has been implicated in multiple physiological pathways (3Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 4Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (569) Google Scholar, 5Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1358) Google Scholar). SMT3, as well as the genes encoding the SUMO E1, E2, and Ulp1 protease, are all essential for viability in budding yeast, and cells with a conditional allele of either UBC9 or ULP1 are defective in cell cycle progression. The first identified target of SUMO conjugation was vertebrate RanGAP1, a protein required for nucleocytoplasmic trafficking. Modification by SUMO is required for RanGAP1 localization to the nuclear pore complex. Among the many (>50) mammalian targets for SUMO now known are RanBP2, another nuclear pore complex component; PML, a nuclear protein that is altered in certain leukemias; Sp100, an autoantigen in primary biliary cirrhosis; the p53 tumor suppressor; the Sp3 transcription factor; and the NF-κB inhibitor IκB. Even this partial list of in vivo targets makes it clear that the SUMO system must exert a major influence on mammalian growth and regulation. Misregulation of the system is likely to contribute to tumorigenesis, abnormal inflammatory responses, and autoimmune defects. In yeast, a much more limited number of SUMO-linked substrates has been characterized. The first was a subset of the septins, which are proteins essential for cytokinesis; however, eliminating septin sumoylation has no detectable phenotypic effect (10Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (324) Google Scholar, 11Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Yeast topoisomerase II is also sumoylated in vivo, and this modification, while not required for the essential function of the enzyme, may contribute to the cohesive properties of the centromere (12Bachant J. Alcasabas A. Blat Y. Kleckner N. Elledge S.J. Mol. Cell. 2002; 9: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). SUMO is also ligated to the DNA replication/repair protein PCNA (Pol30), and sumoylation inhibits the DNA repair activity of PCNA (13Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1716) Google Scholar, 14Stelter P. Ulrich H.D. Nature. 2003; 425: 188-191Crossref PubMed Scopus (683) Google Scholar). Although all of these substrate proteins are essential for growth, in no case is their sumoylation required for viability. Therefore, the protein or proteins that must be sumoylated for cell division in yeast remain to be identified. Additional proteins involved in chromosome cohesion, Pds5 and Ysc4, have also recently been reported to be transiently sumoylated in yeast, but the sites of sumoylation were not mapped (15Stead K. Aguilar C. Hartman T. Drexel M. Meluh P. Guacci V. J. Cell Biol. 2003; 163: 729-741Crossref PubMed Scopus (134) Google Scholar, 16D'Amours D. Stegmeier F. Amon A. Cell. 2004; 117: 455-469Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Determination of the precise functions of specific SUMO-protein modifications in S. cerevisiae should generally be more facile than in other eukaryotes. However, a much more comprehensive list of sumoylated proteins in this organism must first be obtained. Toward this end, we have taken several independent genetic and proteomic approaches. Our data reveal a surprisingly large and functionally diverse set of SUMO system substrates. Interestingly, the different methods of substrate identification have yielded overlapping but distinct groups of proteins, underscoring the value of using multiple approaches to establish the SUMO proteome. Yeast and Bacterial Methods—Rich (YPD) and minimal (SD) media were prepared as described (17Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1986Google Scholar). Standard methods were used for genetic analysis of yeast (17Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1986Google Scholar). The Escherichia coli strains used in this study were JM101, MC1061, BL21(DE3), and DH10B, and bacterial methods and media were as described (18Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1989Google Scholar). Construction of Yeast Strains—Yeast strains used are listed in Table I. The strain background for all experiments other than the two-hybrid screen was MHY501 (19Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (351) Google Scholar). To make the MHY2811 strain for purification of HFT-SUMO-protein conjugates, MHY1538 was transformed with YRTAG310-HFT-SMT3GG, and transformants were plated on 5-fluoroorotic acid to