The SUMO Isopeptidase Ulp2 Prevents Accumulation of SUMO Chains in Yeast
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m308357200
ISSN1083-351X
AutoresGwendolyn R. Bylebyl, Irina Belichenko, Erica S. Johnson,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2Δ, consistent with the possibility that some ulp2Δ phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae. The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2Δ, consistent with the possibility that some ulp2Δ phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae. SUMOs are ubiquitin-related proteins that function by being covalently attached to other proteins as post-translational modifications. SUMO conjugation is essential for viability of most eukaryotic cells and participates in many cellular processes including transcription, DNA repair, chromatin organization, nuclear transport, signal transduction, and the cell cycle (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (653) Google Scholar, 2Kim K.I. Baek S.H. Chung C.H. J. Cell Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar, 3Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). SUMO conjugation acts by different mechanisms on different substrates but does not directly target proteins for proteasome-dependent proteolysis. SUMO, like ubiquitin (Ub), 1The abbreviations used are: Ub, ubiquitin; E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO ligase; HU, hydroxyurea; TBZ, thiabendazole; HA, hemagglutinin; Ab, antibody; DTT, dithiothreitol; GST, glutathione S-transferase; NEM, N-ethylmaleimide; wt, wild-type; YPD, yeast extract/peptone/dextrose; VSV-G, vesicular stomatitis virus glycoprotein; trSiz1, truncated Siz1.1The abbreviations used are: Ub, ubiquitin; E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; E3, SUMO ligase; HU, hydroxyurea; TBZ, thiabendazole; HA, hemagglutinin; Ab, antibody; DTT, dithiothreitol; GST, glutathione S-transferase; NEM, N-ethylmaleimide; wt, wild-type; YPD, yeast extract/peptone/dextrose; VSV-G, vesicular stomatitis virus glycoprotein; trSiz1, truncated Siz1. is linked to its substrates via an amide bond between its C-terminal carboxyl group and the ϵ-amino group of a lysine residue in the substrate (1Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (653) Google Scholar, 2Kim K.I. Baek S.H. Chung C.H. J. Cell Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar, 3Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). SUMOs share only ∼18% sequence identity with Ub but contain a C-terminal domain with a Ub-fold that is virtually superimposable on the structure of Ub. SUMOs also contain a ∼20-residue non-Ub-related N-terminal extension. Saccharomyces cerevisiae contains a single SUMO protein encoded by the SMT3 gene, while mammals contain three different SUMOs: SUMO-2 and SUMO-3, which are 95% identical to each other, and SUMO-1. SUMO attachment is catalyzed by a three-step enzyme pathway, analogous to the Ub pathway, consisting of the heterodimeric SUMO-activating enzyme (E1) Uba2·Aos1, the SUMO-conjugating enzyme (E2) Ubc9, and several different SUMO ligases (E3s) (4Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (442) Google Scholar, 5Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar, 6Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 7Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (303) Google Scholar, 8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 9Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 10Takahashi Y. Kahyo T. Toh-e A. Yasuda H. Kikuchi Y. J. Biol. Chem. 2001; 276: 48973-48977Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 11Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 12Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). S. cerevisiae contains two known SUMO E3s, Siz1 and Siz2. Ubc9 and the E3s collaborate to confer substrate specificity on sumoylation: SUMO is often attached to the