Activation of the Slx5–Slx8 Ubiquitin Ligase by Poly-small Ubiquitin-like Modifier Conjugates
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m802690200
ISSN1083-351X
AutoresJanet R. Mullen, Steven J. Brill,
Tópico(s)Fungal and yeast genetics research
ResumoProtein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5–Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5–Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5–Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5–Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5–Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5–Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain. Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5–Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5–Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5–Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5–Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5–Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5–Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain. Sumoylation regulates a diverse set of cellular processes and is essential for viability in budding yeast (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar). Sumoylation resembles ubiquitination in that the C terminus of SUMO 2The abbreviations used are: SUMO, small ubiquitin-like modifier; HU, hydroxyurea; SIM, SUMO interacting motif; GST, glutathione S-transferase; wt, wild type; Ulp1UD, Ulp1 Ulp domain; SD, synthetic complete medium; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; aa, amino acid(s); aR, protein in which all lysine residues are changed to arginine; Ub,ubiquitin. 2The abbreviations used are: SUMO, small ubiquitin-like modifier; HU, hydroxyurea; SIM, SUMO interacting motif; GST, glutathione S-transferase; wt, wild type; Ulp1UD, Ulp1 Ulp domain; SD, synthetic complete medium; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; aa, amino acid(s); aR, protein in which all lysine residues are changed to arginine; Ub,ubiquitin. is conjugated to lysine residues of target proteins. In yeast, this occurs through the sequential activity of an activating (E1) enzyme (Aos1/Uba2), a conjugating (E2) enzyme (Ubc9), and one of several SUMO ligases (E3) (e.g. Mms21 and Siz2) (2Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar, 3Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 4Ardley H.C. Robinson P.A. Essays Biochem. 2005; 41: 15-30Crossref PubMed Google Scholar). Although a large class of Ub E3 ligases are RING-domain proteins (5Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Scholar), SUMO E3 ligases often contain a variant domain known as SP-RING (3Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 6Hochstrasser M. Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Sumoylation can be reversed by the Ulp1 and Ulp2 isopeptidases, which catalyze the cleavage of SUMO polypeptides from target proteins (7Li S.J. Hochstrasser M. Mol. Cell Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar, 8Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (601) Google Scholar).In yeast, recombinational DNA repair depends on sumoylation. It has been known for some time that DNA damage tolerance is compromised in yeast strains with defects in Ubc9, Mms21, Ulp1, or Ulp2 (7Li S.J. Hochstrasser M. Mol. Cell Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar, 9Zhao X. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4777-4782Crossref PubMed Scopus (342) Google Scholar, 10Maeda D. Seki M. Onoda F. Branzei D. Kawabe Y. Enomoto T. DNA Repair (Amst.). 2004; 3: 335-341Crossref PubMed Scopus (29) Google Scholar, 11Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1723) Google Scholar). More recently, cells lacking the Srs2 anti-recombinase have been shown to require the Ulp1 isopeptidase for viability (12Soustelle C. Vernis L. Freon K. Reynaud-Angelin A. Chanet R. Fabre F. Heude M. Mol. Cell Biol. 2004; 24: 5130-5143Crossref PubMed Scopus (32) Google Scholar), and cells defective in UBC9 and MMS21 accumulate Rad51-dependent cruciform structures during DNA replication (13Branzei D. Sollier J. Liberi G. Zhao X. Maeda D. Seki M. Enomoto T. Ohta K. Foiani M. Cell. 2006; 127: 509-522Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In the best characterized cases, SUMO has been shown to link Srs2 to proliferating cell nuclear antigen (14Papouli E. Chen S. Davies A.A. Huttner D. Krejci L. Sung P. Ulrich H.D. Mol. Cell. 2005; 19: 123-133Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 15Pfander B. Moldovan G.L. Sacher M. Hoege C. Jentsch S. Nature. 