Abstract Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Δ siz2Δ mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Δ siz2Δ rad52Δ are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Δ and nup133Δ, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Δ siz2Δ mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Δ cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.
At least one essential function of Smt3p, aSaccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub∼Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation. At least one essential function of Smt3p, aSaccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub∼Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation. SMT3 is an essential Saccharomyces cerevisiae gene encoding a member of a family of ubiquitin-like proteins, including the mammalian protein SUMO-1 (1Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar) (also called GMP1, PIC1, UBL1, or sentrin (2Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (952) Google Scholar, 3Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google Scholar, 4Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 37: 183-186Crossref PubMed Scopus (125) Google Scholar, 5Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.F. Chang H.M. Yeh E.T. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar)). SUMO-1 was isolated as a protein covalently linked to the Ran-GTPase-activating protein (RanGAP1), 1The abbreviations used are: RanGAP1, Ran-GTPase-activating protein; Ub, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HF-Smt3p, His6- and FLAG-epitope-tagged mature Smt3p; DTT, dithiothreitol; NTA, nitrilotriacetic acid; EF1α, elongation factor 1α; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 1The abbreviations used are: RanGAP1, Ran-GTPase-activating protein; Ub, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HF-Smt3p, His6- and FLAG-epitope-tagged mature Smt3p; DTT, dithiothreitol; NTA, nitrilotriacetic acid; EF1α, elongation factor 1α; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and it is also conjugated to a number of other, primarily nuclear, proteins (1Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 2Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (952) Google Scholar, 3Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google Scholar, 6Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Smt3p, which is 487 identical to SUMO-1 and 177 identical to Ub, also becomes attached to several proteins posttranslationally, and at least one of its essential functions is mediated by its attachment to another protein (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). Ubiquitin (Ub) conjugation is carried out by a multistep pathway culminating in formation of an isopeptide bond between the C-terminal carboxyl group of Ub and the ε-amino group of a lysine side chain in an acceptor protein (8Pickart C.M. Rechsteiner M. Ubiquitin. Plenum Press, New York1988: 77-99Crossref Google Scholar, 9Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1453) Google Scholar). In the initial step, Ub-activating enzyme (E1) utilizes ATP to adenylate the Ub C terminus, which is then transferred to a conserved Cys residue in the E1, yielding an E1∼Ub thioester, AMP, and pyrophosphate. Ub is transferred from the E1 to a Cys residue in a Ub-conjugating enzyme (E2). Cells contain multiple E2s (13 in yeast by sequence similarity) which are involved in ubiquitinating different proteins. In some cases Ub can be transferred directly from the E2 to the acceptor protein, but more often Ub-isopeptide bond formation is facilitated by a third heterogeneous class of proteins termed Ub-protein ligases or recognins (E3). Several features of the Ub pathway are conserved in the early steps of the Smt3p conjugation pathway (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). Like Ub, the SMT3translation product is proteolytically processed to expose its mature C terminus. Smt3p undergoes ATP-dependent activation by a heterodimeric activating enzyme consisting of Uba2p, a 71-kDa protein with extensive sequence similarity to the C-terminal region of E1s, including the active site Cys residue participating in the thioester (10Dohmen R.J. Stappen R. McGrath J.P. Forrová H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and Aos1p, a 40-kDa protein similar to the N termini of E1s (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). While the Smt3p- and