lysine in the short motif (I/V/L)KXE, and this motif is bound directly by Ubc9 (13Sampson D.A. Wang M. Matunis M.J. J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 14Bernier-Villamor V. Sampson D.A. Matunis M.J. Lima C.D. Cell. 2002; 108: 345-356Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar), while E3s bind other features of the substrate to enhance specificity. Sumoylation is a reversible modification, and a family of SUMO-specific isopeptidases, including the yeast proteins Ulp1 and Ulp2 (also called Smt4), removes SUMO from modified proteins. Ulp1 and Ulp2 have different subcellular localizations and are responsible for desumoylating different proteins (15Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar, 16Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar, 17Schwienhorst I. Johnson E.S. Dohmen R.J. Mol. Gen. Genet. 2000; 263: 771-786Crossref PubMed Scopus (107) Google Scholar). Ulp1 also cleaves a SUMO precursor to generate mature SUMO that can be conjugated. In S. cerevisiae many of the genes encoding SUMO pathway proteins are essential, including SMT3, UBA2, AOS1, UBC9, and ULP1, but it is not known what the essential function of SUMO conjugation is. SUMO is attached to many yeast proteins, but only five have been characterized to date: three members of the septin family of cytoskeletal proteins, the multifunctional replication processivity factor PCNA (proliferating cell nuclear antigen) and topoisomerase II (Top2) (18Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar, 19Takahashi Y. Iwase M. Konishi M. Tanaka M. Toh-e A. Kikuchi Y. Biochem. Biophys. Res. Commun. 1999; 259: 582-587Crossref PubMed Scopus (85) Google Scholar, 20Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1734) Google Scholar, 21Bachant J. Alcasabas A. Blat Y. Kleckner N. Elledge S.J. Mol. Cell. 2002; 9: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Mutants lacking the SUMO attachment sites on any of these proteins have quite subtle phenotypes, indicating that the substrate(s) involved in the essential function of SUMO remains to be discovered. The other SUMO pathway genes SIZ1, SIZ2, and ULP2 are not essential. ulp2Δ mutants grow poorly, are sensitive to a variety of stress conditions, and have defects in chromosome segregation and recovery from cell cycle checkpoint arrest (16Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar, 17Schwienhorst I. Johnson E.S. Dohmen R.J. Mol. Gen. Genet. 2000; 263: 771-786Crossref PubMed Scopus (107) Google Scholar, 22Strunnikov A.V. Aravind L. Koonin E.V. Genetics. 2001; 158: 95-107Crossref PubMed Google Scholar). Intriguingly some of the ulp2Δ phenotypes are suppressed by elimination of the major SUMO attachment sites in Top2, suggesting that excessive SUMO attachment to Top2 may cause some of the phenotypes of ulp2Δ (21Bachant J. Alcasabas A. Blat Y. Kleckner N. Elledge S.J. Mol. Cell. 2002; 9: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The siz1Δ and siz2Δ single mutants grow well and do not have obvious phenotypes, although they are each deficient for SUMO attachment to different classes of substrates (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 10Takahashi Y. Kahyo T. Toh-e A. Yasuda H. Kikuchi Y. J. Biol. Chem. 2001; 276: 48973-48977Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Strunnikov A.V. Aravind L. Koonin E.V. Genetics. 