select for cells that had lost the two original URA3-marked plasmids. Other strains listed were generated by standard molecular genetic methods. The library of yeast strains with genes fused to the TAP tag was obtained from Open Biosystems (Huntsville, AL).Table IYeast strainsStrainGenotypeMHY501α his3-Δ200 leu2-3,112 lys2-801 trp1-1 ura3-52MHY1380α his3-Δ200 leu2-3,112 lys2-801 trp1-1 ura3-52 ulp2Δ::HIS3MHY1538α his3-Δ200 leu2-3,112 lys2-801 trp1-1 ura3-52 smt3Δ::HIS3 ulp1Δ::HIS3[pVT102U-SMT3gg YCp50-ULP1]MHY2809α his3-Δ200 leu2-3,112 lys2-801 trp1-1 ura3-52 smt3Δ::HIS3 ulp1Δ::HIS3[YRTAG310-H6-SMT3gg]MHY2811α his3-Δ200 leu2-3,112 lys2-801 trp1-1 ura3-52 smt3Δ::HIS3 ulpΔ::HIS3[YRTAG310-HFT-SMT3gg]PJ69-4Aa trp1-901 leu2-3,112 ura3-52 his3-Δ200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ Open table in a new tab Plasmid Constructions—The plasmid pGBD-U-C1-SMT3 for the two-hybrid screen was constructed by ligating a BamHI, SalI-cut PCR product that contained the entire SMT3 open reading frame (ORF) into pGBD-U-C1 (20James P. Halladay J. Craig E. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Derivatives of this plasmid that lacked either N-terminal or C-terminal coding sequences of SMT3 were made by EcoRI digestion and religation of the plasmid to make pGBD-SMT3-C30 or by BglII digestion and religation to make pGBD-SMT3-N30, respectively. Plasmid pGBD-SMT3ΔGG was made by PCR amplification of SMT3 sequence encoding its first 96 residues and ligation into BamHI, SalI-cut pGBD-U-C1. Plasmid pQE30-FLAG-TEV-SMT3GG was made by PCR amplification of the SMT3 ORF using a 5′ primer that included a BamHI site and sequences encoding a FLAG epitope (DYKDDDDK) followed by the tobacco etch virus recognition sequence (TEV; ENLYFQ/G) and a 3′ primer with sequences for SMT3 codons 90–98 followed by a stop codon and a SalI recognition site. The PCR product was cut with BamHI and SalI and ligated to the identically digested pQE30 (Qiagen) His6-tagging vector. A DNA fragment encoding His6-FLAG-TEV-SMT-GG was then amplified by PCR, digested with SalI and SacI, and cloned into the yeast CUP1-driven expression vector YRTAG310 that had been cut with XhoI and SacI, creating YRTAG310-HFT-SMT3GG. SMT3 and FLAG-TEV-tagged SMT3 alleles encoding mature SUMO were also amplified by PCR and were cloned into pRS426GAL for galactose-inducible expression. Insert sequences were confirmed by DNA sequencing. Two-hybrid Screen with SUMO Bait—Full-length SMT3 (mature domain) was fused to the sequence encoding the Gal4 DNA-binding domain. This was used as bait for a two-hybrid screen of yeast plasmid libraries made of partial digested S. cerevisiae genomic sequences ligated to a sequence encoding the Gal4 activation domain (20James P. Halladay J. Craig E. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Purified DNA from the libraries was transformed into yeast strain PJ69–4A carrying the bait plasmid pGBD-U-C1-SMT3, and transformants were grown on triple dropout plates (SD-trp-leu-his) for 5 days at 30 °C. The PJ69–4A strain carries three reporter genes (HIS3, ADE2, and lacZ), each under the control of a different GAL promoter. Transformants that grew on the triple dropout plates (his+ and therefore able to activate GAL-HIS3) were rescreened on SD-trp-leu-ade dropout plates to test ADE2 expression and on X-gal-containing plates to test lacZ expression. Plasmids were isolated from yeast transformants that were his+ ade+ and blue on X-gal. The plasmids were retransformed into the two-hybrid tester strain to confirm the two-hybrid signal, and the plasmid insert junctions were sequenced using the suggested upstream and downstream primers (20James P. Halladay J. Craig E. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). We tested the positive clones for interactions with the SUMO deletion mutant pGBD-SMT3ΔGG and in a tester strain that also carried ULP1 on a 2-μm plasmid. Selection for High Copy Suppressors of ulp2Δ—Selection of dosage suppressors of ulp2Δ temperature-sensitive growth at 37 °C was done by transformation of the ulp2Δ strain MHY1380 (21Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (308) Google Scholar) with a YEp24 (2 μm, URA3)-based yeast genomic library. Transformants were plated on uracil dropout plates and incubated at 37 °C and, as a control, 30 °C. Approximately one in 104 to 105 ura+ transformants appeared as colonies on the 37 °C plates after 7 days. The ura+ thermoresistant colonies were restreaked at 37 °C to confirm the phenotype. Plasmid DNA was isolated