Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m104214200
ISSN1083-351X
AutoresMichael H. Tatham, Ellis Jaffray, O. Anthony Vaughan, Joana Desterro, Catherine H. Botting, James H. Naismith, Ronald T. Hay,
Tópico(s)RNA modifications and cancer
ResumoConjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences. Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence ψKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detectedin vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences. small ubiquitin-like modifier type 1, 2, and 3, respectively ubiquitin-conjugating enzyme 9 SUMO-activating enzyme subunit 1 and subunit 2, respectively Ran GTPase-activating protein 1 histone deacetylase 4 consensus SUMO modification site where ψ represents a large hydrophobic amino acid and X represents any amino acid full-length SUMO pro-protein construct SUMO protein that terminates in the diglycine motif matrix-assisted laser desorption ionization-time of flight dithiothreitol glutathione S-transferase hemagglutinin polymerase chain reaction reverse transcriptase-PCR wild type promyelocytic leukemia protein tobacco etch virus amino acids ubiquitin-like protein-activating enzyme ubiquitin-like protein carrier protein ubiquitin-like protein isopeptide ligase The small ubiquitin-like modifier SUMO-11 (also known as SMT3C, Sentrin, GMP1, UBL1, and PIC1) is a member of the ubiquitin-like protein family (1Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). SUMO-1 is known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway analogous to that of ubiquitin conjugation. The E1-like enzymes for both SUMO-1 and the yeast homologue Smt3p exist as heterodimers known as SAE1/SAE2 and Uba2p/Aos1p, respectively (2Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 3Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (442) Google Scholar, 4Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar, 5Gong L. Li B. Millas S. Yeh E.T. FEBS Lett. 1999; 448: 185-189Crossref PubMed Scopus (131) Google Scholar). In the first step the SAE1/SAE2 heterodimer utilizes ATP to adenylate the C-terminal glycine of SUMO-1. Formation of a thioester bond between the C-terminal glycine of SUMO-1 and a cysteine residue in SAE2 is accompanied by the release of AMP. The second step is a transesterification reaction, which transfers SUMO-1 from the E1 to a cysteine residue within the SUMO-specific E2-conjugating enzyme (Cys93 in Ubc9). In the third step, Ubc9 catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ε-amino group of lysine in the target protein. In contrast to the ubiquitin conjugation pathway no activity equivalent to an E3 ligase is required for SUMO-1 conjugationin vitro (2Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 4Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar), suggesting that the specificity for target proteins is conferred by Ubc9 itself or the Ubc9·SUMO-1 thioester complex. This is supported by the observations that almost all SUMO-1-conjugated proteins bind Ubc9 in two-hybrid assays, and the acceptor lysine residues on target proteins appear to exist within the consensus motif ψKXE (where ψ represents a large hydrophobic amino acid, and X represents any amino acid) (6Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar, 7Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 8Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). Furthermore, SUMO-1 is thought not to form SUMO-1-SUMO-1 polymers, which are characteristic of ubiquitination. Unlike the majority of ubiquitinated proteins, acceptors of SUMO-1 modifications are not targeted for degradation. In fact, in the case of the transcriptional inhibitor IκBα the target lysine for SUMO-1 modification is the same as that of ubiquitin conjugation, thus blocking ubiquitination at that residue and stabilizing the protein (8Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). Transcriptional activity of specific proteins appears to be affected by SUMO-1 modification. For example, conjugation at a single site in the C terminus of p53 activates its transcriptional response (9Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (559) Google Scholar, 10Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (437) Google Scholar). Furthermore, SUMO-1 modification of certain substrates is