Rad23 and Rpn10 Serve as Alternative Ubiquitin Receptors for the Proteasome
2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês
10.1074/jbc.m404020200
ISSN1083-351X
AutoresSuzanne Elsasser, Devin Chandler-Militello, Britta Müller, John Hanna, Daniel Finley,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains. The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains. The breakdown of ubiquitin-protein conjugates by the proteasome is a major mechanism for biological regulation in eukaryotes (1Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (611) Google Scholar). The conjugative and degradative machineries are coupled at the ubiquitin chain recognition step. This step, although intensively studied, has remained problematic (1Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (611) Google Scholar, 2Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. 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Among the putative shuttling factors having affinity for both the proteasome and ubiquitin conjugates is a family of proteins exemplified by the Rad23 protein of Saccharomyces cerevisiae (27Hartmann-Petersen R. Seeger M. Gordon C. Trends Biochem. Sci. 2003; 28: 26-31Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Madura K. Cell Cycle. 2002; 1: 235-244Crossref PubMed Scopus (43) Google Scholar). Proteasome binding by Rad23-like proteins involves their N-terminal ubiquitin-like (UBL) 1The abbreviations used are: UBL, ubiquitin-like; UBA, ubiquitin associated; UIM, ubiquitin interacting motif; GST, glutathione S-transferase; suc, succinyl; AMC, 7-amido-4 methylcoumarin. domains (8Schauber C. Chen L. Tongaonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar, 9Funakoshi M. Sasaki T. Nishimoto T. Kobayashi H. Proc. Natl. Acad. Sci. U. S. 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Commun. 2002; 293: 986-992Crossref PubMed Scopus (114) Google Scholar, 16Hiyama H. Yokoi M. Masutani C. Sugasawa K. Maekawa T. Tanaka K. Hoeijmakers J.H. Hanaoka F. J. Biol. Chem. 1999; 274: 28019-28025Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 17Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (351) Google Scholar, 18Walters K.J. Lech P.J. Goh A.M. Wang Q. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12694-12699Crossref PubMed Scopus (128) Google Scholar, 19Mueller T.D. Feigon J. EMBO J. 2003; 22: 4634-4645Crossref PubMed Scopus (111) Google Scholar), whereas ubiquitin chain binding is mediated by ubiquitin-associated (UBA) domains (9Funakoshi M. Sasaki T. Nishimoto T. Kobayashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 745-750Crossref PubMed Scopus (212) Google Scholar, 15Saeki Y. Saitoh A. Toh-e A. Yokosawa H. Biochem. Biophys. Res. Commun. 2002; 293: 986-992Crossref PubMed Scopus (114) Google Scholar, 17Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (351) Google Scholar, 20Bertolaet B.L. Clarke D.J. Wolff M. Watson M.H. Henze M. Divita G. Reed S.I. Nat. Struct. Biol. 2001; 8: 417-422Crossref PubMed Scopus (275) Google Scholar, 21Chen L. Madura K. Mol. Cell. Biol. 2002; 22: 4902-4913Crossref PubMed Scopus (253) Google Scholar, 22Rao H. Sastry A. J. Biol. Chem. 2002; 277: 11691-11695Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 23Mueller T.D. Kamionka M. Feigon J. J. Biol. Chem. 2004; 279: 11926-11936Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) (see Fig. 1A). The UBL domain of Rad23 binds proteasomes with a higher affinity than does the full-length protein (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar), and for the human Rad23 homolog hHR23a binding of ubiquitin via the UBA domain disrupts the intramolecular interactions between the UBL and UBA domains (18Walters K.J. Lech P.J. Goh A.M. Wang Q. