Global Analyses of Sumoylated Proteins in Saccharomyces cerevisiae

Abstract

We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells. We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells. Protein ubiquitination is well known to be involved in protein degradation through the proteasome and intracellular protein sorting (1Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Google Scholar, 3Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Google Scholar). A family of ubiquitin-like proteins has also been identified to be attached to substrate proteins through enzymatic processes similar to that of ubiquitination (4Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 5Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 6Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Google Scholar). However, the function of these ubiquitin-like proteins is unclear. Among them, SUMO, 1The abbreviations used are: SUMO, small ubiquitin-like modifier; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin-derived epitope; DTT, dithiothreitol; Ni-NTA, nickel-nitrilotriacetic acid. a small ubiquitin-like modifier, is the most widely studied to date. In mammals, SUMO was first identified to be conjugated to substrate protein such as Ran-GAP1 (7Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Google Scholar, 8Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Google Scholar). The Smt3 gene, in the budding yeast Saccharomyces cerevisiae, is essential for cell viability and is the only budding yeast homolog of mammalian SUMO. Smt3 was originally identified as a suppressor of Mif2 mutation, and its function in the nucleus has been suggested (9Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Google Scholar). With an approach similar to that used in the identification of ubiquitination enzymes, the enzymes of protein sumoylation in budding yeast were identified (10Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Google Scholar, 11Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Google Scholar). In the yeast S. cerevisiae, Smt3 is first proteolytically processed to expose its Gly-Gly repeat at the C terminus by a SUMO-specific protease, Ulp1 (12Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Google Scholar). In an ATP-dependent reaction, the C terminus of mature Smt3 forms a thioester bond with the cysteine residue at the active site of the E1 enzyme, which is a heterodimeric Aos1/Uba2 complex (10Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Google Scholar). Through a trans-esterification reaction, Smt3 is transferred to the SUMO-conjugating E2 enzyme Ubc9, and the C terminus of Smt3 forms a thioester bond with the active site cysteine residue of Ubc9 (11Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Google Scholar). In the presence or absence of E3 SUMO ligases, the C terminus of Smt3 is then covalently attached to lysine residue of substrate proteins via formation of a stable isopeptide bond. In yeast, two SUMO E3 ligases were identified and are known as Siz1 and Siz2 (13Takahashi Y. Kahyo T. Toh-E A. Yasuda H. Kikuchi Y. J. Biol. Chem. 2001; 276: 48973-48977Google Scholar, 14Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Google Scholar). No other E3 enzyme for Smt3 in the budding yeast has been identified. A number of SUMO substrate proteins have also been identified in yeast, and these include the septin components, PCNA/Pol30 and Top2 (15Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google Scholar, 16Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar, 17Bachant J. Alcasabas A. Blat Y. Kleckner N. Elledge S.J. Mol. Cell. 2002; 9: 1169-1182Google Scholar). However, the function of protein sumoylation remains unclear, and questions remain about whether sumoylation plays a broad role in regulating cellular functions like the ubiquitination pathway. In mammals, a larger number of sumoylated proteins have been identified, and most are involved in nuclear processes such as gene transcription (18Sapetschnig A. Rischitor G. Braun H. Doll A. Schergaut M. Melchior F. Suske G. EMBO J. 2002; 21: 5206-5215Google Scholar, 19Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Google Scholar, 20Girdwood D. Bumpass D. Vaughan O.A. Thain A. Anderson L.A. Snowden A.W. Garcia-Wilson E. Perkins N.D. Hay R.T. Mol. Cell. 2003; 11: 1043-1054Google Scholar). Yeast is a very useful model organism, because it allows both facile genetic and biochemical studies. Yet only a limited number of sumoylated proteins in yeast have been found so far (15Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google Scholar, 16Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar, 17Bachant J. Alcasabas A. Blat Y. Kleckner N. Elledge S.J. Mol. Cell. 2002; 9: 1169-1182Google Scholar). Identification of new sumoylated proteins in yeast would immediately reveal the cellular processes in which protein sumoylation plays a role. In addition, it could lead to new target proteins in which the function of sumoylation could be studied in detail. With these goals, we undertook a global analysis of sumoylated proteins in yeast cells under various cell growth conditions. This has revealed the autosumoylation of the enzymes involved in protein sumoylation. Furthermore, the newly identified sumoylation substrates have revealed many diverse cellular processes in which sumoylation plays a role. Materials—Expression plasmid pYES2/NT was from Invitrogen. Plasmid pFA6a-3HA-kanMX6 was obtained from Mark Longtine (21Longtine M.S. Yeast. 1998; 14: 953-961Google Scholar). Yeast was grown in YPD (1% yeast extract, 2% peptone, and 2% dextrose) or in synthetic minimal defined medium with appropriate sugar at 30 °C. Ni+-NTA resin was from Qiagen; anti-FLAG antibody and conjugated-agarose were from Sigma. Anti-HA antibody and anti-HA affinity matrix were from Roche Applied Science. Protease inhibitor mixture for purification of histidine-tagged proteins was from Sigma. Plasmid Construction—The S. cerevisiae SMT3 gene was obtained by PCR using yeast genomic DNA as template, then cloned into the BamHI and XhoI sites of pYES2/NT plasmid. The Xpress epitope in pYES2/NT was mutated to the FLAG epitope, generating pYES-6HisFLAG-SMT3 plasmid, which contains Smt3 with an N-terminal extension MGGSHHHHHHGMASMTGGQQMGRDYKDDDDKVPGS. The pYES-2HA-SMT3 plasmid was created by replacing the 6HisFLAG tag in the pYES-6HisFLAG-SMT3 plasmid with two copies of HA epitope (amino acid sequence: MGYPYDVPDYAGSYPYDVPDYAGVPGS). To make pFA6a-6HisFLAG-KanMX6 plasmid for C-terminal tagging of genes of interest, the 3HA tag in pFA6a-3HA-kanMX6 plasmid was replaced by the 6HisFLAG tag (amino acid sequence: GGSHHHHHHGMASMTGGQQMGRDYKDDDDKVPGSSVVE), yielding the pFA6a-6HisFLAG-kanMX6 plasmid. DNA sequences in these plasmids were verified by sequencing. Genetic Methods Used in the Yeast S. cerevisiae—To tag the SMT3 gene with 6HisFLAG at the N terminus chromosomal locus, the DNA sequence of 6HisFLAG-SMT3 from the above pYES-6HisFLAG-SMT3 plasmid was subcloned into the pFA6a-3HA-kanMX6 plasmid, generating the pFA6a-6HisFLAG-SMT3-kanMX6 plasmid. The chromosomal SMT3 was then replaced by 6HisFLAG-SMT3 through PCR-based homologous recombination. The forward primer sequence was 5′-GGACAGAAGGACCCAGTTCAGTTCTAGTTTTACAAATAAATACACGAGCGatggggggttctcatcatcat, and the reverse primer sequence was 5′-TGGGGGGAAGGGAGAGGTTTGTTGGCGTTTTTTAGGCATTGTTAAGAGTCgcagatgatgtcgaggcg. Uppercase letters in the primers correspond to 50 bp immediately upstream and downstream of SMT3 in the chromosome, respectively. Using PCR-based homologous recombination and pFA6a-6HisFLAG-KanMX6 as template, we tagged a few of the candidate sumoylated proteins at their C termini by using the 6HisFLAG epitope. The yeast SOD1 gene was cloned into the pFA6a-kanMX6 plasmid, resulting in pFA6a-Sod1-kanMX6 plasmid from which mutation of a specific lysine residue of Sod1 was carried out. The chromosomal SOD1 was then replaced with mutant SOD1 through homologous recombination. Correct integration of the epitope tag in each case was verified by DNA sequencing. All the yeast strains and their relevant genotypes are listed in Table I.Table IThe yeast S. cerevisiae strains used in this studyNameGenotypeSourceBY4741MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0ATCCHZY1017MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 6HisFLAG-smt3::kanMX6This studyHZY1205MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 elp5-6HisFLAG::kanMX6This studyHZY1206MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 sod1-6HisFLAG::kanMX6This studyHZY1207MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pol30-6HisFLAG::kanMX6This study Open table in a new tab Purification of Sumoylated Proteins in Yeast—Two liters of HZY1017 cells were grown in YPD and harvested at log phase (A600 ∼ 1) by centrifugation. Collected cells were broken by glass beads using a beads-beater in denatured lysis buffer (100 mm NaH2PO4, 10 mm Tris, pH 8.0, 0.1% SDS, 8 m urea, and protease inhibitor mix). Cell debris was removed by centrifugation at 30,000 × g for 30 min. The resulting was with of Ni+-NTA resin for and then a The resin was first by denatured buffer (100 mm NaH2PO4, 10 mm 8 m urea, pH