Key points are not available for this paper at this time.
SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence “ΨKXE,” where Ψ is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates. SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence “ΨKXE,” where Ψ is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates. small ubiquitin-like modifier-1 SUMO-1 consensus sequence SUMO-1 conjugating enzyme Ran GTPase activating protein 1 glutathione S-transferase polyacrylamide gel electrophoresis promyelocytic leukemia protein ubiquitin activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase. SUMO-11 is a member of a family of ubiquitin-like proteins that are post-translationally conjugated to other proteins (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). The specific effects of SUMO-1 modification appear to be substrate dependent, but they are clearly distinct from the effects of ubiquitination in mediating protein degradation. In a number of cases, SUMO-1 modification regulates the subcellular localization of specific substrates. For example, SUMO-1 modification targets RanGAP1 from the cytoplasm to the nuclear pore complex (2Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (239) Google Scholar, 3Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (381) Google Scholar) and PML from the nucleoplasm to PML nuclear bodies (4Muller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (581) Google Scholar). SUMO-1 modification of certain other substrates may play a role in antagonizing ubiquitin-mediated proteolysis. IκBα and MDM2, for example, are both modified by SUMO-1 on lysine residues that also function as sites for ubiquitination (5Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, 6Buschmann T. Fuchs S.Y. Lee C.G. Pan Z.Q. Ronai Z. Cell. 2000; 101: 753-762Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). SUMO-1 modification of these lysines has been proposed to stabilize the substrates by blocking ubiquitin modification. For a growing list of other substrates, the exact effects of SUMO-1 modification remain to be determined. A majority of these substrates, including p53 (7Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 8Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (438) Google Scholar, 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 (561) Google Scholar), c-Jun (7Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), and topoisomerases I and II (10Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar) are nuclear proteins that function in regulating transcription or chromatin structure. Immunofluorescence analysis and cell fractionation studies further indicate that the majority of proteins modified by SUMO-1 are nuclear and that they correspond to only a small subfraction of all cellular proteins (12Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar). The specific subfraction of proteins modified by SUMO-1 also varies throughout the cell cycle (13Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (608) Google Scholar), and possibly in response to cellular growth conditions, indicating that SUMO-1 modification and de-modification are dynamic processes. However, the precise mechanisms involved in substrate selection and in regulating the timing of modification or demodification are poorly defined. Many steps involved in SUMO-1 modification parallel those involved in ubiquitination. Like ubiquitin, SUMO-1 is synthesized as a precursor that is proteolytically processed to generate the mature, active polypeptide (13Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (608) Google Scholar, 14Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar). Once processed, SUMO-1 is activated in an ATP-dependent reaction that creates a thioester intermediate between the active-site cysteine of the SUMO-1 activating enzyme (E1) and the carboxyl terminus of SUMO-1. The SUMO-1 E1 enzyme, a heterodimer consisting of Aos1 and Uba2, is structurally and functionally related to the ubiquitin E1 enzyme (14Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar, 15Gong L. Li B. Millas S. Yeh E.T. FEBS Lett. 1999; 448: 185-189Crossref PubMed Scopus (132) Google Scholar, 16Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (184) Google Scholar). Following activation, SUMO-1 is transferred from the E1 enzyme to Ubc9, a protein similar in structure and function to ubiquitin E2 enzymes (17Gong L. Kamitani T. Fujise K. Caskey L.S. Yeh E.T. J. Biol. Chem. 1997; 272: 28198-28201Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 19Lee G.W. Melchior F. Matunis M.J. Mahajan R. Tian Q. Anderson P. J. Biol. Chem. 1998; 273: 6503-6507Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 20Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (304) Google Scholar, 21Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). How SUMO-1 is subsequently transferred from Ubc9 to specific protein substrates is the most poorly defined step in the SUMO-1 conjugation pathway. Although SUMO-1 modification must be quite specific by virtue of the limited number of cellular proteins that are modified, there is very little, if any, homology among the currently known substrates. Probably all ubiquitination reactions involve E3 ligases, factors that mediate the transfer of ubiquitin from E2 enzymes to specific protein substrates (22Yamao F. J. Biochem. ( Tokyo ). 