also known to have implications upon subcellular localization. The interaction of Ran GTPase-activating protein 1 (RanGAP1) with the Ran-GTP-binding protein 2 at the cytoplasmic face of the nuclear pore complex is dependent on SUMO-1 conjugation of RanGAP1 (11Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 12Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (955) Google Scholar). Modification of the promyelocytic leukemia protein (PML) targets it to distinct nuclear bodies (13Duprez E. Saurin A.J. Desterro J.M. Lallemand-Breitenbach V. Howe K. Boddy M.N. Solomon E. de The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; 112: 381-393Crossref PubMed Google Scholar, 14Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (578) Google Scholar, 15Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (289) Google Scholar) and is required for Daxx recruitment to these structures (16Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T. Strauss III, J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-234Crossref PubMed Scopus (677) Google Scholar, 17Zhong S. Muller S. Ronchetti S. Freemont P.S. Dejean A. Pandolfi P.P. Blood. 2000; 95: 2748-2752Crossref PubMed Google Scholar). Two ubiquitin-like proteins, known as SUMO-2 (SMT3A, Sentrin-3) and SUMO-3 (SMT3B, Sentrin-2), have been identified that are related to SUMO-1 but are apparently functionally distinct (18Lapenta V. Chiurazzi P. van der Spek P. Pizzuti A. Hanaoka F. Brahe C. Genomics. 1997; 40: 362-366Crossref PubMed Scopus (102) Google Scholar, 19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 20Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). SUMO-2 and SUMO-3 are very similar (95% sequence identity) but are relatively different from SUMO-1 (50% sequence identity). In vivostudies have indicated that PML is modified by SUMO-1 and SUMO-2/-3, although the functional significance of SUMO-2/-3 conjugation has not been revealed (21Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Whether or not SUMO-2/-3 conjugates to RanGAP1in vivo may depend on the expression levels of the SUMO proteins (19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 20Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Recent evidence indicates that SUMO-2/-3 is more abundant than SUMO-1 in COS-7 cells and that pools of free SUMO-2/-3 decrease when these cells are exposed to heat, ethanol, or hydrogen peroxide (19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Thus SUMO-2/-3 may be involved in the cellular response to environmental stresses. Here we demonstrate that functional SUMO modification sites present in the N-terminal regions of SUMO-2 and SUMO-3 are utilized by SAE1/SAE2 and Ubc9 to form polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1. Thus, although all SUMO species share the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences. HA-SUMO-1, HA-SUMO-2, HA-K11R-SUMO-2, and HA-SUMO-3 were detected in Western blot experiments using monoclonal antibody 12CA5 (at a 1:5000 dilution), which recognizes YPYDVPDYA from influenza HA, obtained from BAbCO. Sheep anti-mouse horseradish-peroxidase-conjugated IgG (Amersham Pharmacia Biotech) was used to detect primary antibodies at a 1:5000 dilution. The wild type (wt) full-length (FL) 2The full-length versions of the SUMO proteins are the pro-protein forms that contain inhibitory C-terminal extensions (4, 11, and 2 residues for SUMO-1, SUMO-2, and SUMO-3, respectively). These are removed in vivo by specific proteases to yield mature, active protein products that terminate in a diglycine motif. cDNAs of SUMO-2 (309 nucleotides) and SUMO-3 (285 nucleotides) were cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) using HeLa poly(A)+ RNA as template in the TitanTMone-tube RT-PCR system (Roche Molecular Biochemicals) according to the manufacturer's instructions. wt-SUMO-2 was cloned using primers 5′-TCCCCGCGCCGCTCGGAATCCATGTCCGAG-3′ and 5′-CCCGAATTCGGGACGGGCCCTCTAGAAACT-3′, and wt-SUMO-3 was cloned using primers 5′-GAGGAGACTCCGGCGGGATCCATGGCCGACGAA-3′ and 5′-GTAGAATTCCAGGTTCCCTTTTCAGTAGAC-3′. Shorter DNA constructs that code for proteins terminated at the diglycine (276 nucleotides for wt-SUMO-2-GG and 279 nucleotides for wt-SUMO-3-GG) were amplified by PCR using a single downstream primer (5′-CCCGAATTCCTAACCTCCCTGCTGCTGTTGGAACAC-3′) for both with the respective upstream primer as described above. The restriction sites introduced into the primers allowed the cleavage of the cDNAs by the enzymes BamHI and EcoRI and subsequent ligation into both pCDNA3-HA (22Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar) and pGEX-2T. Note that our sequences for SUMO-2 and SUMO-3 cloned from poly(A)+ HeLa RNA were consistent with those cloned recently from a human B-lymphocyte library (23Ahn J.H. Xu Y. Jang W.J. Matunis M.J. Hayward G.S. J. Virol. 