Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9

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. 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) to at a 1:5000 The wild type full-length full-length of the SUMO proteins are the pro-protein that contain C-terminal and 2 residues for SUMO-1, and are in vivo by specific to protein that in a diglycine of SUMO-2 and SUMO-3 were by reverse chain reaction using as in the to the using and and using and that for proteins at the diglycine for and for were by using a single for both with the as The sites the the of the by the enzymes and and both J.M. J. Hay R.T. FEBS Lett. 1997; PubMed Scopus Google Scholar) and that sequences for SUMO-2 and SUMO-3 from were with from a Matunis M.J. J. 2001; PubMed Scopus Google Scholar) that from by residues to the terminus 2 The SUMO-2 and SUMO-3 were by using the same single for the wild type with and with and the of for protein expression and for the which not form from the wild type protein using the and and the for the of the protein J.M. J. Hay R.T. FEBS Lett. 1997; PubMed Scopus Google Scholar). for expression of SAE2 by of SAE2 (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 and The with and for expression of by of (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 a site sites and The as a in to form the expression a a single SUMO modification to an acid sequence amino acids of PML M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). and the acid modification site were by a motif for the tobacco etch virus J.F. R.J. PubMed Scopus Google Scholar). to in the of the site and a both a site and a sequence for the motif were in using as were to the expression the acid of PML as M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). wild type histone deacetylase 4 in from T. of C. E. J. T. EMBO J. 1999; 18: PubMed Scopus Google which with an N-terminal to a as with the and with the as by the This the construct in were by on an cells were in modified with For of of cells were with of as indicated in the protein and Western were as proteins were from cells by and as M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). from the were by in Western as protein and by using as E. Hay R.T. Mol. Cell. Biol. PubMed Google Scholar). The protein with and to and the by on a of SUMO-1 to as (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). of both proteins by Western and the activity of protein by thioester using and Ubc9 not Ubc9 and as J.M. J. Hay R.T. FEBS Lett. 1997; PubMed Scopus Google Scholar). SUMO proteins were in E. and as E. Hay R.T. Mol. Cell. Biol. PubMed Google Scholar). proteins were to and with glutathione or by proteins were 1 dithiothreitol and using to at SUMO proteins were 1 were and in 1 at a of SUMO protein were by on a of use in the The protein and as for the SUMO The protein to as 1 and to 4 using protein were using both the Biochem. PubMed Scopus Google Scholar) or for at proteins were by of of as in the of (Amersham Pharmacia proteins were as for the In of proteins using 1 of and a to the by the (Amersham Pharmacia Biotech) in the to and were with using the as for J.M. J. Hay R.T. FEBS Lett. 1997; PubMed Scopus Google Scholar) that the protein 1 of a SUMO conjugation were in between and of SUMO proteins, an and 2 and in the or of 1 of or promyelocytic leukemia or of or as indicated in the of SAE1/SAE2 and of were at for between and 4 as with reaction were by in and by were in reaction with Ubc9 of 4 of and with no or of of to to or to were for 4 at with by in a were and and to a for The proteins and were to from SUMO-1-SUMO-1 and the formation of and a a of the SUMO-2 conjugation in the of or in the of on a This not the but also ATP thus the of 1 to the and the to at The by of to the target with of acid to the and of acid in acid) and to The using a in the of SUMO-1 conjugation an in vitro has been that The SUMO-1, as to an acid sequence the SUMO modification site between amino acids of PML M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). of the in a and as by SUMO-1 by the of to the In with that of and in vitro protein the utilized as a for modification with SUMO-1 in 1 The specificity of the reaction by modification of with that of the and of the by Although lysine of to the SUMO modification consensus ψKXE, and modification of than of that for the 1 The of modification that by the