2005; 436: 428-433Crossref PubMed Scopus (488) Google Scholar) and to be conjugated to Rad52 (16Torres-Rosell J. Sunjevaric I. De Piccoli G. Sacher M. Eckert-Boulet N. Reid R. Jentsch S. Rothstein R. Aragon L. Lisby M. Nat. Cell Biol. 2007; 9: 923-931Crossref PubMed Scopus (289) Google Scholar, 17Sacher M. Pfander B. Hoege C. Jentsch S. Nat. Cell Biol. 2006; 8: 1284-1290Crossref PubMed Scopus (151) Google Scholar).Sumoylation is distinct from ubiquitination in that target proteins are typically modified by single SUMO moieties that result in mono- or multisumoylated products as opposed to targets bearing poly-SUMO chains (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar). Nonetheless, poly-SUMO conjugates are commonly observed in in vitro sumoylation reactions (3Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 18Bencsath 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 (109) Google Scholar, 19Tatham 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 (634) Google Scholar), and they accumulate in yeast cells deficient in the Ulp2 SUMO protease or Ub-mediated proteolysis (20Bylebyl G.R. Belichenko I. Johnson E.S. J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The polymerization of SUMO in yeast (called Smt3) occurs preferentially through its three N-terminal lysine residues (lysines 11, 15, and 19) that are found in a domain of 20 amino acids (aa) unique to SUMO (18Bencsath 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 (109) Google Scholar, 19Tatham 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 (634) Google Scholar, 20Bylebyl G.R. Belichenko I. Johnson E.S. J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The function of such poly-sumoylation is unknown, although in the case of ulp2Δ cells, it appears to be toxic (20Bylebyl G.R. Belichenko I. Johnson E.S. J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar).SLX5 and SLX8 are required for the viability of yeast cells lacking the Sgs1 DNA helicase (22Mullen J.R. Kaliraman V. Ibrahim S.S. Brill S.J. Genetics. 2001; 157: 103-118Crossref PubMed Google Scholar). These genes encode a heterodimeric Ub ligase that links sumoylation to recombinational DNA repair (21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 23Ii T. Fung J. Mullen J.R. Brill S.J. Cell Cycle. 2007; 6: 2800-2809Crossref PubMed Scopus (35) Google Scholar, 24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 25Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). On their own, slx5Δ and slx8Δ null mutants display similar phenotypes, including slow growth, sensitivity to hydroxyurea (HU), and increased rates of gross chromosomal rearrangements and mitotic recombination (22Mullen J.R. Kaliraman V. Ibrahim S.S. Brill S.J. Genetics. 2001; 157: 103-118Crossref PubMed Google Scholar, 26Burgess R.C. Rahman S. Lisby M. Rothstein R. Zhao X. Mol. Cell Biol. 2007; 27: 6153-6162Crossref PubMed Scopus (115) Google Scholar, 27Zhang C. Roberts T.M. Yang J. Desai R. Brown G.W. DNA Repair (Amst.). 2006; 5: 336-346Crossref PubMed Scopus (81) Google Scholar). Like mutants deficient in sumoylation, slx5Δ and slx8Δ mutants display a clonal lethality that is dependent on the 2μ circle (9Zhao X. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4777-4782Crossref PubMed Scopus (342) Google Scholar, 28Ii T. Mullen J.R. Slagle C.E. Brill S.J. DNA Repair (Amst.). 