2001; 75: 3859-3872Crossref PubMed Scopus (83) Google Scholar) that only differ from one another by 3 residues close to the N terminus (see Fig. 2 A). The mutant SUMO-2 and SUMO-3 K11R DNAs were amplified by PCR using the same single downstream primer described above for the wild type GG constructs along with mutant upstream primers 5′-ATCGATGGATCCATGTCCGAGGAGAAGCCCAAGGAGGGTGTGAGGACAGAGAAT-3′ and 5′-ATCGATGGATCCATGGCCGACGAAAAGCCCAAGGAAGGAGTCAGGACTGAGAAC-3′, respectively. Cleavage with BamHI and EcoRI allowed the cloning of PCR products into pGEX-2T for GST fusion protein expression and pCDNA3-HA for transient cell transfection. DNA encoding the C52A-SUMO-1 mutant, which does not form disulfide dimers, was PCR-amplified from the wild type protein using the internal primers 5′-GAATCATACGCTCAAAGACAG-3′ and 5′-CTGTCTTTGAGCGTATGATTC-3′ and the external primers described previously for the cloning of the wt-SUMO-1-GG protein (22Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar). pGEX-4T-SAE2 plasmid for expression of SAE2 was constructed by PCR amplification of SAE2 cDNA (2Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) with the primers 5′-ACGCGTCGACATGGCACTGTCGCGGGGGCTG-3′ and 5′-GAATGCGGCCGCTCAATCTAATGCTATGACATC-3′. The PCR product was cleaved with SalI and NotI before insertion into similarly cleaved pGEX-4T. A plasmid for translationally linked expression of GST-SAE2/SAE1 was constructed by PCR amplification of SAE1 cDNA (2Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) with primers containing a ribosome binding site andNotI restriction sites (5′-ATAAGAATGCGGCCGCATTAAAAGGAGAAATTAACTATGGTGGAGAAGGAGGAG-3′ and 5′-TATCAAATGCGGCCGCTCACTTGGGGCCAAGGCACTC-3′). The PCR product was cloned as a NotI insert in pGEX-4T-SAE2 to form the GST-SAE2/SAE1 expression plasmid. To generate a recombinant substrate containing a single SUMO modification site, GST was fused to an 11-amino acid sequence (PRKVIKMESEE) representing amino acids 485–495 of PML (7Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). GST and the 11-amino acid modification site were separated by a recognition motif (EPVYFQG) for the tobacco etch virus (TEV) protease (24Dougherty W.G. Parks T.D. Cary S.M. Bazan J.F. Fletterick R.J. Virology. 1989; 172: 302-310Crossref PubMed Scopus (115) Google Scholar). An upstream primer complementary to pGEX-2T in the region of the BstBI restriction site (5′-GCTGAAAATGTTCGAAGATCGTTTATGTCA-3′) and a downstream primer containing both a BamHI restriction site and a sequence coding for the TEV protease recognition motif (5′-CAGGGATCCTTGGAAATAGACTGGTTCATCCGATTTTGGAGGATGGTC-3′) were used in PCR reactions using pGEX-2T as template. BstBI- andBamHI-cleaved PCR products were subsequently ligated into pGEX-2T to give the pGEX-2T-TEV plasmid. Into this expression plasmid DNA coding the 11-amino acid fragment of PML was inserted as described previously (7Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Human wild type histone deacetylase 4 (wt-HDAC4) in pCDNA3.1 (a gift from T. Kouzarides, University of Cambridge (25Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (465) Google Scholar)), which encodes HDAC4 with an N-terminal Myc epitope tag linked to a 6-histidine (His6) peptide, was used as template with the internal primers 5′-GGCGTGCAGGTGAGGCAGGAGCCCATT-3′ and 5′-AATGGGCTCCTGCCTCACCTGCACGCC-3′ with the TransformerTMsite-directed mutagenesis kit (CLONTECH) as directed by the manufacturer. This generated the K559R-HDAC4 mutant DNA construct in pCDNA3.1. All DNA constructs were verified by automated DNA sequencing on an ABI PRISMTM 377 DNA Sequencer (St. Andrews University DNA Sequencing Unit). 