Ub-activating enzymes are related, they do not interact with each other's substrates, suggesting that the two pathways are distinct. One candidate to be the Smt3p-conjugating enzyme is Ubc9p, a member of the E2 sequence family whose Xenopus leavis homolog co-immunoprecipitates with a complex including SUMO-1-conjugated RanGAP1 (11Saitoh H. Pu R. Cavenagh M. Dasso M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3736-3741Crossref PubMed Scopus (163) Google Scholar) and whose human homolog interacts in a two-hybrid screen with SUMO-1 (4Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 37: 183-186Crossref PubMed Scopus (125) Google Scholar). UBC9 is an essential gene. Conditionalubc9 mutants arrest in the cell cycle at G2/M and are impaired in proteolysis of both B-type cyclins (12Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (425) Google Scholar) and G1 cyclins (13Blondel M. Mann C. Nature. 1996; 384: 279-282Crossref PubMed Scopus (34) Google Scholar). However, Ubc9p does not seem to be the E2 involved in cyclin B ubiquitination (14Yu H. King R.W. Peters J.M. Kirschner M.W. Curr. Biol. 1996; 6: 455-466Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4299Crossref PubMed Scopus (121) Google Scholar). While it has been suggested that Ubc9p functions as an E2 in the Ub pathway, there is no clear biochemical data to support this hypothesis. We have found that Ubc9p is a Smt3p-conjugating enzyme and that it is likely to constitute the only Smt3p-conjugating activity in yeast. Furthermore, Ubc9p does not seem to be a Ub-conjugating enzyme, suggesting that the ubc9 cell cycle defect actually results from impairment of Smt3p conjugation. Standard techniques were used (16Ausubel 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 York1994Google Scholar). S. cerevisiae strains used were DF5α (MATα trp1–1 ura3–52 his3-Δ200 leu2–3,112 lys2–801) (17Finley D. Özkaynak E. Varshavsky A. Cell. 1987; 48: 1035-1046Abstract Full Text PDF PubMed Scopus (630) Google Scholar) and the DF5-derived strain YWO102 (MATα ubc9Δ::TRP1 leu2::ubc9Pro-Ser::LEU2) (12Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (425) Google Scholar), which was a generous gift of S. Jentsch (Heidelberg, Germany). pET21b or pET11a (Novagen)-based Escherichia coli expression plasmids expressing N-terminally His6- (18Hoffman A. Roeder R.G. Nucleic Acids Res. 1991; 19: 6337-6338Crossref PubMed Scopus (249) Google Scholar) and FLAG-tagged (19Brizzard B.L. Chubet R.G. Vizard D.L. BioTechniques. 1994; 16: 730-734PubMed Google Scholar) Smt3p (HF-Smt3p) and His6-tagged Aos1p and Uba2p have been described (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). pET21b-based plasmids for expressing Ubc9p-, Ubc2p-, and Pex4p-tagged C-terminally with His6 were produced by polymerase chain reaction. The Ubc2p construct expresses Ubc2p truncated after Met153, deleting most of the acidic C-terminal domain. A pET21b-based plasmid expressing N-terminally His6- and FLAG-tagged ubiquitin bearing Lys48→ Arg, Lys63 → Arg and Gly76 → Ala mutations (HF-Ub(A76)) was also produced by polymerase chain reaction. The A76 mutation alters the kinetics of thioester formation (20Pickart C.M. Kasperek E.M. Beal R. Kim A. J. Biol. Chem. 1994; 269: 7115-7123Abstract Full Text PDF PubMed Google Scholar). Construction details are available on request. Proteins were expressed in E. coli and purified by Ni-NTA chromatography as described (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). The H-Uba2p·Aos1p complex was further purified by Smt3p affinity and gel filtration chromatography (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). Purified recombinant Ubc4p was a generous gift of V. Chau (Wayne State University School of Medicine, Detroit, MI). His6-tagged Uba1p was purified from yeast lysate of strain JD77–1A (MAT a uba1Δ::HIS3) (10Dohmen R.J. Stappen R. McGrath J.P. Forrová H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) bearing plasmid pJD325, which expresses H-Uba1p from PCUP1, both gifts of J. Dohmen (Heinrich-Heine Universität, Düsseldorf, Germany). Uba1p was purified by Ni-NTA chromatography as described for Uba2p (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar) followed by Ub affinity chromatography (21Ciechanover A. Elias S. Heller H. Hershko A. J. Biol. Chem. 1982; 257: 2537-2542Abstract Full Text PDF PubMed Google Scholar). Ni-NTA-purified recombinant Aos1p and Uba2p were applied to an HF-Smt3p-Affi-Gel 