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12694-12699Crossref PubMed Scopus (128) Google Scholar). In vivo, modulating the interactions between the UBL and UBA domains might serve to potentiate the binding of the Rad23-conjugate complex to the proteasome. Despite the logical appeal of the shuttling hypothesis, it remains controversial. A positive role for UBL-UBA proteins in degradation is supported by the observations that some model substrates are stabilized in yeast cells lacking Rad23 and in cells lacking Dsk2, another UBL-UBA family member (9Funakoshi M. Sasaki T. Nishimoto T. Kobayashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 745-750Crossref PubMed Scopus (212) Google Scholar, 17Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (351) Google Scholar, 29Lambertson D. Chen L. Madura K. Genetics. 1999; 153: 69-79Crossref PubMed Google Scholar). Additionally, depletion of hHR23 by small interfering RNA has been shown to lead to the stabilization of p53 in mammalian cells (30Glockzin S. Ogi F.X. Hengstermann A. Scheffner M. Blattner C. Mol. Cell. Biol. 2003; 23: 8960-8969Crossref PubMed Scopus (81) Google Scholar). At variance with a role for Rad23 and similar molecules in promoting degradation is the finding that the influence of Rad23 on the in vitro proteasomal degradation of artificial and native substrates appears to be exclusively inhibitory (25Raasi S. Pickart C.M. J. Biol. Chem. 2003; 278: 8951-8959Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 30Glockzin S. Ogi F.X. Hengstermann A. Scheffner M. Blattner C. Mol. Cell. Biol. 2003; 23: 8960-8969Crossref PubMed Scopus (81) Google Scholar). In addition, Rad4 (a Rad23-binding protein) is destablized in rad23Δ cells, although this has been attributed to Rad23-dependent inhibition of Rad4 ubiquitination (31Lommel L. Ortolan T. Chen L. Madura K. Sweder K.S. Curr. Genet. 2002; 42: 9-20Crossref PubMed Scopus (69) Google Scholar, 32Ng J.M. Vermeulen W. van der Horst G.T. Bergink S. Sugasawa K. Vrieling H. Hoeijmakers J.H. Genes Dev. 2003; 17: 1630-1645Crossref PubMed Scopus (202) Google Scholar). When overexpressed, Rad23 stabilizes Pds1 and model proteasome substrates (33Clarke D.J. Mondesert G. Segal M. Bertolaet B.L. Jensen S. Wolff M. Henze M. Reed S.I. Mol. Cell. Biol. 2001; 21: 1997-2007Crossref PubMed Scopus (76) Google Scholar, 34Ortolan T.G. Tongaonkar P. Lambertson D. Chen L. Schauber C. Madura K. Nat. Cell Biol. 2000; 2: 601-608Crossref PubMed Scopus (161) Google Scholar) and causes the accumulation of undefined ubiquitinated species (21Chen L. Madura K. Mol. Cell. Biol. 2002; 22: 4902-4913Crossref PubMed Scopus (253) Google Scholar). However, such overexpression results do not necessarily imply that Rad23 antagonizes protein degradation when present at wild-type levels. The capacity of proteasomes to recognize substrates directly through covalently attached ubiquitin chains has long been known (4Beal R. Deveraux Q. Xia G. Rechsteiner M. Pickart C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 861-866Crossref PubMed Scopus (218) Google Scholar, 35Hough R. Pratt G. Rechsteiner M. J. Biol. Chem. 1986; 261: 2400-2408Abstract Full Text PDF PubMed Google Scholar). S5a was the first proteasome subunit implicated in conjugate binding (2Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). However, cells lacking Rpn10, the yeast ortholog of S5a, proved to be viable, implying that other ubiquitin receptors must exist (3van Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar). rpn10Δ cells are slightly sensitive to canavanine, an arginine analog whose toxicity is typically enhanced in proteolysis-deficient mutants (3van Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar). Surprisingly, the canavanine sensitivity does not map to the ubiquitin interacting motif (UIM) but rather to the von Willebrand factor type A domain (36Fu H. Reis N. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; 20: 7096-7107Crossref PubMed Scopus (212) Google Scholar), which is required for proteasome stability under conventional purification conditions (37Glickman M.H. Rubin D.M. Coux O. Wefes I. Pfeifer G. Cjeka Z. Baumeister W. Fried V.A. Finley D. Cell. 1998; 94: 615-623Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar) (see Fig. 1A). This result and the observation that free Rpn10 cross-links to ubiquitin chains whereas proteasome-associated Rpn10 does not have led to the view that Rpn10 is not a true proteasomal ubiquitin receptor (6Lam Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (370) Google