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For analysis of SUMO-1/-2-modified His6-HDAC4, 75-cm2 flasks of subconfluent 293 cells were transfected with 12 μg of total plasmid DNA as indicated in the figures. Lysates, protein purification, and Western blots were prepared as indicated. His6-HDAC4 proteins were purified from transfected cells by guanidine cell lysis and nickel-nitrilotriacetic acid-Sepharose (Qiagen) purification as described previously (7Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Proteins eluted from the nickel-nitrilotriacetic acid-Sepharose were fractionated by electrophoresis in 8% polyacrylamide gels containing SDS before anti-HA Western blotting as described above. GST-SAE2/SAE1 fusion protein was expressed inEscherichia coli B834 and purified by affinity chromatography using glutathione-Sepharose as described previously (26Jaffray E. Wood K.M. Hay R.T. Mol. Cell. Biol. 1995; 15: 2166-2172Crossref PubMed Google Scholar). The GST fusion protein was cleaved with thrombin and dialyzed to remove glutathione, and the SAE1/2 was purified by covalent affinity chromatography on a column of SUMO-1 linked to agarose as described previously (2Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Expression of both proteins was confirmed by Western blotting, and the activity of recombinant protein was tested by thioester assay using 125I-SUMO-1 and Ubc9 (data not shown). Recombinant Ubc9 was expressed and purified as detailed previously (22Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar). All SUMO proteins were expressed in E. coli strain B834, extracted, and purified as described previously (26Jaffray E. Wood K.M. Hay R.T. Mol. Cell. Biol. 1995; 15: 2166-2172Crossref PubMed Google Scholar). GST-SUMO proteins were bound to glutathione-Sepharose beads and either eluted with buffer containing 10 mm glutathione or cleaved by 17 units·ml−1 thrombin while bound. Eluted GST-SUMO proteins were dialyzed against 50 mm Tris (pH 7.5), 1 mm dithiothreitol (DTT) and concentrated using 30-kDa molecular mass cut-off microconcentrators to 10 mg·ml−1 before storage at −70 °C. Thrombin-cleaved SUMO proteins were dialyzed against 20 mm ammonium bicarbonate (pH 8.2), 1 mm DTT. Protein samples were lyophilized and taken up in 50 mm Tris/HCl (pH 7.5), 1 mm DTT at a concentration of 10 mg·ml−1. All cleaved SUMO protein masses were verified by MALDI-TOF mass spectrometry on a Micromass TofSpec 2E mass spectrometer (Micromass, Manchester, UK; University of St. Andrews Mass Spectrometry Service) before use in the assays. The GST-TEV-PML protein was expressed and extracted as described for the GST fusion SUMO proteins. The resultant GST-TEV-PML protein (referred to herein as GST-PML) solution was dialyzed against 50 mm Tris/HCl (pH 7.5), 1 mm DTT and concentrated to 4 mg·ml−1 using 10-kDa molecular mass cut-off microconcentrators. All recombinant protein concentrations were determined using both the Bradford method (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar) or calculated ε280 extinction coefficients for absorbance measurements at 280 nm. wt-SUMO-GG proteins were labeled by incorporation of [35S]methionine/cysteine during isopropyl-1-thio-β-d-galactopyranoside induction of bacterial cultures as described above in the presence of 35.75 mCi/liter [35S]methionine/cysteine (Amersham Pharmacia Biotech). Expressed proteins were then purified as outlined above for the unlabeled proteins. In vitrotranscription/translation of proteins was performed using 1 μg of plasmid DNA and a wheat germ-coupled transcription/translation system according to the instructions provided by the manufacturer (Promega). [35S]Methionine (Amersham Pharmacia Biotech) was used in the reactions to generate radiolabeled proteins. C52A-SUMO-1, GST-SUMO-2-FL, and GST-PML were radiolabeled with 125I using the chloramine-T method as described previously for wt-SUMO-1 (22Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar) except that the labeled protein was dialyzed against 50 mm Tris (pH 7.5), 1 mm DTT instead of passage over a P2-acrylamide gel column. Noncompetitive SUMO conjugation assays were performed in 10-μl volumes containing between 1.4 and 10 μg (as indicated) of either unlabeled or35S/125I-labeled SUMO proteins, an ATP-regenerating system, and buffer (50 mm Tris (pH 7.5), 5 mm MgCl2, 2 mm ATP, 10 mm creatine phosphate, 3.5 units·ml−1creatine kinase) and 0.6 units·ml−1 inorganic