then by buffer mm NaH2PO4, mm mm pH Proteins were by using 10 of buffer mm NaH2PO4, mm mm pH and with of anti-FLAG for at the beads were by then by to the sumoylated proteins were with then Purification of Sumoylated liters of with pYES-2HA-SMT3 plasmid, were grown in with ∼ was to a of cells were grown for an The cells were harvested then broken by glass beads in lysis buffer mm NaH2PO4, mm 10 mm 0.1% pH Cell debris was removed by centrifugation at 30,000 × g for 30 min. The resulting was with of Ni+-NTA resin for at then with buffer mm NaH2PO4, mm mm pH Sod1 was by of buffer mm NaH2PO4, mm mm pH The of the was to by an then with 50 of anti-HA affinity matrix Applied for at the anti-HA affinity matrix was with Sumoylated Sod1 was by the resin two with 50 of 0.1% The of the was to by to the SDS, then with 50 of anti-FLAG for purification as The sumoylated Sod1 was by acid and Protein were in buffer mm DTT, for then a the proteins were transferred a The was by mm Tris, pH mm and with anti-FLAG or anti-HA antibody for at °C. The was then with 0.1% and with a of the for The was and then with the was used in the In was and the was in protein was to or was in m urea, and of 50 mm pH 8.0, was to the to was then to the for was to a A. F. M. P. A. H. M. Sci. S. A. 1996; Scholar). were by tandem mass a mass A × with the resin was and the was a originally by the for was used in analysis J. H. R. Nat. 2001; Scholar). Identification of Sumoylated Proteins in Yeast in purification of sumoylated proteins in yeast, we first replaced the chromosomal SMT3 gene by N-terminal yielding the that replacing the SMT3 gene by 6HisFLAG-SMT3 protein sumoylation in yeast cells. Smt3 is essential for cell We in cell growth cells and HZY1017 cells. J. J. and H. In addition, it is well known that the C terminus of Smt3 is processed and covalently attached to the lysine of substrate N-terminal Smt3 in a plasmid has been widely used for analysis of protein and its to substrate proteins to be (11Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Google Scholar, E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google Scholar). as in the sumoylation in HZY1017 cells found by using antibody (12Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Google Scholar, 14Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Google Scholar). it that the sumoylation in the cell is by protein The sumoylated proteins from the HZY1017 cells were by tandem affinity purification under as in the The results are in The sumoylated proteins in are (15Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google Scholar). to purification and lead to of their J. J. and H. in to in the anti-FLAG were from the and to The were from the and by mass spectrometry. from Smt3 were found in each In addition, a number of sumoylated protein were identified Table of Smt3 to a substrate protein is to to its mass by at of the of a candidate SUMO substrate to its an whether it is a sumoylated this and the that Smt3 were found in of these of these candidate proteins are to be sumoylated proteins identified in yeast cells grown in mass identified Ubc9 and Sod1 and and and and and and and and and and and and Open table in a new tab of of and whether of these candidate proteins in Table could be verified by other mass spectrometry. is of a that with the of Mol. Cell. Biol. 2001; 21: Scholar). To as a sumoylated the C-terminal cells were with plasmid. was by Ni+-NTA resin under and anti-FLAG The was by anti-FLAG the sumoylated was by anti-HA The sumoylated in the as in which was originally identified by mass spectrometry. The in by anti-FLAG to with the sumoylated revealed by anti-HA in that the sumoylation of is We also under in which and of the complex were only was found to be To the sumoylation of Sod1 in yeast, C-terminal Sod1 was from cells with in the Sod1 was by an to the of sumoylated Sod1 could be the protein was by anti-HA in the sumoylated Sod1 was at a that to the in as well as in Sod1 is sumoylated in yeast under these growth conditions. To the sumoylation of we the sumoylated Sod1 by of affinity purification as of the protein in that the sumoylated Sod1 was with The of the sumoylated Sod1 was by anti-HA Furthermore, from only Sod1 and Smt3 were found in the to the sumoylated Sod1 by mass spectrometry. J. J. and H. Additionally, two sumoylated from Sod1 were identified by tandem mass analysis of a sumoylated protein sumoylated an lysine by a from the C terminus of Smt3 with the sequence via an isopeptide bond. The mass the lysine residue to sumoylation is which was used in tandem mass mutation