Scholar). Recently, rpn10Δ proteasomes were purified in an intact state and found to be partially defective in conjugate recognition (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). However, the participation of the UIM in conjugate recognition has remained an open question especially as it cannot be excluded that rpn10Δ proteasomes (even when intact) have structural defects that could affect chain recognition by other proteasome subunits. Paralleling the uncertainty over chain binding by the UIM element in the proteasome in vitro is the question of whether it has a positive role in protein degradation in vivo. These issues are addressed below. Using purified systems, we provide evidence that proteasomes recognize ubiquitin chains directly via the UIM element of Rpn10 but also indirectly through association with UBL-UBA proteins. Furthermore, in vivo experiments identify specific genetic interactions between the rpn10-uim mutation and deletions of either RAD23 or DSK2, supporting the model that both direct and indirect pathways exist for chain recognition by the proteasome. Strain Construction—Yeast strains were constructed according to standard methods and are listed in Table I. For the preparation of the rpn10-uim and control strains, fragments were excised from pEL92e and pEL91b, respectively, and transformed into yeast. To prepare these plasmids, the 1.2-kb EcoRI-BamHI fragment from pAG25 (38Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1397) Google Scholar) bearing the natMX cassette was cloned into pBluescript SK+. A 640-bp fragment corresponding to the RPN10 open reading frame beginning 85 bp past the start codon and ending 64 bp past the stop codon was cloned upstream of the natMX cassette. To generate the mutant allele, bases 674–698 were substituted with 5′-GGAT CCG GAA AAC AAC AAT AAT AAC-3′ corresponding to a change at residues 228–232 from LAMAL to NNNNN and generating a silent BamHI site upstream of the target mutation. A 320-bp fragment of the RPN10 3′-untranslated region beginning 65 bp past the stop codon was cloned downstream of the natMX cassette. This fragment overlapped precisely with the 3′ end and stop codon of the adjacent PPX1 gene. Integrants were verified by PCR, and the presence of the mutation was determined by restriction mapping.Table IYeast strainsStrainRelevant genotypeSY73MATα rpt1::HIS3 (pEL35 [ProA-TEV-RPT1-TRP1]) rpn10::RPN10-natMXSY74MATα rpt1::HIS3 (pEL35 [ProA-TEV-RPT1-TRP1]) rpn10::rpn10-uim-natMXSDL135MATa pre1::PRE1-TEV-ProA-HIS3SDL145MATa rpn11::RPN11-TEV-ProA-HIS3 ubp6::URA3SY290bMATa rpn11::RPN11-TEV-ProA-HI3SSY293eMATa rpn11::RPN11-TEV-ProA-HIS3 rad23::kanMX dsk2::klTRP1SY294bMATa rpn11::RPN11-TEV-ProA-HIS3 rpn10::rpn10-uim-natMXSY295aMATa rpn11::RPN11-TEV-ProA-HIS3 rad23::kanMX dsk2::klTRP1 rpn10::rpn10-uim-natMXSY316kMATα rpn10::RPN10-natMXSY317fMATα rpn10::RPN10-natMX dsk2::klTRP1SY318jMATα rpn10::RPN10-natMX rad23::kanMXSY319iMATα rpn10::RPN10-natMX rad23::kanMX dsk2::klTRP1SY304bMATα rpn10::rpn10-uim-natMXSY305cMATα rpn10::rpn10-uim-natM X dsk2::klTRP1SY306aMATα rpn10::rpn10-uim-natMX rad23::kanMXSY307fMATα rpn10::rpn10-uim-natMX rad23::kanMX dsk2::klTRP1 Open table in a new tab For the preparation of rad23::kanMX, dsk2::klTRP1, and ubp6::uraMX deletions, precise substitutions of the open reading frames were made with the markers indicated (39Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2248) Google Scholar, 40Goldstein A.L. Pan X. McCusker J.H. Yeast. 1999; 15: 507-511Crossref PubMed Scopus (107) Google Scholar). Integration was verified by PCR. Expression and Purification of Recombinant Proteins—Recombinant Ubp6 was purified as described previously (41Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). GST fusion proteins of Rad23 and Rpn1 were expressed in BL21(DE3) cells and purified in the presence of 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% Triton X-100, and 1 mm dithiothreitol. Where indicated, proteins were labeled with [γ-32P]ATP as described (42Kaelin Jr., W.G. Krek W. Sellers W.R. DeCaprio J.A. Ajchenbaum F. Fuchs C.S. Chittenden T. Li