2001; 158: 95-107Crossref PubMed Google Scholar). The siz1Δ siz2Δ double mutant is also viable, although it is cold-sensitive, delays in the cell cycle at G2/M, and has very low levels of SUMO conjugates. Thus, Siz1 and Siz2 have an overlapping function probably involving promoting SUMO conjugation to the same substrate(s). A critical property of Ub is its ability to form poly-Ub chains, in which successive Ub moieties are linked to an internal lysine of the previous Ub in the chain. Ub chains can have branch sites at several different lysines, and chains with different branch sites have different functions (23Johnson E.S. Nat. Cell Biol. 2002; 4: E295-E298Crossref PubMed Scopus (41) Google Scholar). In contrast, most substrates of SUMO are modified with only one copy of SUMO per attachment site lysine, although a polypeptide may be multiply sumoylated by attachment of mono-SUMO at several sites (18Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar). However, it has been found recently that yeast SUMO and mammalian SUMO-2 and -3 readily form chains in in vitro reactions (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 24Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 25Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The major branch sites are lysines in the N-terminal non-Ub-like domain: Lys15 in yeast SUMO and Lys11 in SUMO-2/3 (24Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar, 25Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These lysines are in sequences that conform to the sumoylation consensus motif, although the yeast sequence contains a proline after the lysine, which is unusual. SUMO-1 does not have such a sequence but also forms chains in vitro with an as yet uncharacterized linkage (11Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). SUMO-2/3 also forms chains in cells, as the histone deacetylase HDAC4 is disumoylated in cells expressing wt SUMO-2 but monosumoylated if SUMO-2-K11R is expressed (24Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). There is also evidence that generation of amyloid β peptide from the amyloid precursor protein involves SUMO chain formation (26Li Y. Wang H. Wang S. Quon D. Liu Y.W. Cordell B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 259-264Crossref PubMed Scopus (128) Google Scholar). However, the function of SUMO chains, as well as their prevalence in SUMO-dependent processes, is unknown. To determine the physiological function of SUMO chain formation in yeast, we generated a series of yeast strains in which the genomic SUMO (SMT3) gene was replaced with mutant versions lacking combinations of its nine lysines. These experiments showed that SUMO chain formation is not a major feature of SUMO activity in yeast. However, the evidence suggests that SUMO chains do form but that their quantities are restricted by the activities of SUMO-specific isopeptidases. Media and Genetic Techniques—Standard techniques were used (27Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2000Google Scholar). Rich yeast medium containing 2% glucose (YPD) was prepared as described previously (28Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). Cells were arrested at G2/M in the cell cycle by incubating with 15 μg/ml nocodazole (Acros) for 3 h. Growth assays were performed by making 10-fold serial dilutions of logarithmically growing cells that had been normalized to A 600 1.0. A 2.5-μl sample of each dilution was spotted onto YPD plates or YPD plates supplemented with 0.1 m hydroxyurea (Acros); 10 mm caffeine (Acros); 1% dimethylformamide; or 75 μg/ml thiabendazole (Sigma), 1% dimethylformamide. Plasmids and Yeast Strain Construction—S. cerevisiae strains used are listed in Table I. All strains are derivatives of JD51 (29Dohmen R.J. Stappen R. McGrath J.P. Forrova H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The SUMO lysine mutant alleles (smt3-R11,15,19, smt3-R15, smt3-R27, smt3-R38,40,41, smt3-R54,58, and smt3-allR) were constructed using an overlapping PCR strategy described previously (18Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar) to produce PCR products containing 500 bp of the SMT3 5′-flank followed by SMT3 containing Lys to Arg mutations followed by the TRP1 marker and then 500 bp of the SMT3 3′-flank. These PCR products were transformed into JD51, Trp+ diploids were sporulated, and the tetrads were dissected. Mutant alleles were reamplified and sequenced to confirm that they did not contain any additional mutations. smt3-(K to R) ulp2Δ double mutants were made by crossing SUMO mutants to GBY6, a MATα strain containing the ulp2Δ allele from IS30 (17Schwienhorst I. Johnson E.S. Dohmen R.J. Mol. Gen. Genet. 