15 column and washed as described (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). A few milligrams of each protein bound. Yeast nuclear extract, consisting of the soluble fractions (primarily the load) below the nuclear envelopes in a Nycodenz/sucrose flotation gradient, prepared as described (22Strambio-de-Castillia C. Blobel G. Rout M.P. J. Cell Biol. 1995; 131: 19-31Crossref PubMed Scopus (60) Google Scholar), was a generous gift of R. Beckmann and D. Peter. Extract containing ∼20 mg of protein was dialyzed against 50 mm BisTris (pH 6.5), 50 mmNaCl, 1 mm MgCl2, and 1 mmॆ-mercaptoethanol (ॆ-ME), brought to final concentrations 50 mm BisTris (pH 6.5), 75 mm NaCl, 5 mm MgCl2, 2 mm ATP, and 0.5 mm ॆ-ME and applied to the Aos1p/Uba2p-preloaded HF-Smt3p column. The column was washed with 10 column volumes of the same buffer except containing 1 m NaCl and eluted as described (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). The eluate was exchanged into 50 mm Tris (pH 8.0), 300 mm NaCl, 1 mm MgCl2, and 1 mm ॆ-ME using a Biomax-10 ultrafiltration unit (Millipore), bound to 0.5 ml of Ni-NTA-agarose, and washed with 5 column volumes of 50 mm sodium phosphate (pH 6.0), 300 mm NaCl, and 107 glycerol. The unbound and wash fractions were pooled and fractionated by SDS-PAGE on a 4–207 gradient gel (Novex) followed by Coomassie Blue staining. Bands were excised from the gel and identified by direct analysis of a Lys-C endoproteinase digest by MALDI-TOF mass spectrometry at the Rockefeller University Protein/DNA Technology Center as described (23Gharahdaghi F. Kirchner M. Fernandez J. Mische S.M. Anal. Biochem. 1996; 233: 94-99Crossref PubMed Scopus (97) Google Scholar). The six major peaks, with measured masses of 856.68, 1080.54, 1702.28, 1829.39, 2892.92, and 3463.25 Da, are within 1.6 mass units of the peptide masses predicted by a theoretical Lys-C digest of Ubc9p (using the ProFound and MS-Fit programs: http://chait-sgi.rockefeller.edu/cgi-bin/ProFoundandhttp://prospector.ucsf.edu/htmlucsf/msfit.htm), assuming the N terminus is acetylated and cysteine residues are modified by acrylamide. Thioester formation reactions contained 50 mm BisTris (pH 6.5), 100 mm NaCl, 10 mm MgCl2, 0.1 mm DTT and some of the following: 5 mm ATP, 30 ॖg/ml HF-Smt3p or HF-Ub(A98), 15 ॖg/ml Aos1p/Uba2p heterodimer or Uba1p, and ∼15 ॖg/ml Ubc9p, Ubc4p, Pex4p, or Rad6p (Ubc4p and Pex4p contained noticeable amounts of contaminants). HF-Smt3p-containing reactions were incubated 30 min and HF-Ub(A76)-containing reactions 90 min at 25 °C and stopped by addition of SDS-containing loading buffer either lacking reducing agent or containing 100 mm DTT, followed by a 10-min incubation at 37 °C, SDS-PAGE (6–157 acrylamide gradient) and either Coomassie Blue staining or immunoblotting using the M2 anti-FLAG antibody (IBI/Kodak). Whole yeast cell lysate from DF5α or YWO102 (both after 90-min incubation at 37 °C) were prepared as described (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar) and exchanged into 50 mm Tris (pH 7.5), 150 mm NaCl, 1 mm MgCl2, and 0.1 mm DTT using a Sephadex G-25 column. Reactions were incubated for 90 min at 25 °C and contained 25 mm Tris (pH 7.5), 75 mm NaCl, 10 mm MgCl2, 50 ॖm DTT, 10 mg/ml whole yeast lysate, 30 ॖg/ml HF-Smt3p and, where indicated, 5 mm ATP and/or 5 ॖg/ml Ubc9p. E1s and E2s can be readily purified by 舠covalent affinity chromatography舡 (21Ciechanover A. Elias S. Heller H. Hershko A. J. Biol. Chem. 1982; 257: 2537-2542Abstract Full Text PDF PubMed Google Scholar) by adding ATP to a protein mixture containing these enzymes and incubating with an affinity column to which Ub has been coupled. E1s and E2s form thioester bonds with the column-bound Ub and then can be gently eluted in buffer containing a thiol reducing agent, such as DTT, which breaks the thioester bonds. We attempted an analogous approach to purifying the Smt3p-conjugating enzyme(s) using a column linked to His6 (18Hoffman A. Roeder R.G. Nucleic Acids Res. 1991; 19: 6337-6338Crossref PubMed Scopus (249) Google Scholar)- and FLAG-tagged (19Brizzard B.L. Chubet R.G. Vizard D.L. BioTechniques. 