Y. Farnham P.J. Blanar M.A. Livingston D.M. Flemington E.K. Cell. 1992; 70: 351-364Abstract Full Text PDF PubMed Scopus (692) Google Scholar). Rad23 fusion proteins were cleaved from the GST moiety with thrombin, which was subsequently inhibited with 4 μm aminoethyl-benzene sulfonyl fluoride. His6-Cdc34 was purified in 20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm β-mercaptoethanol, and 0.05% Triton X-100 and eluted from nickel-nitrilotriacetic acid resin with 250 mm imidazole in purification buffer. The following plasmids were used for the expression of recombinant proteins: pDL74 (GST-Ubp6), pRG22 (GST-Rad23), pRG63 (GST-Rad23-UBL (residues 1–77)), pDC4 (GST-ΔUBL-Rad23 (residues 78–398)), and pEL1 (His6-Cdc34). Synthesis of Cdc34 Conjugates—All conjugates were prepared in the presence of 1.2 μm Uba1-His6 and conjugation buffer (20 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 0.1 mm dithiothreitol, and 2 mm ATP) for 19 h at 30 °C and with components indicated in Table II. Decreasing the ratio of ubiquitin to Cdc34 in the conjugation reaction produces progressively shorter conjugates. Additionally, native Cdc34 autoubiquitinates somewhat more efficiently than His6-Cdc34. Cdc34 and Uba1-His6 were purified from yeast as described previously (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar), and bovine ubiquitin was purchased from Sigma.Table IIConditions for ubiquitin-conjugate synthesisConjugatesFigs.Cdc34 (Type)Cdc34UbiquitinμmμmLong1, 3BHis6450Short1His6820Long3ANative450Medium3ANative850Short3ANative1250 Open table in a new tab Native Gel Electrophoretic Mobility Assays—Ligands were mixed with proteasome and incubated for 40 min at 30 °C. All of the binding reactions contained fixed volumes: 3 μl of proteasome, 4 μl of Cdc34 conjugates, and 4 μl of each Rad23 species. To make this possible, the molarity of each protein was adjusted to the necessary value with buffers identical to the original stock. Complexes were resolved by nondenaturing PAGE (3.5%) in a solution of 90 mm Tris base, 90 mm boric acid, 5 mm MgCl2, 0.5 mm EDTA, and 0.5 mm ATP and were electrophoresed for 3.5 h at 100 V and 4 °C. Proteasomes were visualized by suc-LLVY-AMC hydrolysis as described previously (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). Proteasome Purification—For the native gel electrophoretic mobility assays, wild-type and rpn10-uim proteasomes were purified from SY73 and SY74 essentially as described and carried out in the absence of ATP (41Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). For the competition assays, core particle and proteasome lacking Ubp6 were purified in the presence of ATP from SDL135 and SDL145, respectively (41Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). For the endogenous conjugate association assay, proteasome was purified from SY290b, SY293e, SY294b, and SY295a. Purifications were carried out in the presence of 1 mm ATP, and salt was omitted from the wash. Competition Assays—IgG resin was charged with proteasome and core particle as described above and resuspended as a 25% slurry in purification buffer. An aliquot was removed, treated with tobacco etch virus protease, and the protein concentration was measured and used to calculate the quantities present on the resin. Bovine serum albumin was added to the slurries at 0.5 mg/ml. Aliquots of 400 μl were mixed with 25-μl mixtures containing 10 pmol of radiolabeled Rad23 (10,000 cpm/pmol) and cold competitor as indicated. After 15 min of binding at room temperature, resin was washed five times with 5 volumes of buffer per wash. Bound material was quantified by scintillation counting. Canavanine Sensitivity—Strains SY304 through SY307 and SY316 through SY319 were grown overnight, diluted 25- or 50-fold, grown 4 h at 30 °C, diluted to an A600 of 0.2, prepared as 3-fold serial dilutions, and spotted on plates. Plates lacking and containing canavanine sulfate were grown for 3 and 4 days, respectively, at 30 °C. Docking of Ubiquitin Conjugates to the Proteasome by Rad23—One possible argument against the shuttling hypothesis is that the proteasome has a