reaction be by these the of the reaction a single SUMO-1 modification with no evidence for the formation of SUMO-1 1, and Inspection of the SUMO-1, and SUMO-3 sequences revealed that a consensus SUMO modification site is present in the N-terminal regions of SUMO-2 and SUMO-3 but is in the sequence of SUMO-1 This the that SUMO-2 and SUMO-3 be as substrates for SUMO modification and thus form SUMO-1, and SUMO-3 were and as both the full-length pro-protein and the the C-terminal diglycine and of protein were by and not The proteins were and which by to that from the the that SUMO-2 and SUMO-3 be conjugated to the proteins were in an SAE1/SAE2 and of the reaction by and revealed that in the of all the a large of more species are that are with the formation of 2 and 2 In case the formation of these dependent on the of SAE1/SAE2 and the ability of SUMO-2 and SUMO-3 to as acceptors for SUMO modification with that of SUMO-1 and of the be conjugated to protein of and and were in to a modification reaction SUMO-1 and of of SUMO-1 conjugated and as a of 2 relatively utilized as a of in the of of than of in the of of not a for SUMO-1 modification of in the of of modification at levels than of in the of of Although the of species with the formation of it to that the case and to demonstrate that these chains be to In vitro SAE1/SAE2 and Ubc9 were in the of or and of the reaction by and revealed that in the of and of SUMO-2 were present In the of but with no no were and SUMO-2 were in the conjugation a of more were that are with the formation of chains to the identity of lysine residues involved in the formation of chains and to the from the reaction in were with and by of the SUMO-2 but no to the by at the of lysine and residues In a detected at that to of This which these to be to with a very small of the a of were detected at and to in which of SUMO-2 are covalently by an isopeptide bond to of SUMO-2 and are by an isopeptide bond to these the of SUMO-2 with a between the C-terminal and of the both SUMO-2 and with the indicated and a of to of SUMO-2 covalently to of via an isopeptide bond to the and of SUMO-2 to of small at to of SUMO-2 and of that it is in that the isopeptide bond and not the of lysine in the formation of chains these residues were to which the on the but be utilized for the formation of an isopeptide and were and in an to that of the wild type type and of SUMO were for the of chains in the of and the reaction were by in a by of SUMO-2 and SUMO-3 were with the wild type proteins, these were when the were with and in the wild type SUMO-2 and SUMO-3 to the formation of to SUMO and chains the same were with and the formation of to SUMO formation of to chains 4 modification by the SUMO in the of a full-length protein in PML with the wild type of SUMO-1, and SUMO-3 in the of SAE1/SAE2 and As PML modified at sites by SUMO-1 (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), but a more of more modified when the were with SUMO-2 and SUMO-3 4 the same were with and the more species were no and the to that for SUMO-1 Thus it is that chains of SUMO-2 and SUMO-3 are on protein substrates the formation of isopeptide between the C-terminal glycine residue of SUMO and lysine of of SUMO-2 or Although the in 1, 2, 3, and that SAE1/SAE2 and Ubc9 SUMO-1, and SUMO-3 for conjugation to protein it not the enzymes a form of a in with in the of SAE1/SAE2 and Ubc9 and a of of SUMO-1, and the to the formation of of SUMO-1 and the of SUMO-2 and which form were the reaction were by in a and the conjugated to by The of of SUMO-1, and SUMO-3 to the reaction in a decrease in the conjugated to of the indicated that form of SUMO for formation of Thus SAE1/SAE2 and Ubc9 SUMO-1, and SUMO-3 for conjugation to protein substrates with evidence that these enzymes have the ability to between the of evidence that formation of chains in vivo to use a SUMO that a single modification site and demonstrate that although SUMO-1 a single modified modified species be detected with a single site of modification at lysine and residue to an SUMO-1 vitro the modification of with SUMO in vivo with modified of were on and SUMO-1 modification detected by Western with an antibody to the single form of detected when both and were This form not detected when the of with of with and of the to by Western with an antibody revealed the of an more species of that is with the formation