2007; 6: 1679-1691Crossref PubMed Scopus (45) Google Scholar, 29Chen X.L. Reindle A. Johnson E.S. Mol. Cell Biol. 2005; 25: 4311-4320Crossref PubMed Scopus (61) Google Scholar), and this can be suppressed by eliminating genes in the RAD51-independent recombination pathway (26Burgess R.C. Rahman S. Lisby M. Rothstein R. Zhao X. Mol. Cell Biol. 2007; 27: 6153-6162Crossref PubMed Scopus (115) Google Scholar). The SLX5 and SLX8 genes were also isolated in a screen for suppressors of a mot1–301 allele, along with mutations in most of the enzymes involved in the sumoylation pathway (30Wang Z. Jones G.M. Prelich G. Genetics. 2006; 172: 1499-1509Crossref PubMed Scopus (63) Google Scholar). Importantly, there is an accumulation of sumoylated proteins in slx5Δ and slx8Δ cells that correspond to high molecular weight SUMO conjugates (21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 30Wang Z. Jones G.M. Prelich G. Genetics. 2006; 172: 1499-1509Crossref PubMed Scopus (63) Google Scholar).The Slx5–Slx8 complex interacts directly with SUMO via SUMO interacting motifs in each subunit (21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 28Ii T. Mullen J.R. Slagle C.E. Brill S.J. DNA Repair (Amst.). 2007; 6: 1679-1691Crossref PubMed Scopus (45) Google Scholar, 31Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar). The complex appears to be functionally conserved in Schizosaccharomyces pombe, where the related RING-finger proteins Rfp1 and Rfp2 also interact with SUMO and Slx8 to suppress the build-up of sumoylated proteins (21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 32Sun H. Leverson J.D. Hunter T. EMBO J. 2007; 18: 4102-4112Crossref Scopus (240) Google Scholar, 33Prudden J. Pebernard S. Raffa G. Slavin D.A. Perry J.J. Tainer J.A. McGowan C.H. Boddy M.N. EMBO J. 2007; 18: 4089-4101Crossref Scopus (278) Google Scholar, 34Kosoy A. Calonge T.M. Outwin E.A. O'Connell M.J. J. Biol. Chem. 2007; 282: 20388-20394Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The idea that the Ub ligase activity of the complex is directed toward sumoylated targets is supported by the finding that substrates containing a SUMO sequence are preferentially ubiquitinated (24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 32Sun H. Leverson J.D. Hunter T. EMBO J. 2007; 18: 4102-4112Crossref Scopus (240) Google Scholar, 33Prudden J. Pebernard S. Raffa G. Slavin D.A. Perry J.J. Tainer J.A. McGowan C.H. Boddy M.N. EMBO J. 2007; 18: 4089-4101Crossref Scopus (278) Google Scholar).In this study we investigated the specificity of the Slx5–Slx8 Ub ligase toward sumoylated substrates. We found that its activity on free SUMO or a multisumoylated test substrate was low, whereas ubiquitination of a poly-sumoylated substrate was high. Characterization of this reaction revealed that Slx5–Slx8 primarily mono-ubiquitinated the N-terminal end of the SUMO chain. Although there is no known physiological role for poly-sumoylation, we found increased levels of these conjugates in cells lacking the Sgs1 DNA helicase. Thus, it appears that one essential function of the Slx5–Slx8 Ub ligase is to ubiquitinate poly-sumoylated proteins that arise in sgs1Δ cells.EXPERIMENTAL PROCEDURESYeast Strains and Plasmids—Standard methods and media were used for the propagation, transformation, and culturing of Saccharomyces cerevisiae (35Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Strain JD194 (MATα ura3Δ5 his3–11,15 leu2–3,112 pre1–1 pre2–2) was kindly provided by Dr. Kiran Madura. Additional genotypes and plasmid construction details are available on request.Expression and Purification of Recombinant Proteins—GST- and His6-tagged proteins were produced in Escherichia coli BL21-RIL cells (Stratagene) using the T7 expression system of Studier (36Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar). His6-tagged proteins were expressed and purified as described previously (37Yang L. Mullen J.R. Brill S.J. Nucleic Acids Res. 2006; 34: 5541-5551Crossref PubMed Scopus (35) Google Scholar). GST-tagged proteins were expressed similarly but purified following lysis in Buffer A (25 mm Tris-HCl (pH 7.5), 10% glycerol, 1 mm EDTA, 0.01% Nonidet P-40, 1 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride) containing 200 mm NaCl and protease inhibitors. The extract was applied to a 1-ml GST-TRAP column (GE Healthcare), washed with 20 ml of Buffer A