SUMO-1 Conjugation in Vivo Requires Both a Consensus Modification Motif and Nuclear Targeting

Abstract

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the ΨKXE motif, where Ψ represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IκBα, containing the ΨKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivorequires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation. SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the ΨKXE motif, where Ψ represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IκBα, containing the ΨKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivorequires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation. small ubiquitin-like modifier 1 RanGTPase-activating protein promyelocytic leukemia protein PML oncogenic domain inhibitor of (nuclear factor) κBα SUMO-1-activating enzyme nuclear localization signal pyruvate kinase hemagglutinin simian virus type 5 SUMO-11 is a small ubiquitin-related modifier (also known as sentrin, GMP1, UBL1, PIC1, or SMT3 in yeast) that has been found covalently conjugated to various cellular proteins (for reviews see Refs. 1Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 2Saitoh H. Pu R.T. Dasso M. Trends Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 3Hodges M. Tissot C. Freemont P.S. Curr. Biol. 1998; 8: R749-R752Abstract Full Text Full Text PDF PubMed Google Scholar). Several substrates for SUMO-1 have been reported: the RanGTPase-activating protein (RanGAP1) (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (955) Google Scholar) and Ran-binding protein 2 (6Saitoh 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 (160) Google Scholar) implicated in nucleocytoplasmic trafficking; the promyelocytic leukemia protein (PML) and Sp100 (7Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (289) Google Scholar) found in subnuclear structures known as PML oncogenic domains or PODs; the IκBα inhibitor of the transcription factor nuclear factor κB, implicated in the control of immune and inflammatory responses (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar); and the tumor suppressor protein p53 (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). Consequently, “SUMOylation” plays a role in multiple vital cellular processes such as oncogenesis, cell cycle control, apoptosis, and response to virus infection. SUMO-1 is conjugated to a target protein by a pathway that is distinct from but analogous to ubiquitin conjugation. Like ubiquitin, SUMO-1 is proteolytically processed to expose its mature C terminus by recently described SUMO-1-specific proteases variously called Ulp1 and Ulp2 in yeast (11Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar, 12Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar) or SENP1 and SUSP-1 in vertebrates (13Suzuki T. Ichiyama A. Saitoh H. Kawakami T. Omata M. Chung C.H. Kimura M. Shimbara N. Tanaka K. J. Biol. Chem. 1999; 274: 31131-31134Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14Gong L. Millas S. Maul G.G. Yeh E.T. J. Biol. Chem. 2000; 275: 3355-3359Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 15Kim K.I. Baek S.H. Jeon Y.J. Nishimori S. Suzuki T. Uchida S. Shimbara N. Saitoh H. Tanaka K. Chung C.H. J. Biol. Chem. 2000; 275: 14102-14106Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Ulp1, Ulp2, and SENP1, but not SUSP-1, are capable of both deconjugating SUMO-1 from modified proteins and removing four amino acids from the C terminus of the 101-amino acid SUMO-1 precursor to generate the mature 97-amino acid form. SUMO-1 addition is accomplished by a thioester cascade, with SUMO-1 first being activated by a heterodimeric SUMO-1-activating enzyme (SAE) that adenylates the C-terminal glycine of SUMO-1 (16Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (442) Google Scholar, 17Desterro J.M.P. 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, 18Gong L. Li B. Millas S. Yeh E.T. FEBS Lett. 1999; 448: 185-189Crossref PubMed Scopus (131) Google Scholar, 19Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar) before catalyzing the formation of a thioester bond between the C terminus of SUMO-1 and a cysteine residue in SAE. In a transesterification reaction SUMO-1 is transferred from the SAE to the SUMO-1-conjugating enzyme Ubc9, which catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ε-amino group of a lysine residue of the target protein (6Saitoh 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 (160) Google Scholar, 20Desterro J.M.P. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar, 21Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 22Schwarz S.E. Matuschewski K. Liakopoulos D. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (189) Google Scholar, 23Lee 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 (119) Google Scholar). Ubc9 is required for cell cycle progression in yeast (24Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar). Unlike ubiquitin conjugation, SUMO-1 modification of target proteins in vitrois not dependent on the equivalent of an E3 protein ligase (17Desterro J.M.P. 