of these sumoylation sites revealed that Sod1 was at site by Smt3 it is many lysine of could be are to reveal the function of the sumoylation of Sod1 in identified by tandem mass sequence identified by site is a sequence to a and an or is the site is a sequence to a and an or is the site is a sequence to a and an or is the site is a sequence to a and an or is the site is a sequence to a and an or is the The site is a sequence to a and an or is the (15Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google The Open table in a new tab enzymes that are known to sumoylation in yeast, including the heterodimeric E1 enzyme of and and the E2 enzyme Ubc9, were found to be sumoylated Table Ubc9 was found to be M.S. J. Biol. Chem. 2002; Scholar). The sumoylation of E1 and E2 enzymes are to thioester formation E1 and E2 enzymes with because are by mm under conditions. known sumoylation substrate was also identified in this Additionally, and and were identified, with metabolic that sumoylation is to protein in the and Identification of Sumoylated Proteins in Yeast under whether protein sumoylation is by various cellular including oxidative stress and in growth We found that the sumoylation was under these as in is a of sumoylation in yeast cells in the presence of mm or many proteins cells were with the of a of protein sumoylation was We to the sumoylated proteins from cells with hydrogen and and the proteins were by in were from the analysis in the in was into The proteins from each were identified, and the results are in Table was found in that is a of sumoylation of the other proteins identified in the their most of these proteins have their that are These sumoylated protein to be involved in many diverse cellular as in Table of the sumoylated proteins from HZY1017 cells hydrogen and ethanol were from the and to and mass The the of the proteins identified in yeast cells under oxidative and ethanol identified in cells identified in ethanol-treated cells and and and and Ubc9 Ubc9 and Sod1 and and and and and and and and and and and and and and and and and and and and and and Siz2 and Siz2 and and Siz1 and Siz2 and Siz1 and and Siz1 and and and Siz1 Open table in a new tab Table of sumoylated proteins identified in yeast cells under Ubc9, and of the and and and and and and and Open table in a new tab the known enzymes involved in the SUMO were found to be Table only were E1 and E2 enzymes identified, Siz1 and the known E3 enzymes for SUMO were also identified in this to be a that the enzymes involved in the SUMO are auto-sumoylated. a protein be by both ubiquitin and SUMO C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar, M.S. Hay R.T. Mol. Cell. 1998; 2: ubiquitin was identified in these sumoylated proteins that are Siz1 was identified in from the of Table ethanol-treated that Siz1 is A number of known sumoylated proteins were identified include and was identified in both and Table that was by Smt3 an known C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar). two sumoylated of were identified by tandem mass analysis as in Table and correspond to the two known sumoylation sites in Yeast was to be sumoylated cells were with the C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar). We whether the sumoylation of is by the cellular stresses used C-terminal cells were with pYES-2HA-SMT3 plasmid, and the tagged was in the sumoylated could be cells are to oxidative and ethanol it is in cells stress These results show that sumoylation of has a role in stress in to its role in the DNA A large number of sumoylated proteins were identified and Among them, a was identified with Smt3 from cells with hydrogen The mass of is about with by sumoylation of was found in cells grown in YPD or ethanol-treated cells by mass Table The function of Smt3 in chromosome was (9Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Google Scholar). a protein involved in the function J. Sci. S. A. 1999; was identified in to from to that is of oxidative because it was only found in cells with hydrogen in cells grown in YPD or ethanol-treated cells. sumoylation substrates in mammalian cells were found to be involved in gene transcription J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Google Scholar). with this of the of nuclear proteins found to be sumoylated are involved in gene transcription Table identification of the role of sumoylation in these The sumoylated in Table were their and Smt3 with the is to the mass of substrate protein by at it is that many of the proteins in Table are sumoylated proteins identified in of the Furthermore, we to