containing 200 mm NaCl, and eluted with the same buffer containing 10 mm glutathione. Peak protein fractions were dialyzed in Buffer A containing 100 mm NaCl. Additional versions of GST–Smt3 and GST-Ub fusion proteins were constructed with protease 3C and cAMPdPK consensus sites downstream of the GST domain. Following treatment with cAMPdPK and γ-[32P]ATP, proteins were re-bound to glutathione beads and washed free of ATP prior to eluting the radiolabeled protein via protease 3C cleavage. Expression and purification of Ulp1UD (38Li S.J. Hochstrasser M. J. Cell Biol. 2003; 160: 1069-1081Crossref PubMed Scopus (163) Google Scholar) has been described (28Ii T. Mullen J.R. Slagle C.E. Brill S.J. DNA Repair (Amst.). 2007; 6: 1679-1691Crossref PubMed Scopus (45) Google Scholar).SUMO Binding Assay—Physical interactions between His6/FLAG-tagged Smt3 (HF-Smt3) (2Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar) and GST-Slx5 or GST-Slx8 were detected following incubation on ice for 1 h in a final volume of 0.1 ml using Buffer A with 50 mm NaCl as the incubation buffer. This reaction was then diluted with 0.3 ml of incubation buffer and mixed with 20 μl of glutathione beads (40 μl of 50% slurry) for 1 h at 4 °C. The beads were recovered by low speed spin and washed three times with incubation buffer. Bound proteins were eluted with 25 μl of SDS sample buffer and detected by immunoblotting as described before (37Yang L. Mullen J.R. Brill S.J. Nucleic Acids Res. 2006; 34: 5541-5551Crossref PubMed Scopus (35) Google Scholar).In Vitro Sumoylation Assay—The standard sumoylation reaction was performed in the presence of 20 mm HEPES (pH 7.5), 5 mm MgCl2, 2 mm ATP, 5 μm ZnSO4, and 0.1 mm dithiothreitol. Unless otherwise stated, reactions were incubated at 30 °C for 60 min and contained 2 nm Aos1/Uba2, 30 nm Ubc9, 100 nm Siz2-V5, and 2 μm HF-Smt3-G98 in a total volume of 20 μl. Immunoblotting was carried out essentially as described (37Yang L. Mullen J.R. Brill S.J. Nucleic Acids Res. 2006; 34: 5541-5551Crossref PubMed Scopus (35) Google Scholar) except that proteins were transferred to polyvinylidene difluoride or a 0.22-μm nitrocellulose membrane. Where indicated, mature HF-Smt3-G98 was substituted with HF-Smt3-G97 or HF-Smt3-G98A mutants, whereas latter experiments employed Smt3 and variants that were simply His6-tagged.In Vitro Ubiquitination Assay—Ubiquitination was performed using the same buffer conditions as the sumoylation assay. Reactions were incubated at 30 °C for 15 min and contained 2 nm Uba1, 30 nm Ubc5, 10 nm unlabeled ubiquitin, and 350,000 cpm 32P-Ub in a total volume of 30 μl. The products of this reaction were resolved by SDS-PAGE (typically 15% acrylamide). The gel was then fixed and visualized on a PhosphorImager (Amersham Biosciences).RESULTSMultiple SUMO Binding Domains Are Required for Slx5–Slx8 Function—To locate potential SUMO-binding domains within the two subunits of the Ub ligase, we fused portions of the N and C termini of Slx5 or Slx8 to the C terminus of GST. Purified recombinant proteins were then assayed for their ability to bind SUMO following incubation with glutathione beads. In contrast to GST alone, GST-Slx5 bound strongly to SUMO (Fig. 1A). Two small N-terminal truncations (ΔN100 and ΔN200) showed diminishing affinity to SUMO, whereas deletion of 300 aa or more eliminated this activity. Analysis of C-terminal deletions revealed a strong SUMO-binding domain within the first 100 aa of Slx5, although additional mapping identified a second larger domain between aa 101 and 300 (Fig. 1B). Thus, the first 300 aa of Slx5 contains at least two SUMO-binding domains. When the N-terminal deletions were examined for their ability to complement sgs1Δ synthetic lethality, a correlation was found between loss of SUMO binding and loss of complementation (Fig. 1A). And consistent with its role in promoting Ub ligase activity (23Ii T. Fung J. Mullen J.R. Brill S.J. Cell Cycle. 