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,19Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar). Here, we demonstrate that a short sequence containing the consensus ΨKXE, where Ψ represents a large hydrophobic amino acid, constitutes a transferable signal that confers the ability to be modified with SUMO-1 on proteins to which it is linked. The predominantly nuclear localization of both subunits of the SAE, Ubc9 and SUMO-1, suggest that SUMOylation is a nuclear process. We demonstrate that heterologous proteins carrying the SUMO-1 consensus modification sequences present in IκBα and p53 are only conjugated to SUMO-1 in vivo when a nuclear localization signal (NLS) is also present. These data suggest that protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation and allow us to propose that this modification may be involved in regulating multiple processes in the nucleus. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected by electroporation as described previously (25Arenzana-Seisdedos F. Turpin P. Rodriguez M. Thomas D. Hay R.T. Virelizier J.L. Dargemont C. J. Cell Sci. 1997; 110: 369-378Crossref PubMed Google Scholar). For immunofluorescence analysis 2 μg of plasmid were transfected in 1 × 106 HeLa cells. For nickel bead purification, 10 μg of each plasmid DNA encoding pyruvate kinase (PK) fusions and His6-SUMO-1 were transfected in 1 × 107 HeLa cells. To increase efficiency of protein expression, no DNA carrier was used in cotransfections. After transfection, cells were seeded in 75 cm2 flasks. One-twentieth of transfected cells were seeded in a separated plate (to control protein input), and incubation was continued for 24 h. Plasmids encoding His6-SUMO-1, HA-SUMO-1, SV5-SAE1, HA-SAE2, and SV5-Ubc9 were reported previously (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) 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 (559) Google Scholar, 17Desterro J.M.P. 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). pcDNA3 plasmids encoding Myc-tagged PK and NLS-PK were described previously (26Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (621) Google Scholar). cDNA encoding the 1–26 fragment of IκBα was obtained by polymerase chain reaction using as template IκBα wild type and IκBαK21R,K22R-encoding plasmids (27Rodriguez M.S. Wright J. Thompson J. Thomas D. Baleux F. Virelizier J.L. Hay R.T. Arenzana-Seisdedos F. Oncogene. 1996; 12: 2425-2435PubMed Google Scholar) to generate PK-IκBα-(1–26) and PK-IκBα-(1–26)-KR. cDNAs encoding 361–393 and 361–393 KR of p53 were subcloned from previously described constructs (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). Polymerase chain reaction fragments and synthetic oligonuclotides encoding the 381–391 fragment of the fragment of IκBα, the fragment of the fragment of type 2 and the fragment of as as to were and of the PK The cDNAs were subcloned of the NLS-PK that a to generate NLS-PK immunofluorescence analysis was as described previously M.S. Thompson J. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). R. of and were for by a incubation with were in were on a with a In and SUMO-1 conjugation were as reported (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). Cells were in of for analysis (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). His6-SUMO-1 were as described (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). Proteins were by in containing transferred to by and processed for as reported previously (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). was obtained from R. E. was from was used to has been previously reported that Ubc9 the nuclear localization of a protein (24Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar). Ubc9 has been predominantly in the nucleus of cells R. N. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). To the of the of the SUMO-1 conjugation we transfected HeLa cells with of SUMO-1 and and of Ubc9 and SUMO-1 nuclear and in nuclear structures S. P. Cell Biol. 2000; 2: PubMed Scopus Google Scholar) 1 Ubc9 was in the nucleus but also in the of transfected cells 1 when cells were with Ubc9 was the nuclear not and 23Lee 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 (119) Google Scholar). The and and subunits of the SAE an nuclear that was not when both subunits were or when cells were by not Thus, the nuclear localization of enzymes implicated in the SUMO-1 modification pathway that this ubiquitin-like modification may in the nucleus. To the that SUMO-1 modification in the we an in which a SUMO-1 modification to a heterologous is located in the nucleus or the by of the presence or of an were such that be for SUMO-1 conjugation in vitro, where is no of or in vivo in the nucleus or the of the sequence of SUMO-1 conjugation in multiple proteins that a short ΨKXE represents the of SUMO-1 modification (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) 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 (559) Google Scholar, E. Desterro J.M. V. K. E. The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; PubMed Google Scholar, E.S. Blobel G. J. Cell Biol. 1999; PubMed Scopus Google