of SUMO-2 to a single site in This supported by of and HA-SUMO-2, which in of both species and by of and which the more that SUMO-2 chains be conjugated to protein substrates in vivo. almost the for the of very similar ubiquitin-like proteins, SUMO-1, and has been Although a of has to SUMO-1, on SUMO-2 and SUMO-3 have been for SUMO-1 and SUMO-2 have from using cells with SUMO-1 and SUMO-2/-3 in which of conjugated proteins are when by Western (19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). H. and R. T. In the species by modification with SUMO-2/-3 appear to be of a than from SUMO-1 Furthermore, it that in cells with SUMO-1 and SUMO-2 with proteins, the in PML nuclear although SUMO-1 is with the nuclear (19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). A. that although SUMO-1 and SUMO-2/-3 may share of the same the functional of modification by SUMO-1 and SUMO-2/-3 may be As has between ubiquitin and the SUMO proteins have revealed the of but functional it appears that SUMO be by (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, 19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, R. Matunis M.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). Although SUMO and ubiquitin are conjugated and by distinct the enzymes involved in of and functional E.T. L. Kamitani T. 2000; PubMed Scopus Google Scholar). is evidence to the that the E1 enzymes for both ubiquitin and the SUMO proteins share M. Cell Biol. 2000; 2: PubMed Scopus Google Scholar). has been that in contrast with family member SUMO-1, SUMO-2 and SUMO-3 share the to form polymeric chains with relatively family member and of SUMO-2 conjugation both in the and of a that via lysine which within a SUMO modification motif where ψ is a large hydrophobic is the target and is Furthermore, of lysine in SUMO-2 and SUMO-3 the of polymeric chains in or in the of or in vitro full-length PML This is also in vivo as the histone deacetylase protein that is modified by SUMO-1 at a single lysine residue is modified by SUMO-2 Although SUMO-2 and SUMO-3 are SUMO-2 appears to be a for chain formation than SUMO-3 2 Thus the acid in the N-terminal appears to be to conjugation of SUMO-3 in with Whether is to structures of the in or of specific amino acid with Ubc9 to be The cellular and significance of these polymeric of SUMO-2 and SUMO-3 are not is that in a analogous to the of proteins by complex proteins Trends Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), proteins may a more distinct to proteins that bind Furthermore, the that SUMO-1, and SUMO-3 form is not In is conjugated by all SUMO proteins in vitro 2 and 4 Thus it is that SUMO-1 may as a SUMO chain This is supported by the that SUMO-1 and SUMO-2 appear to in and although of the identified SUMO-1 substrates are of relatively the majority of SUMO-2 conjugated proteins in are of in (19Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (687) Google sequence between proteins of the SUMO family from different species that although the ψKXE sequence required for polymeric chain formation is present in the N-terminal it is not in all the of SUMO-1, and SUMO-3 conjugation we an in vitro that conjugates SUMO-1, or SUMO-3 a the acid of the targeted for SUMO-1 conjugation in PML The Ubc9 and and ATP or an with for and of using as revealed the specificity of the for the protein and that the consensus lysine as the target for modification. the for to and in vitro protein an is an for the and of the enzymes of the SUMO conjugation In with using and in these conjugation of of from 1, C and in a reaction of and of and thus reaction be by although represents a these relatively with a Ubc9 of in assays, 2 Thus it is that although SUMO conjugation in vitro in the of and may be required to the reaction in vivo. Ubc9 as a in the SUMO an of Ubc9 to the specificity of Ubc9 for SUMO-2/-3 in with SUMO-1. The that and to the same for conjugation to the that the cellular of SUMO-1 and SUMO-2/-3 are to upon conjugation in vivo. In to these the of SUMO with apparently for SUMO-1 and SUMO-2/-3 T. H. Yasuda H. J. Biochem. 2000; PubMed Scopus Google Scholar, L. Millas S. Maul G.G. Yeh E.T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) that the conjugation of and proteins may in be at the of than conjugation. we functional of SUMO-1 in with SUMO-2/-3, the of SUMO-2 and SUMO-3 a of to the SUMO conjugation Hay for with and for