2007; 6: 2800-2809Crossref PubMed Scopus (35) Google Scholar), the RING domain of Slx5 was required to complement sgs1Δ synthetic lethality. However, deletion of a single N-terminal SUMO-binding domain was tolerated in sgs1Δ cells. A similar analysis revealed a SUMO binding activity in Slx8 that was localized to the C-terminal 111 aa (Fig. 1C). As with Slx5, removal of the one SUMO-binding domain in Slx8 was tolerated in sgs1Δ cells. Consistent with the mapping of multiple SUMO interacting motifs within these subunits (21Uzunova K. Gottsche K. Miteva M. Weisshaar S.R. Glanemann C. Schnellhardt M. Niessen M. Scheel H. Hofmann K. Johnson E.S. Praefcke G.J. Dohmen R.J. J. Biol. Chem. 2007; 282: 34167-34175Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 24Xie Y. Kerscher O. Kroetz M.B. McConchie H.F. Sung P. Hochstrasser M. J. Biol. Chem. 2007; 282: 34176-34184Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), we conclude that there are at least three SUMO-binding domains within the Slx5–Slx8 Ub ligase.The Severity of slx5 and slx8 Phenotypes Correlates with in Vivo Sumoylation Levels—To correlate the sumoylation defects of slx5 and slx8 mutants with their other phenotypes, we characterized a collection of point and truncation alleles with respect to their effect on in vitro Ub ligase activity, accumulation of hyper-sumoylated proteins, HU sensitivity, and sgs1Δ synthetic lethality. Among point mutation alleles that map to the RING domains of Slx5 and Slx8, four were shown to eliminate Ub ligase activity in vitro (slx5-6, slx5-8, slx8-1, and slx8-3) and those displayed lethality in the sgs1Δ background (Fig. 2A) (23Ii T. Fung J. Mullen J.R. Brill S.J. Cell Cycle. 2007; 6: 2800-2809Crossref PubMed Scopus (35) Google Scholar, 37Yang L. Mullen J.R. Brill S.J. Nucleic Acids Res. 2006; 34: 5541-5551Crossref PubMed Scopus (35) Google Scholar)). Those retaining Ub ligase activity (slx5-5, slx5-7, and slx8-2) displayed intermediate complementation in the sgs1Δ background (Fig. 2A) (37Yang L. Mullen J.R. Brill S.J. Nucleic Acids Res. 2006; 34: 5541-5551Crossref PubMed Scopus (35) Google Scholar). To assay hyper-sumoylation, a denatured extract from each strain was immunoblotted with α-Smt3 antibody. All mutations that were synthetically lethal with sgs1Δ showed extreme hyper-sumoylation, whereas the intermediate alleles displayed partially elevated sumoylation levels (Fig. 2B). In these and subsequent experiments the 5% stacking gel was retained on the blot, because it has been shown that highly poly-sumoylated proteins are retained in the stacking gel (20Bylebyl G.R. Belichenko I. Johnson E.S. J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Interestingly, removing one SUMO-binding domain (slx5-ΔN100, -ΔN200, and slx8-ΔN200) resulted in intermediate levels of hyper-sumoylation, whereas deleting an additional domain (slx5-ΔN300 and slx5-ΔN400) or a RING domain (slx5-ΔC126 and slx8-ΔC74) produced a null phenotype. Consistently, strains with high sumoylation levels grew poorly on HU (Fig. 2C) and required SGS1 to survive (Fig. 2B). Strains with slightly elevated sumoylation levels grew like wild type on HU and survived loss of SGS1. We conclude that the in vivo sumoylation levels and HU sensitivities of slx5 and slx8 alleles correlate well with their sgs1Δ synthetic-lethal phenotypes.FIGURE 2Synthetic-lethal phenotype of SLX5 and SLX8 mutations correlates with the accumulation of hyper-sumoylated proteins. A, alleles of SLX5 containing the indicated RING-finger mutations were tested for complementation of sgs1Δ slx5Δ synthetic lethality as in Fig. 1. B, the indicated SLX5 or SLX8 alleles were integrated into strain JMY1699 (slx5Δ) or SIY778 (slx8Δ), and N-ethylmaleimide extracts were analyzed for Smt3-protein conjugates by 8% SDS-PAGE and immunoblotting using antibodies against Smt3 (upper panel) or Rfa1 (lower panel). A bracket marks the stacking gel. Also shown is the alleles' ability to complement the relevant slxΔ sgs1Δ synthetic lethality. To determine sgs1Δ viability, strains JMY1464 or VCY1525 [(sgs1Δ slx8Δ plus pJM500 (SGS1/URA3/ADE3)] were transformed with LEU2/CEN/ARS plasmids carrying the indicated SLX5 or SLX8 alleles and evaluated for growth on plates containing 5-fluoro-orotic