Scholar, T. Jensen K. B. Will H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). To define the sequence required for conjugation with SUMO-1, we a of constructs containing IκBα and p53 C-terminal modification to the C terminus of a Myc-tagged of PK or an equivalent containing the PK and NLS-PK fusions by in transcription and were for SUMO-1 conjugation in the previously described (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). PK and NLS-PK to amino acids 1–26 and of IκBα or amino acids 361–393 and 381–391 of p53 were conjugated with SUMO-1, PK or NLS-PK were not and lysine of IκBα and of p53 were to SUMO-1-modified of and fusions were not that SUMO-1 was conjugated to the previously described lysine (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) 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 (559) Google Scholar, 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). To the acid sequence required for conjugation with SUMO-1 be a of synthetic that the amino amino amino and amino amino acid sequences was to the C terminus of PK to generate the constructs 2 C and SUMO-1 conjugation was with PK encoding and amino the efficiency of conjugation was but with only 5 amino acids 2 Thus, SUMO-1 modification a of amino the efficiency of conjugation. In SUMO-1 is found in with target and as such the of SUMO-1 is (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). cellular substrates reported to be conjugated with SUMO-1 a nuclear and between the nucleus and the F. Thompson J. Rodriguez M.S. Bachelerie F. Thomas D. Hay R.T. Mol. Cell. Biol. 1995; PubMed Google Scholar, M.J. Blobel G. J. Cell Biol. 1998; PubMed Scopus Google or are to the nuclear and Ran-binding protein (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (955) Google Scholar, 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 (160) Google Scholar). Cell analysis that of proteins have a nuclear and data not To SUMO-1 conjugation nuclear the and constructs were with and constructs for SUMO-1 The ability of the NLS-PK constructs to be conjugated with SUMO-1 in is to the PK and PK constructs were in the and NLS-PK constructs were in the nucleus of transfected cells To of PK and NLS-PK modified by SUMO-1 in constructs proteins were HeLa cells with an plasmid for His6-SUMO-1 (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). proteins were on nickel and proteins were by with a the and as as and are conjugated with SUMO-1 and the KR were not In to NLS-PK PK were not These that nuclear localization is required for conjugation of proteins with a short SUMO-1 modification when transferred to a heterologous the efficiency of SUMO-1 modification be a of the of the and the of the in the To the of a of SUMO-1 modification we additional PK constructs encoding amino acids of PML E. Desterro J.M. V. K. E. The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; PubMed Google Scholar, T. K. T. Yeh J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and amino acids of the protein and R. T. which the lysine residue that is the of SUMO-1 conjugation and were used as substrates for SUMO-1 conjugation in of is with and being modified and being modified and being modified were also in vivo when NLS-PK fusions encoding IκBα, and p53 sequences in where PK were modified or not modified not The in the conjugation of SUMO-1 to this of substrates that the sequence of the SUMO-1 modification motif, when from its the efficiency of SUMO-1 conjugation. To the role of each amino acid the ΨKXE in SUMO-1 conjugation, a of PK constructs encoding in the sequence of p53 were (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). of residue not conjugation with SUMO-1 residue Ψ was to conjugation with SUMO-1 was of the and conjugation with SUMO-1 5 These that and are the of the consensus and suggest that the Ψ and may the efficiency of SUMO-1 conjugation. To this a of was in which the Ψ residue of the modified was to each of the hydrophobic and the efficiency of conjugation was in Whereas the to or the efficiency of SUMO-1 conjugation, to or the efficiency of conjugation. of with or not the efficiency of conjugation 5 To the role of amino acid, the residue this in was to or Whereas of with a small in of the this were described modification in proteins the sequence ΨKXE, it was of to a residue was required for The to was the of the and the was The is modified when with a containing the wild type 5 Thus, SUMO-1 modification is by a ΨKXE motif, where the of the hydrophobic amino acid the lysine residue has a on the efficiency with which SUMO-1 is conjugated to the target protein protein by protein or the ability to with or by localization of the modified with SUMO-1 has been to protein of a short amino acid sequence required for the of the to be conjugated with SUMO-1 to a heterologous protein that this is for by the SUMO-1 modification of the for SUMO-1 modification are and it is that is by The C-terminal of Ubc9, which is to be involved in to the and the of SUMO-1 to proteins Q. C. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acids in the consensus sequences are and Whereas the residue be be by to generate a is conjugated with SUMO-1 To the only reported modification containing in a protein is in the yeast it was not this was for modification E.S. Blobel G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). In addition to the and the large hydrophobic residue to the efficiency of SUMO-1 transferable sequences from various protein substrates modified with SUMO-1 to be conjugated with The conjugated short sequence the sequence from the sequence that is modified the sequence from is of the cellular substrates for conjugation with SUMO-1 (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (955) Google that or may its efficiency of conjugation. to the C terminus of a heterologous the acid sequence containing the ΨKXE as an for SUMO-1 modification in when an amino acid is from each of this sequence to generate a acid sequence containing the ΨKXE motif, its is the transferable sequence we have amino additional to the ΨKXE that to is that the acid sequence is a the acid the sequences were to the C-terminal of a carrier protein such that the in the ΨKXE is the C terminus of the the ΨKXE to be by additional C-terminal amino acid for by the SUMO-1 modification factor is the is to the of the it is located in an it may be to the modification analysis of that sequences the domain containing the lysine are required for SUMO-1 modification in vitro, the of additional domains has to be M.J. Blobel G. J. Cell Biol. 1998; PubMed Scopus Google Scholar). additional for Ubc9 in the target protein may in of Ubc9 to the protein with a increase in the efficiency of conjugation. may be the with in which the is required for with Ubc9 in a yeast PML proteins containing that the are vivo but modified in E. Desterro J.M. V. K. E. The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; PubMed Google Scholar). Whereas SUMO-1 conjugation of and IκBα be with SAE and Ubc9 (17Desterro J.M.P. 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, 19Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google it is that additional protein may the efficiency of conjugation. The of the modification is is by the of the protein which a to the consensus for SUMO-1 modification and is not modified by SUMO-1 in T. of the of J. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) that the is not and not be to the modification The nuclear of SUMO-1, SAE, and Ubc9, as as the SUMO-1 modification of NLS-PK that SUMO-1 modification nuclear Ubc9 is the nuclear we SUMO-1 modification with SUMO-1 of substrates the nucleus or modification the nuclear the nucleus. has previously been that or of the nuclear localization of and Sp100 that in of proteins also in of SUMO-1 modification in vivo E. Desterro J.M. V. K. E. The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; PubMed Google Scholar, T. Jensen K. B. Will H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Blobel G. J. Cell Biol. 1998; PubMed Scopus Google Scholar). In a of for is located in the also to undergo SUMO-1 The is modified by SUMO-1 in E. Desterro J.M. V. K. E. The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; PubMed Google Scholar). Thus, substrates be targeted to the where be modified by the of and proteins may be subnuclear structures such as In SUMO-1 modification of the PML is required for both formation of the S. S. S. Freemont P.S. A. 2000; 95: PubMed Google Scholar) and for of proteins such as the D. N. T. Yeh E.T. Maul G.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). SUMO-1-modified proteins as in the of be targeted to the nuclear (4Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 5Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (955) Google Scholar) or between the nucleus and the IκBα and is as no data this is the for the SUMO-1-modified of proteins (8Desterro J.M.P. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (911) 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 (559) Google Scholar, 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). nuclear to be required for SUMO-1 modification of we the that proteins may be modified in cellular may be the for the and which are targeted to the cell and to be SUMO-1-modified F. L. C. S. R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). is that SAE and Ubc9 are to a containing and where modification is with the that and both with the of SAE and Ubc9 are in this this allow a small of the predominantly nuclear SAE and Ubc9 to in the with The recently described proteases Ulp1 and Ulp2 (11Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar) a of proteins in that of substrates be and by multiple SMT3 proteases with SUMO-1 is a factor for conjugation of of SUMO-1 may be a to protein of a protein and increase the of a conjugated when SUMO-1 is the cellular which this are not the cellular localization of SUMO-1-specific proteases has to be SUMO-1 modification of proteins to be by the of the to be targeted to the nucleus and by the of a SUMO-1 on the of the target is that SUMO-1 modification as an control that the of many nuclear We of for DNA We and for and