from individual clones and transformed again into MHY1380 to test for the plasmid dependence of high temperature growth. Nine plasmid-dependent, temperature-resistant clones from ∼106 MHY1380 transformants were identified. Vector-insert junctions in these plasmids were determined by DNA sequencing and data base searching. For the four strongest suppressors, DNA subcloning and deletion was used to identify the genes responsible for the suppression. Immunoblotting—Proteins were electrotransferred to polyvinylidene difluoride membranes using either a GENIE blotter at 20 V for 2 h at 4 °C or a Bio-Rad semidry blotter at 25 V for 30 min at room temperature. The membrane was blocked with 10% skim milk in TBST solution (10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.05% (v/v) Tween 20) for 1 h at room temperature and washed three times with TBST for 10 min. Primary antibodies diluted 1:3,000 to 1:10,000 in TBST with 1% skim milk were incubated with membrane for 1–2 h. The membrane was washed with TBST and incubated for 1 h with secondary antibody diluted 1:4,000 in TBST. After washing with TBST, antibody binding was detected using ECL Western blotting reagents (Amersham Biosciences). SUMO conjugates were visualized with a purified rabbit polyclonal antibody raised against yeast SUMO (22Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (597) Google Scholar). Purification of SUMO-Protein Conjugates from Yeast—MHY2811 yeast cells were grown in 4 liters of YPD (no added copper) at 24 °C to an A600 of 0.5–1.0. Approximately 7–14 g of cells (wet weight) were pelleted, washed twice with deionized water, and shock-frozen in liquid nitrogen. Frozen cells were ground to a powder to facilitate later resuspension in lysis buffer and were stored at –80 °C. Cells were resuspended in 3 volumes of extraction buffer (8 m guanidinium HCl, 100 mm sodium phosphate, pH 8) at room temperature. Cells were lysed at room temperature with three passes through a French press at 18,000 p.s.i. The lysate was cleared at 30,000 × g for 30 min at room temperature, and the supernatant was carefully removed with a pipette from below the cloudy lipid layer on top. The cleared lysate was bound to 2–4 ml of Talon beads for 60 min at room temperature. Protein-bound beads were then loaded into a column and washed three times with 10 volumes of wash buffer (8 m urea, 100 mm sodium phosphate, pH 8). Bound proteins were eluted with 300 mm imidazole in wash buffer, and 2-ml fractions were collected. Eluates were pooled, and the resulting 10–20-ml sample was diluted into 24 volumes of FLAG-binding buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 10% glycerol, 0.1% Nonidet P-40) with protease inhibitors, in which 0.1 volumes of anti-FLAG beads (Sigma) were already present. After binding in batch for 90 min at 4 °C, the beads were transferred to a column, drained, and washed with 100–200 ml of FLAG wash buffer (10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% Nonidet P-40) and then 10–20 ml of TEV cleavage buffer (10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% Nonidet P-40, 0.5 mm EDTA, 1 mm dithiothreitol). The drained beads were resuspended in 1–2 ml of TEV cleavage buffer containing 100 units of recombinant TEV protease (Invitrogen). TEV cleavage was executed in the sealed column on a shaker at 16 °C for at least 16 h. The column was drained and then washed several times with TEV cleavage buffer to collect all of the eluted protein. Samples of 2 ml were collected. Eluate 1 (E1) is the material from the original draining plus one column volume of wash, and E2 the following 2 column volumes of washings. The anti-FLAG beads were stripped with 0.1 m glycine at pH 3.5. Samples were precipitated with 10% trichloroacetic acid at –20 °C for 2 h and spun down at 16,000 × g at 4 °C. Pellets were washed four times with ice-cold acetone to remove residual Nonidet P-40. Protein samples were checked by anti-SUMO immunoblot analysis and by Gel Code Blue (Pierce) staining. Mass Spectrometry and Computational Methods—The final purified protein fractions were reduced with 1 mm dithiothreitol and then carboxyamidomethylated with 5 mm iodoacetamide. The samples were diluted with 50 mm ammonium bicarbonate, pH 8.5, and digested with 1:20 (v/v) Poroszyme-immobilized trypsin (Applied Biosystems, Streetsville, Canada) for 48 h at 30 °C with rotation. The resulting peptide mixtures were solid phase-extracted with SPEC-Plus PT C18 cartridges (Ansys Diagnostics; Lake Forest, CA) according to the manufacturer's instructions. The resulting peptide mixtures were sequenced by shotgun tandem mass spectrometry using the multidimensional protein identification technology (MudPIT) of Yates and colleagues (23Washburn M.P. Wolters D. Yates J.R. II I Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4029) Google Scholar), as adapted by Kislinger et al. (24Kislinger T. Rahman K. Radulovic D. Cox B. Rossant J. Emili A. Mol. Cell Proteomics. 