high intrinsic affinity for conjugates (43Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1332) Google Scholar), and thus, its affinity does not need to be strengthened by extrinsic factors. We recently developed a direct assay for ubiquitin conjugate binding by the proteasome, employing nondenaturing gel electrophoresis followed by proteasome visualization via an in-gel activity stain (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). We have used in vitro synthesized conjugates (Fig. 1B) in this assay to test whether Rad23 can promote the binding to proteasomes of conjugates that would otherwise not show stable binding. As shown in Fig. 1C, complexes of low electrophoretic mobility can be formed when Rad23, the proteasome, and ubiquitin conjugates are mixed. All three components must be added for the low mobility species to form, indicating that the electrophoretically retarded species is a ternary complex as further verified below. Formation of the retarded ternary complex requires the UBL domain of Rad23 (Fig. 1C) as well as the UBA-containing C-terminal domain (Fig. 1D). Additionally, if the N-terminal UBL domain of Rad23 and the C-terminal UBA domain are added as separate polypeptides to the reaction, the retarded complex does not form (Fig. 1E). This result indicates that ternary complex formation is critically dependent on the ability of Rad23 to bind the proteasome and ubiquitin conjugates simultaneously as expected if conjugates are joined to proteasomes through a Rad23 linker. In the absence of a linked UBL domain, the C-terminal domain effectively competes with proteasomes for ubiquitin conjugate binding (Fig. 1, C–E, and data not shown). In both S. cerevisiae and Schizosaccharomyces pombe, proteasome subunit Rpn1 docks Rad23 onto proteasomes through recognition of its UBL domain (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar, 11Seeger M. Hartmann-Petersen R. Wilkinson C.R. Wallace M. Samejima I. Taylor M.S. Gordon C. J. Biol. Chem. 2003; 278: 16791-16796Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 14Saeki Y. Sone T. Toh-e A. Yokosawa H. Biochem. Biophys. Res. Commun. 2002; 296: 813-819Crossref PubMed Scopus (121) Google Scholar). Interestingly, another proteasome-associated protein, deubiquitinating enzyme Ubp6, also binds proteasomes by virtue of an interaction between its UBL domain and Rpn1 (41Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). Affinity-purified proteasomes are heavily if not stoichiometrically loaded with Ubp6 (41Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). These data suggest that Rad23 and Ubp6 may compete with each other for binding to proteasomes and that only a minor fraction of proteasomes (those lacking Ubp6) therefore may be available for conjugate docking via Rad23. To test this scenario, the binding of 32P-labeled Rad23 to immobilized proteasome was measured in the presence and absence of unlabeled Ubp6. As seen in Fig. 2, Ubp6 has no influence on Rad23 binding. Thus, although Rad23 and Ubp6 both bind proteasomes via their UBL domains, they do so through distinct binding sites on Rpn1. To confirm this conclusion, we carried out similar binding assays using GST-Rpn1 as the immobilized ligand. As seen for intact proteasomes, Rad23 and Ubp6 bound Rpn1 independently (data not shown). By simultaneously binding two UBL proteins that are active on ubiquitin chains, Rpn1 may play a critical scaffolding role in the proteasome. Previously we localized the Rad23 binding site in Rpn1 using a series of deletions constructed from a GST-Rpn1 fusion (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). When analogous experiments were carried out with Ubp6, the Rpn1 segments that had shown specific and essentially wild-type affinity for Rad23 (10Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar) showed little or no specific binding to Ubp6 despite the fact that the binding of Ubp6 to full-length Rpn1 is quite robust as compared with Rad23 (data not shown). These findings are in agreement with the proposal of a distinct, noncompeti
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