pyrophosphatase in either the absence or presence of 1 μl of [35S]methionine-labeled substrate (HDAC4 or promyelocytic leukemia protein) or varying concentrations of GST, GST-PML, GST-SUMO-1-FL, GST-SUMO-2-FL, or GST-SUMO-3-FL as indicated in the figure legends. Unless otherwise stated, assays contained 120 ng (1.1 pmol) of purified recombinant SAE1/SAE2 and 650 ng (35.9 pmol) of Ubc9. Reactions were incubated at 37 °C for between 45 min and 4 h as described. After termination with SDS sample buffer containing β-mercaptoethanol, reaction products were fractionated by electrophoresis in polyacrylamide gels (8–10%) containing SDS, stained, destained, and dried before analysis by phosphorimaging. Competition assays were performed in 10-μl reaction volumes with limiting Ubc9 (0.55 pmol/10 ng), 5 mm ATP, 5 mm MgCl2, 50 mm Tris (pH 7.5), 120 ng (1.1 pmol) of purified recombinant SAE1/SAE2, 4 μg (143 pmol) of GST-PML, and 0.5 μm125I-C52A-SUMO-1 with either no competitor or one of seven 1:1 serial dilutions of wt-SUMO-1 (from 142 to 2.22 μm), K11R-SUMO-2 (from 80 to 1.25 μm), or K11R-SUMO-3 (from 73 to 1.14 μm). Reactions were incubated for 4 h at 37 °C before termination with SDS sample buffer containing β-mercaptoethanol followed by electrophoresis in a 10% polyacrylamide gel containing SDS. Gels were stained and destained before drying and exposure to a phosphorimaging screen for 10 min. The mutant proteins C52A-SUMO-1 and K11R-SUMO-2/-3 were used to avoid experimental interference from SUMO-1-SUMO-1 disulfide linked dimers and the formation of poly(SUMO-2) and poly(SUMO-3). After a 3-h incubation a portion (10 μl) of the SUMO-2 conjugation assays carried out either in the absence of substrate or in the presence of GST-PML was dialyzed against 50 mm ammonium bicarbonate on a 0.025-μm VS membrane disc (Millipore, Bedford, MA). This procedure not only exchanged the buffer but also removed ATP thus stopping the reaction. 0.5 μl of trypsin (bovine, sequencing grade, Roche Diagnostics, 1 μg·μl−1) was added to the dialyzed sample, and the digestion was allowed to proceed overnight at 37 °C. The tryptic digest was then analyzed by MALDI-TOF mass spectrometry. 0.5 μl of tryptic digest was applied to the target along with 0.5 μl of 0.1% trifluoroacetic acid to acidify the sample and 0.5 μl of α-cyano-4-hydroxycinnamic acid matrix solution (10 mg·ml−1 in 75% acetonitrile, 2.5% formic acid) and allowed to dry. The sample was analyzed using a TofSpec 2E mass spectrometer (Micromass) in reflectron mode. To facilitate the biochemical analysis of SUMO-1 conjugation an in vitro system has been developed that contains recombinant, bacterially produced components. The assay contains SAE1/SAE2, Ubc9, SUMO-1, and, as substrate, GST fused to an 11-amino acid sequence containing the SUMO modification site located between amino acids 485–495 of PML (7Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Each of the components was analyzed in a polyacrylamide gel containing SDS and was highly purified as judged by Coomassie Blue staining (Fig.1 A). SUMO-1 was labeled with35S by the addition of [35S]methionine/cysteine to the bacterial growth medium. In comparison with previous assay methods that used cell extract sources of SAE1/2 and in vitro translated protein substrates, the GST-PML fusion was efficiently utilized as a substrate for modification with 35S-labeled SUMO-1 in this system (Fig. 1 B). The specificity of the reaction was assessed by comparing modification of GST with that of the GST-PML fusion and quantitation of the 35S-labeled products by phosphorimaging. Although GST contains 21 lysine residues, none of them conform to the SUMO modification consensus ψKXE, and modification of GST was less than 1% of that observed for the GST-PML fusion (Fig. 1 C). The extent of modification was such that after fractionation by polyacrylamide gel electrophoresis the reaction products could be easily monitored by Coomassie Blue staining. Under these conditions the product of the reaction was GST-PML bearing a single SUMO-1 modification with no evidence for the formation of SUMO-1 multimers (Fig. 1, D and E). Inspection of the SUMO-1, SUMO-2, and SUMO-3 sequences revealed that a consensus SUMO modification site (ψKXE) is present in the N-terminal regions of SUMO-2 and SUMO-3 