2003; 2: 96-106Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Briefly, a 150-μm inner diameter fused silica capillary microcolumn (Polymicro Technologies; Phoenix, AZ) was pulled to a fine tip using a P-2000 laser puller (Sutter Instruments; Novato, CA) and packed to a 10-cm bed height with 5 μm Zorbax Eclipse XDB-C18 resin (Agilent Technologies, Mississauga, Canada) followed by 6cmof5 μm Partisphere strong ion exchanger (Whatman). The sample was loaded manually onto the column using a pressure vessel and then connected in-line to a quaternary high pressure liquid chromatography pump and interfaced with an LCQ DECA XP quadrupole ion trap tandem mass spectrometer (Thermo Finnigan; San Jose, CA). The eluate was analyzed by electrospray ionization using a fully automated four-buffer, 15-step chromatographic cycle essentially as described (24Kislinger T. Rahman K. Radulovic D. Cox B. Rossant J. Emili A. Mol. Cell Proteomics. 2003; 2: 96-106Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Individual eluting peptides were subject to automated, data-dependent collision-induced fragmentation. The peptide spectra were searched against a locally maintained data base of yeast protein sequences obtained from the Saccharomyces Genome Database (around August 2001) using a distributed version of the SEQUEST algorithm running on a multiprocessor computer cluster (25Eng J.K. McCormack A.L. Yates J.R. II I J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5315) Google Scholar) and filtered for high confidence protein matches (p value <0.05) using the STATQUEST statistical evaluation algorithm (24Kislinger T. Rahman K. Radulovic D. Cox B. Rossant J. Emili A. Mol. Cell Proteomics. 2003; 2: 96-106Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The data sets were arranged into tabular format using the DTASelect and Contrast software programs (26Tabb D.L. McDonald W.H. Yates J.R. II I J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1114) Google Scholar) and subgrouped into select gene ontology functional annotation categories (27Harris M.A. Clark J. Ireland A. Lomax J. Ashburner M. Foulger R. Eilbeck K. Lewis S. Marshall B. Mungall C. Richter J. Rubin G.M. Blake J.A. Bult C. Dolan M. Drabkin H. Eppig J.T. Hill D.P. Ni L. Ringwald M. Balakrishnan R. Cherry J.M. Christie K.R. Costanzo M.C. Dwight S.S. Engel S. Fisk D.G. Hirschman J.E. Hong E.L. Nash R.S. Sethuraman A. Theesfeld C.L. Botstein D. Dolinski K. Feierbach B. Berardini T. Mundodi S. Rhee S.Y. Apweiler R. Barrell D. Camon E. Dimmer E. Lee V. Chisholm R. Gaudet P. Kibbe W. Kishore R. Schwarz E.M. Sternberg P. Gwinn M. Hannick L. Wortman J. Berriman M. de la Wood V. Cruz N. Tonellato P. Jaiswal P. Seigfried T. White R. Nucleic Acids Res. 2004; 32: 258-261Crossref PubMed Google Scholar) using the FunSpec program (28Robinson M.D. Grigull J. Mohammad N. Hughes T.R. BMC Bioinformatics. 2002; 3: 35Crossref PubMed Scopus (323) Google Scholar). We have undertaken a series of approaches to identify sumoylated proteins in yeast. These different methods were expected to yield different but related kinds of information about SUMO-protein ligation and the roles of SUMO modification in the cell. A “Covalent Interaction Trap” for SUMO-linked Proteins—A protein that can be covalently coupled to other proteins offers an interesting type of bait in an otherwise standard two-hybrid screen for protein-protein interactions (Fig. 1A). We fused SUMO (Smt3) to the C terminus of the Gal4 DNA-binding domain (GBD), creating a fusion that can functionally replace normal SUMO (data not shown). Unlike most baits, a positive signal in the two-hybrid screen can be generated by covalent ligation of the bait, GBD-SUMO, to a library substrate protein fused to the Gal4 transcriptional activation domain (AD) (Fig. 1A, I). Alternatively, GBD-SUMO may get attached to an endogenous yeast protein that then can interact with the activation domain fusion (Fig. 1A, II). Finally and more conventionally, GBD-SUMO could interact noncovalently with an activation domain fusion (Fig. 1A, III). As will be described, it is possible to distinguish ligation-dependent association from noncovalent interactions. Thus, the screen could identify both SUMO-conjugated substrates and proteins that bind noncovalently to SUMO or SUMO-protein conjugates. The latter proteins may be SUMO pathway regulators or enzymes of the SUMO system. We screened ∼5 × 105 yeast genomic clones and identified segments of 13 different proteins that gave strong two-hybrid interaction signals with three reporter genes, each under the control of a different GAL promoter (20James P. Halladay J. Craig E. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) (Fig. 1B). To determine whether any of the two-hybrid interactions were likely to depend on SUMO ligation, we tested them against a GBD-SUMOΔGG fusion, which lacks the terminal pair of glycines of SUMO and therefore cannot be attached to other proteins (29Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (439) Google Scholar). Seven of the tested interactions appear to require SUMO ligation because the interaction signal was no longer seen with GBD-SUMOΔGG (minus signs in Fig. 1B), as predicted for binding modes I and II in Fig. 1A. We also tested the sensitivity of these interactions to Ulp1 isopeptidase overexpression, since most SUMO-protein conjugates are depleted in cells overproducing this enzyme (22Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (597) Google Scholar). Of the seven SUMOΔGG-sensitive interactors, five were also sensitive to Ulp1 overproduction; some SUMO conjugates might be poor substrates for Ulp1 in vivo, which might account for the two exceptions (Hex3 and Ufd1). In contrast, all but one of the identified prey proteins that also bound SUMOΔGG still interacted with SUMO in the presence of high Ulp1. ULP1HC-sensitive, SUMO diglycine-dependent interactors represent strong candidates for SUMO-linked substrates or proteins that bind such substrates. Among the 13 identified SUMO-interacting proteins are proteins involved in RNA metabolism, DNA repair, protein degradation, and gene silencing. Two of the proteins, Siz2/Nfi1 and Wss1, had been linked to the SUMO pathway in previous studies (11Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 30Biggins S. Bhalla N. Chang A. Smith D.L. Murray A.W. Genetics. 2001; 159: 453-470Crossref PubMed Google Scholar). Siz2 is an SP-RING-bearing E3-like factor involved in protein sumoylation, although its substrates are not known. The function of Wss1 is unclear, but it is a weak, high copy suppressor of a temperature-sensitive smt3 mutant. Interestingly, Siz2 and Nis1 have both been found to bind to septins, which are the most abundant sumoylated proteins in yeast. The septins localize to the bud neck during cell division, and another two-hybrid hit, Fir1, also localizes to this region. Besides Siz2, two other proteins, Ris1 and Hex3, contain a RING motif, a signature sequence for E3 ligases. Therefore, these proteins might be SUMO E3-like enzymes as well. Both Siz2 and Hex3 require a conjugation-competent form of SUMO for detection of a two-hybrid interaction. RING E3s in the ubiquitin pathway frequently ubiquitinate themselves. Autosumoylation might be occurring with Siz2 and Hex3, but the minimal segments of these two proteins that interacted with GBD-SUMO in the two-hybrid screen lacked the catalytic RING motif, which would suggest that sumoylation was occurring in trans. We chose Siz2 to determine whether it is in fact sumoylated in vivo and to validate the use of the two-hybrid screen to identify substrates for SUMO ligation. Previously, we had developed a gel shift assay to demonstrate in vivo protein ubiquitination (31Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4606-4610Crossref PubMed Scopus (196) Google Scholar); an analogous strategy was used here to test sumoylation. When Myc-tagged Siz2, expressed from the normal chromosomal locus," @default.
• W2091602394 created "2016-06-24" @default.
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• W2091602394 creator A5032098391 @default.
• W2091602394 creator A5050648920 @default.
• W2091602394 creator A5051352544 @default.
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• W2091602394 creator A5076978787 @default.
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• W2091602394 date "2005-02-01" @default.
• W2091602394 modified "2023-10-18" @default.
• W2091602394 title "Defining the SUMO-modified Proteome by Multiple Approaches in Saccharomyces cerevisiae*" @default.
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