1999; 125: 223-229Crossref PubMed Scopus (41) Google Scholar, 23Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). In general, E3s function in substrate recognition and are responsible for the high degree of specificity that is characteristic of most ubiquitination reactions. Currently no E3-like factors have been identified for SUMO-1 conjugation, so how specific proteins are recognized as substrates for SUMO-1 conjugation remains unknown. Relative to ubiquitin-specific E2 enzymes, an unusual feature of Ubc9 is that it interacts directly with many SUMO-1 substrates (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). These interactions suggest that Ubc9 may play a direct role in recognizing SUMO-1 substrates, as well as in modifying them. In this study, we demonstrate that Ubc9 binds to the SUMO-1 consensus sequence (SUMO-1-CS), a motif of conserved residues surrounding the modified lysine of many SUMO-1 substrates. We further demonstrate that the binding of Ubc9 to the SUMO-1-CS is essential for SUMO-1 modification. Expression vectors coding for wild-type mouse RanGAP1, CΔ23, NΔ419/PK, and NΔ502/PK were constructed as previously described (3Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (381) Google Scholar). Site-directed mutagenesis was performed using the GeneEditor Mutatgenesis System (Promega Corp., Madison, WI) and mutations were verified by DNA sequencing. A cDNA coding for human Ubc9 was obtained from a fetal liver cDNA library using the polymerase chain reaction and cloned into the GST expression vector, pGEX-5X-1 (Amersham Pharmacia Biotech, Piscataway, NJ). The carboxyl-terminal domain of mouse RanGAP1 (NΔ419: amino acids 420–589) was similarly amplified from a cDNA clone by polymerase chain reaction and subcloned into pGEX-5X-1. In vitro transcription and translation of RanGAP1 and RanGAP1-pyruvate kinase fusion proteins were performed in rabbit reticulocyte lysate in the presence of 35Smethionine as described by the manufacturer (Promega Corp., Madison, WI). GST-NΔ419 and GST-Ubc9 fusion proteins were expressed in bacteria, purified by affinity chromatography on glutathione-Sepharose beads, and cleaved from the beads by Factor Xa as outlined by the manufacturer (Amersham Pharmacia Biotech). Gel filtration analysis of the carboxyl-terminal domain of RanGAP1 and Ubc9 was performed on a Amersham Pharmacia Biotech Superdex 75 chromatography column. The column was equilibrated and proteins were fractionated with buffer containing 110 mm potassium acetate, 2 mm magnesium acetate, 20 mm HEPES (pH 7.3), and 1 mm dithiothreitol. 20μ of each protein was loaded either individually or together following mixing and incubation for 30 min at room temperature. 0.5-ml fractions were collected, trichloroacetic acid precipitated, and analyzed by SDS-PAGE. GST-Ubc9, or GST alone, was bound to 20 μ l of glutathione-Sepharose beads (1 mg of protein/ml beads) in phosphate-buffered saline containing 1 mmdithiothreitol. Nonspecific protein-binding sites were blocked by incubation with 2% bovine serum albumin for 60 min at 4 °C. An equivalent amount (radioactive counts) of each in vitrotranslated protein was incubated with the beads in 100 μ l of binding buffer (20 mm Tris (pH 7.5), 150 mm NaCl, 0.1% for 30 min at room temperature. were with binding buffer by of the bound proteins with was analyzed by or by in a The Ubc9 binding for each protein to that of wild-type was from the of GST-Ubc9 bound of bound to GST studies using SUMO-1 as a were performed as described that proteins were incubated with Ubc9 in the presence of the of SUMO-1. of the of modified and proteins bound to Ubc9 was by analysis of the proteins by SDS-PAGE. The carboxyl-terminal domain of RanGAP1 is modified by SUMO-1 at lysine residue (3Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (381) Google Scholar, R. T. Gerace L. Melchior F. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We have previously that modification at this is on a acid domain of RanGAP1 from residue to the carboxyl terminus (3Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (381) Google Scholar) in The of this domain to SUMO-1 modification was by in vitro translation in rabbit reticulocyte where SUMO-1 modification is by and Ubc9 (3Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (381) Google Scholar) 1 acid domain the carboxyl terminus of RanGAP1 Ubc9 RanGAP1 the carboxyl-terminal and the kinase fusion proteins and were and in rabbit reticulocyte in the presence of 35Smethionine and incubated with GST-Ubc9 or proteins were with buffer and analyzed by by The amount of protein loaded in is equivalent to the amount of protein in each binding are on the and proteins. RanGAP1 and kinase fusion protein translation all appear as following by possibly to of translation at sites or to A of the proteins modified by SUMO-1 are known to interact directly with the SUMO-1 conjugating enzyme, Ubc9 (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). Although RanGAP1 is of the SUMO-1 substrates, interactions with Ubc9 have been analyzed the of interactions, we for the of Ubc9 to to RanGAP1 and also to the and proteins in GST-Ubc9 GST as a was on glutathione-Sepharose beads and incubated with proteins by translation in rabbit reticulocyte proteins were with buffer and analyzed by SDS-PAGE. RanGAP1 with Ubc9 as both of these proteins are also modified by SUMO-1. both and RanGAP1 and with Ubc9. In and NΔ502/PK interact with Ubc9. 