but is absent in the sequence of SUMO-1 (Fig.2 A). This raises the possibility that SUMO-2 and SUMO-3 could be used as substrates for SUMO modification and thus form poly(SUMO) chains. SUMO-1, SUMO-2, and SUMO-3 were therefore expressed and purified as both the full-length pro-protein precursors and the shorter active forms (exposing the C-terminal diglycine motif). GST fusion and thrombin-cleaved versions of each protein were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (data not shown). The thrombin-cleaved proteins were essentially homogeneous, and their mass, which was determined by mass spectrometry, corresponded to that predicted from the sequence. To test the possibility that SUMO-2 and SUMO-3 could be conjugated to themselves the purified proteins were incubated in an assay containing purified SAE1/SAE2 and Ubc9. Analysis of the reaction products by polyacrylamide gel electrophoresis and Coomassie Blue staining revealed that in the presence of all the assay components a large number of more slowly migrating species are observed that are consistent with the formation of poly(SUMO-2) (Fig. 2 B) and poly(SUMO-3) (Fig. 2 C) chains. In each case the formation of these products was dependent on the presence of SAE1/SAE2 and Ubc9. To compare the ability of SUMO-2 and SUMO-3 to act as acceptors for SUMO modification with that of SUMO-1 and GST-PML, GST fusions of the unprocessed forms (which cannot be conjugated to protein substrates) of SUMO-1-(1–101) (SUMO-1-FL), SUMO-2-(1–103) (SUMO-2-FL), and SUMO-3-(1–95) (SUMO-3-FL) and GST-PML were added in increasing amounts to a modification reaction containing 125I-labeled SUMO-1 and limiting amounts of Ubc9. Incorporation of SUMO-1 into conjugated product was determined and plotted as a function of added substrate (Fig. 2 D). GST-SUMO-2-FL was relatively efficiently utilized as a substrate (0.9 pmol of product formed in the presence of 100 pmol of substrate) albeit less efficiently than GST-PML (1.6 pmol of product formed in the presence of 100 pmol of substrate), whereas GST-SUMO-3-FL was not a good substrate for SUMO-1 modification (0.2 pmol of product formed in the presence of 100 pmol of substrate). GST-SUMO-1-FL modification was at background levels (less than 0.1 pmol of product formed in the presence of 100 pmol of substrate). Although the accumulation of slowly migrating species was consistent with the formation of poly(SUMO-2) chains, it was important to establish that this was indeed the case and to demonstrate that these chains could be linked to substrate proteins. In vitro reactions containing SAE1/SAE2 and Ubc9 were therefore set up in the additional presence of GST-PML, SUMO-2, or GST-PML and SUMO-2. Analysis of the reaction products by polyacrylamide gel electrophoresis and Coomassie Blue staining revealed that in the presence of SUMO-2, dimers and trimers of SUMO-2 were present (Fig. 3 A). In the presence of GST-PML, but with no added SUMO-2, no additional products were generated. When GST-PML and SUMO-2 were incubated in the conjugation assay a series of more slowly migrating products were generated that are consistent with the formation of poly(SUMO-2) chains linked to GST-PML (Fig. 3 A). To establish the identity of lysine residues involved in the formation of poly(SUMO-2) chains and attachment to the GST-PML substrate, samples from the reaction products analyzed in Fig. 3 A were digested with trypsin and analyzed by MALDI-TOF mass spectrometry. Trypsin digestion of the assay mixture, containing SUMO-2 but no substrate, gave signals corresponding to the expected peptides produced by cleavage at the carboxyl side of lysine and arginine residues (Fig. 3 B). In particular a signal was detected at 3873.5 Da that corresponds to aa 59–92 of SUMO-2. This peptide contains Arg60, which under these conditions appeared to be resistant to trypsin cleavage with only a very small portion of the completely digested product (3570.7 Da) being detected. Additionally a pair of signals were detected at 5048.9 and 5352.1 Da. These signals correspond to digestion fragments in which aa 61–92 of SUMO-2 are covalently linked by an isopeptide bond to aa 8–20 of SUMO-2 and aa 59–92 are linked by an isopeptide bond to aa 8–20. Hence, these sign
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