sumoylated by tandem mass Identification of sumoylated from protein would it as an sumoylated Through both and of the sumoylated we identified sumoylated including both known (15Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google and sumoylation sites Table In this we undertook a global analysis of sumoylated proteins by mass using the budding yeast S. cerevisiae as a model In to the identification of known sumoylated proteins in yeast, a large number of new sumoylated substrates was identified. stresses such as oxidative stress and ethanol stress to a in the of protein sumoylation in yeast cells. This in protein sumoylation to be a from a protein; it to be from a large number of including and is to that the sumoylation in mammalian cells and also of sumoylation to cellular stresses H. J. J. Biol. Chem. 2000; Scholar, J. J. S.J. J. Biol. Chem. 2003; Scholar). The of sumoylation under cellular stresses could be to a number of including in the enzymatic of sumoylation and The role of sumoylation in stress and the be of for We identified known sumoylation enzymes in yeast by this mass approach and found of E1, E2, and E3 enzymes to be a of the sumoylation enzyme This be that ubiquitination enzymes are also known to be Biochem. J. Scholar). Furthermore, of the E3 enzymes, Siz1 and was identified ethanol in the cells. of E3 enzymes with the in protein sumoylation in cells. whether and the of Siz1 and Siz2 are under cellular stresses remain we identified a large number of sumoylated proteins involved in many cellular Among them, the sumoylation of was in in to the DNA C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar, P. Nature. 2003; Scholar). We that the sumoylation of is by both oxidative and ethanol the sumoylation of be involved in the stress is an enzyme that that to and of Sod1 in are known to be involved in of T. D. P. A. D. J. Nature. Scholar). is to that Sod1 in yeast is by with an Sod1 and are involved in cells oxidative and sumoylation of these enzymes to their functions in yeast cells would be in sumoylation of a of the in cells was found Y. Sci. S. A. 2003; Scholar). we identified in yeast as a sumoylated substrate Table is known to be by a number of the of lysine including and T. Science. 2001; Scholar). The of sumoylation to the complex of of is involved in function J. Sci. S. A. 1999; Scholar). we found it to be in cells under oxidative stress Table of the regulation of sumoylation of could reveal the role of sumoylation in chromosome segregation, a function of sumoylation (9Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Google Scholar). with the function of Smt3 in the the of proteins found to be sumoylated substrates are involved in regulation of DNA replication, gene transcription, and protein Table is to that proteins were also found in Table that are include and Identification of sumoylation sites of a sumoylated substrate is the first analysis of the function of sumoylation. sumoylated of a protein be found by mass the mass approach is and could reveal new sumoylation sites from the sumoylation sequence we identified a number of sumoylated using tandem mass analysis Table The sumoylation sites to the were found to be Table However, sumoylation of with acid sequences were also Proteins such as and Sod1 have both and sumoylation The of sumoylation sites questions about whether the used lysine to mutation the sumoylation would be J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Google Scholar, E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Google Scholar, 16Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar). many sumoylated proteins to be lysine and each lysine residue could be by a number of other including and is that mutation analysis has been the function of protein sumoylation would of a biochemical and cell for specific The identification of a large number of sumoylated substrates such in the that protein sumoylation is and is involved in a of cellular is involved in gene transcription, protein translation, chromosome segregation, DNA and stress and so The role of for has been from protein degradation and the proteasome to intracellular and nuclear functions L. Nat. Rev. Mol. Cell. Biol. 2001; 2: Scholar). is so that protein sumoylation also has many diverse analysis of the sumoylated proteins identified in would be and to the function of protein sumoylation in these cellular Additionally, of the enzymatic cascade of sumoylation is autosumoylation of E3 enzymes to with the of sumoylation in cells. We for of the and for with yeast genetic and and and Mark Hochstrasser for and