1994; 16: 730-734PubMed Google Scholar) Smt3p (HF-Smt3p) (mature processed Smt3p having C-terminal Gly98 will be referred to as Smt3p). Because the Smt3p-conjugating enzyme would be predicted to bind the column by displacing Uba2p/Aos1p, which is necessary to activate the column-bound Smt3p, the Smt3p column was prebound with recombinant His6-tagged Uba2p and Aos1p to improve the efficiency of conjugating enzyme binding. Next, ATP-supplemented yeast nuclear extract was applied to the column, which was then washed and eluted with DTT. Nuclear extract was used because Uba2p has been reported to be nuclear (10Dohmen R.J. Stappen R. McGrath J.P. Forrová H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and because we had previously found a yeast cytosolic fraction to be inactive in Smt3p conjugation (data not shown). The eluate contained predominantly recombinant Uba2p and Aos1p (data not shown), but as these bore His6 tags, they were selectively removed by Ni-NTA chromatography. The resulting fraction contained four major bands of 90, 50, 40, and 19 kDa and a number of minor bands (Fig.1). The 90- and 40-kDa bands are likely to represent the endogenous Uba2p and Aos1p from the yeast lysate. The other two bands were analyzed by direct mass spectrometric analysis of a protease digestion mixture (see 舠Experimental Procedures舡). The 50-kDa band contained the elongation factor EF1α, and the 19-kDa band contained Ubc9p. The isolation of EF1α in this fractionation was intriguing, as EF1α has been connected to Ub-like systems before, as an isopeptidase involved in Ub-dependent proteolysis ofN-α-acetylated substrates (24Gonen H. Dickman D. Schwartz A.L. Ciechanover A. Adv. Exp. Med. Biol. 1996; 389: 209-219Crossref PubMed Scopus (31) Google Scholar). However, it is also an extremely abundant protein and a frequent contaminant in affinity purifications. 2R. Beckmann, personal communication. We attempted to purify EF1α and add it to some of the reactions described below, but did not obtain any conclusive results. Smt3p-thioester formation assays were performed using purified, His6-tagged recombinant proteins expressed in E. coli. Incubation of the Uba2p/Aos1p heterodimer with ATP and HF-Smt3p promoted formation of the ∼105-kDa HF-Smt3p∼Uba2p thioester (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar) (Fig. 2,lane 6). When purified Ubc9p was also added to this reaction, a different HF-Smt3p-containing band formed at ∼38 kDa (Fig. 2, lanes 2 and 7). In addition to Ubc9p, formation of this product required ATP, Uba2p, and Aos1p. Furthermore, the vast majority of this product could be destroyed by incubation with DTT (Fig. 2, lanes 1–8). The data are consistent with the 38-kDa product being the HF-Smt3p∼Ubc9p thioester. The DTT-sensitive bond cannot be a disulfide bond, as HF-Smt3p does not contain any cysteine residues. Coomassie Blue staining of the reaction products provided additional evidence that Ubc9p was the other component of the 38-kDa product, as the Ubc9p bands were significantly depleted upon ATP incubation and formation of the 38-kDa band and reappeared upon addition of DTT (Fig. 2, lanes 1–3; Ubc9p ran as a ∼20-kDa doublet under nonreducing conditions). Also, formation of the 38-kDa product was entirely dependent on the presence of Uba2p/Aos1p, strengthening the analogy between this reaction and that of Ub, E1s, and E2s. This reaction also formed small amounts of a series of DTT-insensitive HF-Smt3p-containing bands, one of which ran at the same position as the HF-Smt3p∼Ubc9p thioester. These bands could represent a polymer of HF-Smt3p or isopeptide-containing conjugates of Smt3p to Ubc9p. We also asked whether Ubc9p was capable of forming thioesters with Ub as well as with Smt3p. When the yeast E2s Ubc4p, Pex4p (Pas2p/Ubc10p), or Rad6p (Ubc2p), which are involved in bulk proteolysis (25Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (406) Google Scholar), peroxisome biogenesis (26Wiebel F.F. Kunau W.H. Nature. 1992; 359: 73-76Crossref PubMed Scopus (163) Google Scholar), and DNA repair (27Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (545) Google Scholar), respectively, were mixed with Uba1p (the yeast E1) and His6- and FLAG-tagged Ub(A76) (see 舠Experimental Procedures舡), addition of ATP caused these E2s to shift almost quantitatively into higher molecular weight forms (Fig. 3, lanes 1–6). These products were DTT-sensitive and contained HF-Ub(A76) (data not shown), suggesting that they were the corresponding Ub-E2 thioesters. No thioester product between Ubc9p and Ub could be detected under these conditions either by Coomassie staining (Fig. 3, lane 8) or by immunoblotting with the antibody against the FLAG epitope (Fig. 3,lane 9). Conversely, extremely little