acid. Symbols: +, good growth; +/–, slow growth; –, no growth. C, strains JMY1699 or SIY778 were transformed with LEU2/CEN/ARS plasmids carrying the indicated alleles, were assayed for growth as in Fig. 1A but in the presence or absence of 0.1 m hydroxyurea. The plates were photographed after 3 days of growth at 30 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sumoylated Siz2 Is Ubiquitinated by Slx5–Slx8—To examine the specificity of the Slx5–Slx8 ubiquitin ligase toward sumoylated proteins, we took advantage of the fact that the SUMO E3 ligase Siz2, which is known to be sumoylated in vivo (39Hannich J.T. Lewis A. Kroetz M.B. Li S.J. Heide H. Emili A. Hochstrasser M. J. Biol. Chem. 2005; 280: 4102-4110Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), exhibits auto-sumoylation in vitro. Sumoylated Siz2 was then used as a substrate for in vitro ubiquitination. To validate this system, sumoylation reactions were carried out with epitope-tagged Siz2 (Siz2-V5) and a variety of mutant SUMO substrates. These products were analyzed on duplicate immunoblots that were probed with α-V5 or α-Smt3 (Figs. 3, A and B). Using mature HF-Smt3-G98 as substrate (2Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (440) Google Scholar), we observed two major species of Siz2 products (Fig. 3A, lane 1). One product migrated as a doublet at 125–150 kDa and was judged to be conjugated to one or more SUMO groups. The upper band is most likely multisumoylated, because it co-migrated with the single product obtained with Smt3-aR (Fig. 3A, lane 4). All nine lysine residues have been mutated to arginine in Smt3-aR, so it is unable to form SUMO chains (20Bylebyl G.R. Belichenko I. Johnson E.S. J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The second product was a highly poly-sumoylated form of Siz2, which displayed a characteristic mobility just entering the stacking gel (Fig. 3, A and B, lane 1). As expected, Smt3-G97 (lacking the essential C-terminal glycine) gave no sumoylated product, because it cannot form covalent attachments to target proteins (Fig. 3, A and B, lane 2), and Smt3-G98A produced both multi- and poly-sumoylated Siz2 in addition to short multimers of Smt3. Finally, synthesis of these products required functional Smt3, E1, E2 and Siz2 itself (Fig. 3, A and B, lanes 5–8). We conclude that Siz2 has multiple sites for Smt3 attachment, and that it can catalyze the formation of poly-SUMO chains.FIGURE 3Poly-sumoylated Siz2 is preferentially ubiquitinated by Slx5–Slx8. A, standard sumoylation assays were performed in the presence of the indicated Smt3 variants or in the absence of the indicated component. Siz2-V5 products were analyzed by SDS-PAGE and immunoblotting with antibodies against the V5 epitope. B, duplicate sumoylation reactions were analyzed by SDS-PAGE and immunoblotting with antibodies against Smt3. Oligomeric chains of Smt3 are indicated by S2-S5. Note that all Smt3 proteins were N-terminally tagged with His6-FLAG, except for Smt3-aR, which was tagged with His6 only. C, sumoylation reactions were first performed in the presence of wt HF-Smt3 (lanes 1 and 5–14), the indicated HF-Smt3 mutants (lanes 2 and 3), or no Smt3 (lane 4). The reaction products were then subjected to a standard ubiquitination assay in the presence of 32P-Ub and either 10 nm Slx5–Slx8 (lanes 1–6), or 0, 10 pm, 30 pm, 100 pm, 300 pm, 1 nm, 3 nm, or 10 nm Slx5–Slx8 (lanes 7–14). The Ub E1 or E2 was omitted where indicated (lanes 5 and 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To detect Ub E3 ligase activity, Siz2 sumoylation products were incubated with Uba1, Ubc5, ATP, and the Slx5–Slx8 dimer as described (23Ii T. Fung J. Mullen J.R. Brill S.J. Cell Cycle. 2007; 6: 2800-2809Crossref PubMed Scopus (35) Google Scholar), together with radiolabeled ubiquitin (32P-Ub) as substrate. Following a 15-min incubation, the products were analyzed by SDS-PAGE and autoradiography. As shown in Fig. 3C, samples containing sumoylated Siz2 produced a labeled band near the well of the gel (lanes 1–4). In a
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