2000; 263: 771-786Crossref PubMed Scopus (107) Google Scholar), followed by sporulation and dissection of the tetrads. TOP2-HA-tagged strains were made by transforming the smt3-(K to R) ulp2Δ double mutants with a PCR product containing 500 bp of the C-terminal coding sequence of TOP2 followed by the HA epitope tag, the HIS3 marker, and 500 bp of the TOP2 3′-flank.Table IS. cerevisiae strainsNameRelevant genotypeSourceJD51MAT a/MATα trp1-Δ1/trp1-Δ1 ura3-52/ura3-52 his3-Δ200/his3-Δ200 leu2-3,112/leu2-3,112 lys2-801/lys2-801Ref. 29Dohmen R.J. Stappen R. McGrath J.P. Forrova H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (165) Google ScholarJD52MAT a trp1-Δ1 ura3-52 his3-Δ200 leu2-3,112 lys2-801J. DohmenEJY332MAT a CDC3-HA::HIS3 CDC12-HF::TRP1Ref. 8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google ScholarGBY1MAT a smt3-R11,15,19::TRP1This studyGBY2MAT a smt3-R27::TRP1This studyGBY3MAT a smt3-R38,40,41::TRP1This studyGBY4MAT a smt3-R54,58::TRP1This studyGBY5MAT a smt3-allR::TRP1This studyIS30MAT a ulp2Δ::URA3Ref. 17Schwienhorst I. Johnson E.S. Dohmen R.J. Mol. Gen. Genet. 2000; 263: 771-786Crossref PubMed Scopus (107) Google ScholarGBY6MATα ulp2Δ::URA3This studyGBY7MAT a smt3-R11,15,19::TRP1 ulp2Δ::URA3This studyGBY8MAT a smt3-R15::TRP1 ulp2Δ::URA3This studyGBY9MAT a smt3-R27::TRP1 ulp2Δ::URA3This studyGBY10MAT a smt3-R38,40,41::TRP1 ulp2Δ::URA3This studyGBY11MAT a smt3-R54,58::TRP1 ulp2Δ::URA3This studyGBY12MAT a smt3-allR::TRP1 ulp2Δ::URA3This studyGBY13MAT a TOP2-HA::HIS3 ulp2Δ::URA3This studyGBY14MAT a TOP2-HA::HIS3 smt3-R11,15,19::TRP1 ulp2Δ::URA3This studyGBY15MAT a TOP2-HA::HIS3 smt3-R27::TRP1 ulp2Δ::URA3This studyGBY16MAT a TOP2-HA::HIS3 smt3-R38,40,41::TRP1 ulp2Δ::URA3This studyGBY17MAT a TOP2-HA::HIS3 smt3-R54,58::TRP1 ulp2Δ::URA3This studyGBY18MAT a TOP2-HA::HIS3 smt3-allR::TRP1 ulp2Δ::URA3This studyGBY19MAT a smt3-NΔ21::TRP1This studyGBY20MAT a smt3-NΔ21::TRP1 ulpΔ::URA3This study Open table in a new tab Plasmids for expressing His6-tagged Aos1, Uba2, Ubc9, and wt Smt3 and His6-FLAG-tagged full-length Siz1 (Siz1-HF) in Escherichia coli have been described previously (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). A truncated version of Siz1 (trSiz1), tagged with His8 and a vesicular stomatitis virus glycoprotein (VSV-G)-derived epitope tag, was made by ligating a PCR-amplified fragment of Siz1 encoding Met313–Arg508 and bearing the C-terminal extension YTDIEMNRLGKHHHHHHHH into pET21a. Plasmids for expressing His6-tagged Smt3 lysine mutants in E. coli were constructed by amplifying the SMT3 sequence from genomic DNA of the corresponding yeast mutants with primers to add the N-terminal extension MASMHHHHHH and to produce a stop codon after Gly98. Resulting PCR products were ligated into pET21a. All plasmids were sequenced to confirm that they did not contain additional mutations. Oligo sequences and construction details are available upon request. A pGEM-based plasmid for expressing GST-Ulp2 (16Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar) was a generous gift of Alaron Lewis and Mark Hochstrasser (Yale University, New Haven, CT). Antibodies and Immunoblot Analyses—Yeast whole cell lysates were prepared as described previously (30Yaffe M.P. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4819-4823Crossref PubMed Scopus (349) Google Scholar) followed by immunoblotting and chemiluminescent detection as described previously (18Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar). Antibodies used were a rabbit polyclonal Ab against Smt3 (18Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar), the 16B12 monoclonal Ab against the HA epitope (Covance), the M2 monoclonal Ab against the FLAG epitope (Kodak Scientific Imaging Systems), a rabbit polyclonal Ab against Cdc11 (Santa Cruz Biotechnology), and a goat polyclonal Ab against Ubc9 (yC-19) (Santa Cruz Biotechnology). Affinity Purification of Epitope-tagged Proteins—His6-SUMO, Ubc9-His6, and His6-Aos1/Uba2-His6 were expressed in E. coli and purified by nickel-nitrilotriacetic acid affinity chromatography as described previously (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). VSV-G-His8-trSiz1 and Siz1-HF were expressed as described for Siz1-HF (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Cells were extracted with 5 volumes of Y-PER (Pierce) supplemented with 1 mm phenylmethylsulfonyl fluoride. After centrifugation to remove cell debris the supernatant was diluted 10-fold with 50 mm NaPO4 (pH 8.0), 150 mm NaCl plus 0.1 mm ZnCl2 and 5 mm imidazole and bound in-batch for 2 h at 4 °C to HIS-Select HC nickel affinity gel (Sigma). Proteins were eluted with 200 mm imidazole, 45 mm HEPES (pH 8), and 0.9 m NaCl. Septins were purified from EJY332, a yeast strain expressing Cdc12-HF, as described previously (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). GST and GST-Ulp2 were expressed and purified from E. coli JM101 cells using a modification of a described protocol (16Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar). Cells induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C were lysed under high pressure using an Avestin Emulsiflex C-5 in 1× PBS (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4·7H2O, 1.4 mm KH2PO4) plus 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 1 mm DTT. After centrifugation at 35,000 × g for 30 min at 4 °C, supernatant was bound in-batch to immobilized glutathione (Sigma) at 25 °C for 30 min. Beads were washed with 1× PBS, 0.1% Triton X-100, 0.1 mm DTT and eluted with 15 mm glutathione, 60 mm Tris (pH 7.5), 150 mm NaCl, 0.1% Triton X-100, and 0.1 mm DTT. Eluates were dialyzed against 50 mm Tris (pH 7.5), 150 mm NaCl, 1 mm DTT, 30% glycerol for 1 h at 4 °C using mini Slide-A-Lyzers (Pierce). In Vitro Sumoylation Assay—Sumoylation reactions were performed as described previously (8Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar) and contained ∼40 μg/ml wt or mutant Smt3, ∼5 μg/ml Uba2·Aos1, 3.5 μg/ml Ubc9, and ∼3 μg/ml trSiz1 or ∼12 μg/ml of the full-length Siz1 preparation, which contained primarily C-terminal fragments of Siz1 and E. coli proteins. Top2-HA Immunoprecipitations—For immunoprecipitation of Top2-HA, cells were grown to A 600 ∼1 in rich medium at 30 °C followed by incubation with 15 μg/ml nocodazole (Acros) for 4 h at 30 °C. Whole cell lysates were prepared as described for isolation of Smt3 conjugates (16Li S.J. Hochstrasser M. Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar) except that cells were lysed using an Avestin Emulsiflex C-5 instead of by sonication. Clarified lysates were bound to anti-HA-agarose (Covance) overnight at 4 °C. Beads were washed three times with 50 mm Tris (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.1% SDS, 2 mm NEM and eluted by boiling in SDS sample buffer. Ulp2 Cleavage of in Vitro Sumoylation Reactions—Sumoylation reactions containing recombinant Siz1-HF (see above) were incubated for 2.5 h at 30 °C and stopped by incubating with 25 mm glucose and 1 unit of hexokinase (Sigma) in a 26-μl reaction for 5 min at 28 °C. These samples were diluted 4-fold into new reactions to contain 50 mm Tris (pH 7.5), 12.5 mm HEPES (pH 7.0), 175 mm NaCl, 0.1% Triton X-100, 0.125 mm DTT, 2.5 mm MgCl2, 5 mm imidazole, 1.25 μm ZnCl2. Then 2.5 μg/ml GST or the GST-Ulp2 preparation, which contained primarily free GST (∼90%), was added. In reactions with NEM or NEM plus DTT, the GST or GST-Ulp2 was preincubated with 10 mm NEM or 10 mm NEM plus 20 mm DTT for 15 min at 25 °C. Cleavage reactions proceeded for 3 h at 30 °C