or no thioester product was detected between Smt3p and either Ubc4p, Pex4p, or Rad6p (data not shown), demonstrating that the transthiolation reaction is very specific. When HF-Smt3p and ATP were incubated with whole yeast cell lysate from wild-type cells, a series of DTT-resistant high molecular weight HF-Smt3p-containing bands formed (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar) (Fig. 4, lane 2). The pattern of Smt3p-containing bands was similar but not identical to that seen in vivo, with a greater proportion of HF-Smt3p found in very high molecular mass bands >200 kDa (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). Theubc9–1 mutant, which has a Ser residue in place of Pro69, is temperature-sensitive by virtue of the fact that Ubc9(P69S)p is rapidly degraded at 37 °C (28Betting J. Seufert W. J. Biol. Chem. 1996; 271: 25790-25796Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). When yeast lysate made from this mutant strain after incubation at 37 °C was used in the same reaction, no Smt3p conjugation was detected even on long exposure. (Fig. 4, lane 3). Addition of recombinant Ubc9p restored the Smt3p conjugation activity (Fig. 4, lane 4). These results suggest a direct requirement for Ubc9p in HF-Smt3p conjugation to other proteins in these lysates. Thus, Ubc9p is likely to be the only Smt3p-conjugating enzyme in yeast, at least that is expressed under normal growth conditions. Our results show that the Smt3p conjugation pathway is distinct from the Ub pathway, at least up to formation of the thioester with the conjugating enzymes. However, sequence comparisons of of the Smt3p-conjugating enzyme Ubc9p to the Ub-conjugating enzymes Rad6p (Ubc2p), Ubc4p, and Pex4p (Pas2p/Ubc10p) do not reveal any large continuous regions of dissimilarity, and the degree of sequence similarity between Ubc9p and any of these three E2s is comparable with that among the E2s. Ubc4p is 377 identical to Ubc2p, 357 identical to Pex4p, and 347 identical to Ubc9p. Yet the transthiolation reaction involving Aos1p/Uba2p and Smt3p is extremely selective for Ubc9p, and the reaction involving Uba1p and Ub selects strongly against Ubc9p. One possible factor in this discrimination may be that Ubc9p is much more basic than the other proteins, with a pI of 9.2 as compared with 6.3 for Ubc4p, 5.4 for Pex4p, or 4.0 for Rad6p. How this specificity is generated depends on whether conjugating enzymes interact directly with the activating enzyme, the Ub-like protein, or both. Although our inability to detect Ub∼Ubc9p thioesters does not prove that Ubc9p never participates in Ub conjugation in vivo, it is unlikely that the metabolic stabilization of cyclins observed inubc9 mutants (12Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (425) Google Scholar, 13Blondel M. Mann C. Nature. 1996; 384: 279-282Crossref PubMed Scopus (34) Google Scholar) results directly from reduced ubiquitination of these proteins by Ubc9p, both because of our results and because the E2s that participate in cyclin B ubiquitination have been isolated and do not include Ubc9p (14Yu H. King R.W. Peters J.M. Kirschner M.W. Curr. Biol. 1996; 6: 455-466Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4299Crossref PubMed Scopus (121) Google Scholar). It is also very unlikely that Smt3p conjugation substitutes for ubiquitination in targeting cyclins for proteolysis, as cyclins have been heavily studied and never found to be Smt3p-conjugated. Furthermore, Smt3p, which is only 177 identical to Ub, probably does not target its substrates for proteasome-dependent proteolysis. Smt3p conjugation could affect cyclin proteolysis by activating some component of the ubiquitination/proteolysis machinery, or its effect could be several steps removed from ubiquitination. It also has not been excluded that Ubc9p could be required for the conjugation of a different Ub-like protein, which could mediate the cell cycle effect. Smt3p, Aos1p, and Uba2p are all essential genes (7Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 10Dohmen R.J. Stappen R. McGrath J.P. Forrová H. Kolarov J. Goffeau A. Varshavsky A. 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Dohmen for strains, plasmids, and other reagents and members of the Rockefeller University Protein/DNA Technology Center for DNA sequencing and especially F. Gharahdaghi for MALDI-TOF analysis. We also thank M. Matunis and J. Rosenblum for critical reading of the manuscript.
Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae , histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae . We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.
La finalidad del trabajo que aqui se describe fie evaluar el tamizaje comunitario para la deteccion de casos de infeccion por VIHvinculado a un programa de lucha antituberculosa en una poblacion en alto riesgo de ambas infecciones. De mayo de 1990 a agosto de 1992, trabajadores de salud comunitarios se comunicaron con adultos en domicilios y dispensarios de Cite Soleil, Haiti, para ofrecerles servicios institucionales de asesoramiento individual y de deteccion de VIH y de tuberculosis. A todas las personas que aceptaron la prueba se les dio asesoramiento posterior sobre VIH. Las que tenian tuberculosis activa recibieron tratamiento y a las que tenian enfermedad latente mas infeccion por VIH se les dio la oportunidad de participar en un ensayo clinico sobre quimioprofilaxis antituberculosa. Las personas sometidas al tamizaje para la deteccion de VIH, que fueron 10 611, constituyeron lO,O% de la poblacion adulta de Cite Soleil. Se encontro infeccion por VIH en 1629 (15,4%) de ellas y tuberculosis activa en 242 (2,3%). Infeccion latente por Mycobacierium tuberculosis fue detectada en 4800 (65,7%) de los 7309 habitantes de la comunidad quefueron sometidos al tamizaje completo para la deteccion de tuberculosis, y de esos 4800,781(16,3%) tambien estaban infectados por VlH. La elevada prevalencia de infeccion por VIH en la poblacion examinada, al compararsela con la de otros grupos sometidos a tamizaje en la misma comunidad, indica que las personas en alto riesgo de infeccion por VIH buscaron selectivamente o aceptaron someterse a las pruebas de tamizaje ofuecidas en los dispensarios de tuberculosis. Asimismo, a muchas personas se les diagnostico tuberculosis activa en una fase mas temprana de la enfermedad de lo que hubiera sido posible sin un programa de tamizaje. En general, los resultados indican que cuando el tamizaje comunitario para la deteccion de VIH es parte de un programa de lucha antituberculosa, el resultado puede ser una mejorfocalizacion de destinatarios para las pruebas de deteccion de ambas infecciones.
Supplementary Table 1 from Small Ubiquitin-Related Modifier Pathway Is a Major Determinant of Doxorubicin Cytotoxicity in <i>Saccharomyces cerevisiae</i>
Introduction: The etiologies of congenital and developmental cataracts are diverse. Most are not syndromic and have no identifiable cause, thus creating a diagnostic dilemma. We investigated the utility of chromosomal microarray in identifying the etiology of isolated childhood cataracts. Methods: Patients with congenital or developmental cataracts without other associated abnormalities received a single-nucleotide polymorphism (SNP) microarray. copy number variations (CNV) and regions of homozygosity (ROH) were compared with previous literature reports and analyzed for candidate genes to assess pathogenicity. Results: We enrolled 37 patients. The mean age of the patient population was 10.98 years old. Nineteen patients (51.4%) had bilateral cataract. Positive family history was found in 11 patients (29.7%). Eighteen patients (48.7%) had a variant on microarray: 10 (27%) with CNV, 5 (13.5%) with ROH, and 3 patients (8.1%) with both CNV and homozygosity. In five patients (13.5%), we found a potentially causative cataract gene within an ROH. Discussion: There is a high rate of notable findings among the CNV and ROH detected. Three patients were homozygous in a region known to have a cataract gene suggesting a possible autosomal recessive disease. In those with CNV, segregation would help to affirm the pathogenicity of these regions and may lead to the identification of new genes. Conclusion: SNP microarray had a surprisingly high rate of notable findings in patients with isolated cataract and may reveal the opportunities for genetic counseling, lead to discovering new cataract genes and identify additional affected genes that could lead to other clinical abnormalities.
Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