4 and these proteins also to be modified by SUMO-1. These indicate a between the of these proteins to interact with Ubc9 and to be modified by SUMO-1. The binding reactions described were in the presence of rabbit reticulocyte it that the between Ubc9 and RanGAP1 was Ubc9 and the carboxyl-terminal domain of RanGAP1 a complex in the of other we analyzed the proteins by gel filtration chromatography either or together mixing and at room temperature. expressed Ubc9 and were purified and individually fractionated by gel filtration chromatography on a Superdex 75 column (Amersham Pharmacia Biotech). these conditions, both proteins as with of 20 Ubc9 and were incubated together and subsequently fractionated on the column they as an heterodimer with a of This that Ubc9 and the carboxyl-terminal domain of RanGAP1 interact directly to a complex and that the complex is to by gel filtration This also that Ubc9 to RanGAP1 to a with SUMO-1. The precise lysine residues modified by SUMO-1 have been identified in a known substrates (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar). The majority of these modification sites to a consensus sequence that we to as the SUMO-1 consensus or The SUMO-1-CS is defined by amino acids with the sequence “ΨKXE,” were Ψ is a large hydrophobic amino acid, K is the lysine residue modified by X is any amino acid, and E is glutamic acid The of residues surrounding the modified lysine that they may be for substrate recognition for the selection of the specific lysine residue for SUMO-1 modification. all of the substrates containing the SUMO-1-CS also interact with Ubc9, that the SUMO-1-CS may play a role in Ubc9 this we residues surrounding lysine of RanGAP1, including the conserved residues to the consensus sequence were analyzed for to be modified by SUMO-1 following translation in rabbit reticulocyte lysate and by of the wild-type protein was to the in rabbit reticulocyte lysate lysine to or SUMO-1 modification and Mutating to a but on SUMO-1 modification for lysine no on SUMO-1 modification of residues and and and glutamic acid SUMO-1 modification. These demonstrate that conserved residues surrounding the SUMO-1 modification are for SUMO-1 residues of the SUMO-1-CS are essential for SUMO-1 modification. in the SUMO-1-CS were into the RanGAP1 kinase fusion protein and proteins were for to be modified by SUMO-1 by transcription and translation in rabbit reticulocyte were analyzed by by amino acid surrounding the SUMO-1-CS of analysis of modification for wild-type and proteins containing mutations in and the SUMO-1-CS are on the and on the we a between the of proteins to interact with Ubc9 and to be modified by SUMO-1. We for mutations of the conserved residues surrounding the SUMO-1 modification of RanGAP1 Ubc9 GST-Ubc9 was on glutathione beads and incubated with proteins vitro proteins were with buffer and analyzed by or by using a mutations were to the with to to lysine to and glutamic acid to of these mutations the with Ubc9 to of that with the wild-type of and lysine to only on Ubc9 the by and Mutating the modification lysine to no on Ubc9 possibly to the of this amino acid These demonstrate a between Ubc9 binding and SUMO-1 proteins that to interact with Ubc9 were modified by SUMO-1 the of those that Ubc9 were modified by SUMO-1. These further demonstrate that conserved residues surrounding the SUMO-1 modification are essential for Ubc9 the role of the SUMO-1-CS the in mediating Ubc9 we that SUMO-1 modified RanGAP1 bound to Ubc9 as well and possibly SUMO-1 conjugated to RanGAP1, to Ubc9 we for the binding of to Ubc9 in the presence of of SUMO-1. we that SUMO-1 a on the binding of RanGAP1 to Ubc9, but only a on the binding of RanGAP1 We an in the binding of RanGAP1 binding were performed in the presence of SUMO-1 to an 1 and In the the binding of RanGAP1 was by 1 and These findings indicate that Ubc9 interacts with SUMO-1 modified RanGAP1 possibly of direct interactions with SUMO-1 modification protein by protein-protein interactions, protein-DNA interactions, protein subcellular and possibly by directly protein is that SUMO-1 modification is with substrates recognized and modified in response to specific cellular for example, have that proteins with in the cell cycle (13Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (608) Google Scholar) and in response to cellular growth (10Mao Y. Desai S.D. Liu L.F. J. Biol. Chem. 2000; 275: 26066-26073Abstract Full Text Full Text PDF PubMed Scopus (123) Google H. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the mechanisms by which proteins are recognized as substrates for SUMO-1 modification or de-modification and how mechanisms be are We have to how proteins are recognized as substrates for SUMO-1 modification and that direct with the E2 enzyme, Ubc9, is an important of this a domain from a well SUMO-1 RanGAP1, we have that SUMO-1 modification with the to directly interact with Ubc9. This domain of RanGAP1 a consensus the which is in all known SUMO-1 substrates. The SUMO-1-CS the lysine residue to which SUMO-1 is covalently and conserved residues that this Although recognized by other E. Desterro J.M. V. K. E. The H. Hay R.T. J. Cell Sci. 1999; PubMed Google Scholar, M.S. Hay R.T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF Scopus Google Scholar, T. K. B. H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the of the SUMO-1-CS was previously role in SUMO-1 we of the conserved residues in the These mutations were to both Ubc9 binding and SUMO-1 modification. These findings indicate that the SUMO-1-CS a direct role in mediating the binding of Ubc9 to SUMO-1 substrates and that this binding is essential for substrate modification. The lysine in the SUMO-1-CS of RanGAP1 is important for the with Ubc9, as an at this Ubc9 In to this it was also that RanGAP1 and RanGAP1 interact well with Ubc9. using that modified RanGAP1 interacts with Ubc9 the SUMO-1 remains to be the binding of modified RanGAP1 to Ubc9 is interactions with SUMO-1 or a of interactions with RanGAP1 and SUMO-1. studies have that Ubc9 and SUMO-1 interactions Q. Z. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). be to other substrates interact similarly with Ubc9, or this is specific for modified In Ubc9 is at the nuclear at sites that with the localization of RanGAP1 G.W. Melchior F. Matunis M.J. Mahajan R. Tian Q. Anderson P. J. Biol. Chem. 1998; 273: 6503-6507Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Although Ubc9 has been to interact with the H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), it remains to be this is direct or by In to a role for the SUMO-1-CS in Ubc9 we also that amino acids on either of the consensus sequence were for Ubc9 binding and SUMO-1 modification. of the amino acid sequence of this domain with other SUMO-1 substrates no of the remains to be residues of the SUMO-1-CS are directly involved in Ubc9 or they binding by the and of a domain containing the that the precise of the SUMO-1 a protein be an important determinant of to function in SUMO-1 modification is by analysis of the transcription SUMO-1-CS but is modified at a lysine residue in only of these M. M. and K. J. Biol. Chem. in Scholar). The precise of the SUMO-1-CS a protein is to to interact with Ubc9. The presence of a SUMO-1-CS in a protein is an of that protein be a substrate for SUMO-1 modification. analysis of the amino acid residues surrounding the SUMO-1-CS and to Ubc9 binding be to the of a SUMO-1 In this study, we the SUMO-1-CS of RanGAP1, but we that the SUMO-1-CS of other SUMO-1 substrates function similarly to mediate Ubc9 binding and SUMO-1 modification. have been in a large number of known and SUMO-1 substrates. Many of these SUMO-1-CS including the in T. T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), R. S. S. Z. G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), B. P. P. 1997; PubMed Scopus Google Scholar), V. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), p53 Z. 1996; PubMed Scopus Google Scholar), c-Jun M. S. V. E. M. P. 1996; PubMed Scopus Google Scholar), M. S. V. E. M. P. 1996; PubMed Scopus Google Scholar), and polymerase M. J. G. ( 1997; PubMed Scopus Google Scholar). in to the SUMO-1-CS may also mediate Ubc9 Ubc9 also interacts with the domain of PML E. Desterro J.M. V. K. E. The H. Hay R.T. J. Cell Sci. 1999; PubMed Google Scholar). The of this remains as mutations that interactions between Ubc9 and the PML domain have very little on the SUMO-1 modification of PML E. Desterro J.M. V. K. E. The H. Hay R.T. J. Cell Sci. 1999; PubMed Google T. K. H. T. Yeh E.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). has also been proposed that may play a role in regulating the of Ubc9 with at SUMO-1 substrates (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar, Y. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). Although are known for in ubiquitin-mediated M. Biol. Google Scholar), they are in of the known SUMO-1 substrates. The binding of Ubc9 to the SUMO-1-CS a direct for E3 in SUMO-1 modification. However, it is that the modification of substrates with a SUMO-1-CS may be or by the of E3-like factors in of this a analysis of the direct interactions between Ubc9 and the SUMO-1-CS important into the mechanisms involved in the transfer of SUMO-1 to specific substrates. studies of ubiquitin L. E. G. S. J.M. 1999; PubMed Scopus (445) Google Scholar, N. P. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the exact that the transfer of ubiquitin from factors to protein substrates remains poorly defined. the high degree of homology between Ubc9 and ubiquitin E2 enzymes, it is that the transfer of SUMO-1 to protein substrates be very similar to the of ubiquitination. of the interactions between Ubc9 and the SUMO-1-CS important into the mechanisms of both ubiquitination and SUMO-1 modification. also be important to further the role of amino acids surrounding the to they function to an consensus or they are also involved in interactions with Ubc9. We and for and the of this
Sampson et al. (Fri,) studied this question.