and were stopped by boiling in SDS sample buffer. N-terminal Lysines Are Required for SUMO Chain Formation in Vitro—To identify the branch site(s) in SUMO chains that had been assembled in in vitro reactions containing Siz1, high molecular weight SUMO-containing species were excised from the top of an SDS-polyacrylamide resolving gel (much like Fig. 2B, lane 2), digested with trypsin and endoproteinase Lys-C, and analyzed by surface-enhanced laser desorption/ionization-time of flight mass spectrometry (data not shown). A major peak was detected that was the correct size to contain the C-terminal tryptic peptide of SUMO covalently linked to the N-terminal peptide of SUMO up to Lys27. This experiment suggested that SUMO was attached to Lys11, Lys15, and/or Lys19 of SUMO but did not distinguish among them. Neither trypsin nor Lys-C cleaved after any of these lysine residues, which are all followed by prolines (Fig. 1). However, a similar experiment, using chains that were assembled by Uba2·Aos1 and Ubc9 alone and then cleaved with endoproteinase Glu-C, was published recently showing the major branch site to be at Lys15 (25Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).Fig. 1Diagram of lysine residues in SUMO. A, there are nine lysines in Smt3 that fall into four groups. Mutant SUMOs were constructed by mutating one, all but one, or all groups of lysines to arginines. B, alignment of the N termini of Smt3, SUMO-1, SUMO-2, SUMO-3, and Ub. Lysines are highlighted. The arrow above Smt3 designates the start position for the smt3-NΔ21 allele.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Yeast SUMO has a total of nine lysines that fall into four groups (Fig. 1). Three of these, Lys11, Lys15, and Lys19 are in the N-terminal non-Ub-like region and are part of a repeat sequence containing three tetrapeptide units resembling SUMO attachment consensus motifs. We decided to generate mutants lacking each of the nine lysines in yeast SUMO because preliminary experiments indicated that mutating only the N-terminal lysines, which were identified as potential branch sites by mass spectrometry, did not prevent formation of extremely high molecular weight SUMO conjugates in vitro (see below). Also we wanted to test thoroughly whether SUMO chains with different branch sites might have different functions in vivo. A series of SUMO mutants was constructed in which one group, all but one group, or all four groups of lysines were mutated to arginine. These mutants were expressed in E. coli, purified, and assayed in sumoylation reactions containing recombinant Aos1·Uba2 (E1), Ubc9 (E2), and trSiz1 (Fig. 2A). trSiz1 was used because full-length Siz1 was itself sumoylated and generated large amounts of high molecular weight conjugates that complicated analysis. Because these reactions did not contain any additional substrate, all SUMO-containing species larger than free SUMO contained SUMO attached either to itself (SUMO chains) or to the enzymes in the reaction. Wild-type SUMO formed a ladder of bands corresponding to SUMO chains (Fig. 2A, lane 3). This ladder was absent in all reactions containing SUMO mutants lacking Lys11, Lys15, and Lys19 (Fig. 2A, lanes 4 and 9–12). These results confirm that SUMO forms chains in vitro primarily via the N terminus (25Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 31Takahashi Y. Toh-e A. Kikuchi Y. J. Biochem. (Tokyo). 2003; 133: 415-422Crossref PubMed Scopus (53) Google Scholar). Other groups have examined SUMO chain formation in reactions that lack Siz proteins and depend only on the E1 and Ubc9 (25Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 31Takahashi Y. Toh-e A. Kikuchi Y. J. Biochem. (Tokyo). 2003; 133: 415-422Crossref PubMed Scopus (53) Google Scholar), but under our reaction conditions, SUMO chain formation requires trSiz. Thus, these resul
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