Ubiquitylation of BAG-1 Suggests a Novel Regulatory Mechanism during the Sorting of Chaperone Substrates to the Proteasome
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m204196200
ISSN1083-351X
AutoresSimon Alberti, Jens Demand, Claudia Esser, Niels Emmerich, Hansjörg Schild, Jörg Höhfeld,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoBAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus ofHsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70·BAG-1·CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome. BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus ofHsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70·BAG-1·CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome. The control and maintenance of the three-dimensional structure of proteins are prerequisites for cell survival and involve a cooperation of molecular chaperones and energy-dependent proteases (1Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (907) Google Scholar, 2McClellan A.J. Frydman J. Nature Cell Biol. 2001; 3: E1-E3Crossref PubMed Scopus (104) Google Scholar, 3Höhfeld J. Cyr D.M. Patterson C. EMBO Rep. 2001; 2: 885-890Crossref PubMed Scopus (285) Google Scholar, 4Cyr D.M. Höhfeld J. Patterson C. Trends Biochem. Sci. 2002; 27: 368-375Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Molecular chaperones recognize hydrophobic regions exposed on unfolded proteins and stabilize non-native conformations. As a consequence formation of insoluble protein aggregates is prevented, and folding to the native state is promoted. On the other hand, energy-dependent proteases, such as the eukaryotic 26 S proteasome, degrade irreversibly damaged proteins that fail to be folded properly. Selection of proteins for degradation by the proteasome involves ubiquitin conjugation (5Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (511) Google Scholar, 6Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 7Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). A polyubiquitin chain is attached to a protein substrate through the concerted action of a ubiquitin-activating enzyme (E1), 1The abbreviations used for: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; CHIP, carboxyl terminus of Hsc70-interacting protein; HA, hemagglutinin; HEK, human embryonic kidney; Hsc70, 70-kDa heat shock cognate protein; Hsp40, Hsp70, Hsp90, 40-, 70-, and 90-kDa heat shock proteins, respectively; 4K-R, mutant form of BAG-1M with four lysine to arginine substitutions within its ubiquitin-like domain; MOPS, 4-morpholinepropanesulfonic acid; RIPA, radioimmune precipitation assay buffer. 1The abbreviations used for: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; CHIP, carboxyl terminus of Hsc70-interacting protein; HA, hemagglutinin; HEK, human embryonic kidney; Hsc70, 70-kDa heat shock cognate protein; Hsp40, Hsp70, Hsp90, 40-, 70-, and 90-kDa heat shock proteins, respectively; 4K-R, mutant form of BAG-1M with four lysine to arginine substitutions within its ubiquitin-like domain; MOPS, 4-morpholinepropanesulfonic acid; RIPA, radioimmune precipitation assay buffer. a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein isopeptide ligase (E3). In contrast to the presence of only one type of E1 enzyme in the eukaryotic cytosol, E2 and E3 enzymes are recruited from large protein families and mediate a specific recognition of a large repertoire of protein substrates. A polyubiquitin chain generated through the linkage of lysine 48 residues of successive ubiquitin moieties is usually sufficient to target a protein substrate to the proteasome, where finally deubiquitylation, unfolding, and degradation occur. Recent studies shed light onto molecular mechanisms underlying the cooperation of molecular chaperones with the ubiquitin/proteasome system during protein quality control. Two co-chaperones, CHIP and BAG-1, are of central importance in this regard. The CHIP protein was shown to act as a chaperone-associated ubiquitin ligase that directs chaperone substrates to the proteasome (8Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nature Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 10Jiang J. Ballinger C.A. Wu Y. Dai Q. Cyr D.M. Höhfeld J. Patterson C. J. Biol. Chem. 2001; 276: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 11Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nature Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (695) Google Scholar, 12Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (454) Google Scholar). CHIP interacts with the constitutively expressed Hsc70, the stress-inducible Hsp70, or Hsp90 via its amino-terminal tetratricopeptide repeat domain. This interaction negatively regulates ATPase and folding activities of the Hsc/Hsp70 chaperone and induces remodeling of Hsp90 heterocomplexes into a folding incompetent state (8Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nature Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 13Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.-Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (736) Google Scholar). In addition, CHIP possesses a U box domain at its carboxyl terminus which is structurally related to RING domains but lacks the Zn2+-chelating residues (4Cyr D.M. Höhfeld J. Patterson C. Trends Biochem. Sci. 2002; 27: 368-375Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 14Aravind L. Koonin E.V. Curr. Biol. 2000; 10: R132-R134Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). The U box domain provides the structural basis for the ubiquitin ligase activity of CHIP (9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 10Jiang J. Ballinger C.A. Wu Y. Dai Q. Cyr D.M. Höhfeld J. Patterson C. J. Biol. Chem. 2001; 276: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 12Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (454) Google Scholar). In conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family CHIP mediates polyubiquitylation of protein substrates presented by Hsc/Hsp70 and Hsp90. As a consequence, CHIP induces the degradation of known chaperone substrates such as the glucocorticoid hormone receptor or immature endoplasmic reticulum-localized forms of the cystic fibrosis transmembrane conductance regulator in cell culture experiments (8Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nature Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 11Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nature Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (695) Google Scholar). The ubiquitylation factor CHIP apparently mediates the removal of chaperone-selected proteins from the productive folding pathway and targets them for degradation by the proteasome. The Hsc/Hsp70 cofactor BAG-1 acts as a coupling factor between molecular chaperones and the proteasomal complex (15Lüders J. Demand J. Höhfeld J. J. Biol. Chem. 2000; 275: 4613-4617Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). BAG-1 belongs to a family of proteins with an integral ubiquitin-like domain, so called ubiquitin domain proteins (6Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The referred domain appears to serve as a proteasomal targeting signal and enables BAG-1 to recruit Hsc/Hsp70 to the proteasome (15Lüders J. Demand J. Höhfeld J. J. Biol. Chem. 2000; 275: 4613-4617Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). In addition, BAG-1 is able to stimulate the release of chaperone substrates from Hsc/Hsp70 because of its nucleotide exchange activity (16Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 17Sondermann H. Scheufler C. Schneider C. Höhfeld J. Hartl F.-U. Moarefi I. Science. 2001; 291: 1553-1557Crossref PubMed Scopus (358) Google Scholar, 18Gassler C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This chaperone-regulating activity may stimulate a transfer of chaperone substrates to the proteasome. In fact, evidence for a participation of BAG-1 in proteasomal targeting was recently provided when BAG-1 was shown to promote the degradation of the glucocorticoid hormone receptor in conjunction with CHIP (9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Cooperation of the two cofactors appears to reflect their physical interaction. BAG-1 and CHIP are able to associate simultaneously with Hsc/Hsp70 because of binding to different domains of the chaperone, and formation of the ternary chaperone-cofactor complex is promoted further through a direct interaction between the two cofactors (9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Accordingly, a model for the cooperation of Hsc/Hsp70 with the ubiquitin-proteasome system was proposed which integrated BAG-1 and CHIP activity (3Höhfeld J. Cyr D.M. Patterson C. EMBO Rep. 2001; 2: 885-890Crossref PubMed Scopus (285) Google Scholar, 4Cyr D.M. Höhfeld J. Patterson C. Trends Biochem. Sci. 2002; 27: 368-375Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 19Wiederkehr T. Bukau B. Buchberger A. Curr. Biol. 2002; 12: R26-R28Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Association with CHIP seems to convert the Hsc/Hsp70 chaperone into a substrate recognition factor of a functional ubiquitin ligase complex, whereas BAG-1 supports binding of the ligase complex to the proteasome and triggers the release of ubiquitylated substrates from Hsc/Hsp70 for their transfer to the proteasome. However, central mechanistic and regulatory aspects of the proposed cooperation remained elusive. Here, we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP cooperates with ubiquitin-conjugating enzymes of the Ubc4/5 family during ubiquitylation of BAG-1. The efficiency of ubiquitylation is increased strongly upon formation of the ternary Hsc70·BAG-1·CHIP complex. In this situation, a single polyubiquitin chain with linkages involving lysine 11 is assembled on BAG-1. Notably, CHIP-mediated ubiquitylation does not induce the degradation of BAG-1 but stimulates binding to the proteasome in a degradation-independent fashion. We provide evidence that the integrated ubiquitin-like domain of BAG-1 and the attached polyubiquitin chain are recognized by the proteasome in a cooperative manner. Our data reveal a novel regulatory step during the sorting of chaperone substrates to the proteasome. The following antibodies were used: mouse anti-Hsc/Hsp70 (Stressgen), rabbit anti-BAG-1 (C-16, Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc.), rabbit anti-ubiquitin (Sigma), and mouse anti-human C-8 (Affiniti). Purified bovine ubiquitin was purchased from Sigma, and the ubiquitin K48R mutant,N-methylated ubiquitin, and rabbit E1 were from Affiniti. Other point mutants of ubiquitin were expressed recombinantly inEscherichia coli BL21 (DE3) pJY2 cells using corresponding pET3a plasmids (20You J. Cohen R.E. Pickart C.M. BioTechniques. 1999; 27: 950-954Crossref PubMed Scopus (43) Google Scholar). Cells were lysed by sonication in 20 mm MOPS, pH 7.2, 100 mm KCl containing Complete protease inhibitor (Roche Molecular Biochemicals). The lysate was centrifuged for 30 min at 30,000 × g, and the resulting supernatant was used as a soluble extract. Rat Hsc70, human BAG-1M, and BAG-1S were purified as described after recombinant expression in baculovirus-infected Sf9 cells (16Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 21Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. Hoppe-Seyler. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 22Lüders J. Demand J. Papp O. Höhfeld J. J. Biol. Chem. 2000; 275: 14817-14823Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Wheat E1 was also expressed recombinantly in insect cells and purified as described for bacterially expressed E1 (23Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1939) Google Scholar). The C130 fragment of BAG-1 (covering the carboxyl-terminal 130 amino acids of the co-chaperone) was expressed in E. coli using a corresponding pTYB2 construct and purified as described via its self-cutting intein/chitin binding domain (22Lüders J. Demand J. Papp O. Höhfeld J. J. Biol. Chem. 2000; 275: 14817-14823Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Human Hsp40 (Hdj-1) and human UbcH5b were expressed in bacteria and purified as described (9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 16Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar). A mutant form of BAG-1M with arginine substitutions of all lysine residues within the ubiquitin-like domain of the co-chaperone (4K-R) was generated by PCR using corresponding primers, subcloning into pET3a (Novagen), and expression in E. coli BL21 (DE3). The ATPase domain of Hsc70 was purified and immobilized as described previously (16Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar). For initial ubiquitylation experiments His-tagged CHIP was used, which was purified as described (13Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.-Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (736) Google Scholar). For purification of untagged CHIP thechip cDNA was subcloned into pET3a (Novagen). The protein was expressed in E. coli BL21 (DE3) pLysS cells. Cells were lysed by sonication in buffer A (20 mm MOPS, pH 7.2, 50 mm KCl, 0.5 mm EDTA) containing 1 mm β-mercaptoethanol and Complete protease inhibitor. After centrifugation of the lysate at 100,000 × g for 30 min at 4 °C, the supernatant was applied to a DEAE-Sepharose column (Amersham Biosciences). Bound protein was eluted by a linear gradient of 50–300 mm KCl. CHIP-containing fractions were pooled, and the potassium concentration was adjusted to 50 mm. The fractions were loaded onto an HT Gel hydroxyapatite column (Bio-Rad), and elution of bound protein occurred in a linear gradient from 20 to 200 mm potassium phosphate at pH 7.0. CHIP-containing fractions were pooled, and the potassium concentration was adjusted below 120 mm. The fractions were applied to a Source 30Q column (Amersham Biosciences), and bound CHIP was eluted by a linear gradient of 120–300 mm KCl. CHIP-containing fractions were pooled and stored at −80 °C. 26 S proteasomes were purified from human red blood cells as described previously (24Emmerich N. Nussbaum A.K. Stevanovic S. Priemer M. Toes R.E. Rammensee H.G. Schild H. J. Biol. Chem. 2000; 275: 21140-21148Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Protein concentrations were determined using the Bio-Rad Bradford reagent with purified IgG (Sigma) as the standard. Mammalian cells were transiently transfected with a CalPhos transfection kit (Clontech) using pcDNA3.1-hbag-1S, pcDNA3.1-hbag-1M, pcDNA3.1-chip, and pcDNA3.1-HA-ubiquitinaccording to the protocol of the manufacturer. As control, plasmid pcDNA3.1-myc/his-lacZ was used. Usually, cell extracts were prepared 48 h post-transfection. Expression of BAG-1 isoforms in HeLa cells was analyzed after lysis of cells in RIPA buffer (25 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 10% glycerol, 2 mm EDTA) containing Complete protease inhibitor for 15 min on ice. Samples were centrifuged at 30,000 × g at 4 °C for 20 min, and the soluble fraction was used as cell lysate. Effects of CHIP overexpression on the ubiquitylation of BAG-1 in HEK 293 cells were analyzed with stably transfected cells. For this purpose the CHIP-expressing plasmid was linearized before transfection, and stably transfected cells were finally selected in the presence of 1,200 μg/ml Geneticin (Invitrogen). After selection stably transfected cells were cultivated in medium containing 600 μg/ml Geneticin. For immunoprecipitations of BAG-1 isoforms under denaturing conditions, HEK 293 cells were resuspended in buffer B (50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1% SDS, 5 mm dithiothreitol). The cell suspension was boiled for 5 min and diluted with 9 volumes of buffer C (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm EDTA, 1% Triton X-100) containing Complete protease inhibitor. The diluted lysate was passaged repeatedly through a thin needle to reduce the viscosity of the solution and cleared by ultracentrifugation at 100,000 × g at 4 °C for 30 min. To the resultant supernatant 2 μg of anti-BAG-1 antibody or control IgG was added, and samples were incubated for 1 h at 4 °C. After the addition of protein G-Sepharose, incubation was continued for an additional hour. Protein G-Sepharose-absorbed immunocomplexes were collected by centrifugation and washed five times with RIPA buffer followed by elution of immunocomplexes in SDS-PAGE loading buffer. Samples were analyzed by SDS-PAGE and immunoblotting. For coimmunoprecipitation of Hsc/Hsp70-associated BAG-1, transiently transfected HeLa cells were lysed in 25 mm Tris-HCl, pH 7.5, 100 mm KCl (buffer D) containing 0.5% Tween 20, 2 mm EDTA, and Complete protease inhibitor. Lysates were centrifuged for 10 min at 30,000 × g at 4 °C, and to the supernatant fractions 0.04 μg/μl anti-Hsc/Hsp70 or control IgG was added. Samples were incubated for 1 h at 4 °C, followed by the addition of protein G-Sepharose and further incubation for 1 h. Immunocomplexes were collected by centrifugation, washed four times with buffer D containing 0.5% Tween 20, once with buffer D lacking detergent, and bound BAG-1 was eluted by incubation for 15 min at 30 °C in buffer D containing 1 mm ATP, 2 mm MgCl2, and protease inhibitors. Protein G-Sepharose was collected by centrifugation, and ATP-eluted proteins were precipitated from the supernatant fraction by the addition of trichloroacetic acid. Precipitated protein was analyzed by SDS-PAGE and immunoblotting. Purified BAG-1M (0.6 μg in a 10-μl reaction) was incubated for 2 h at 30 °C in the presence of 20% rabbit reticulocyte lysate (Promega) in 20 mm MOPS, pH 7.2, 50 mm KCl, 0.1 mmdithiothreitol, containing 10 mm MgCl2, 5 mm ATP, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin. When indicated, 10 μg of purified ubiquitin and 0.4 μg of ubiquitin aldehyde (Affiniti) were added. In initial ubiquitylation reactions that explored the ubiquitylation activity of CHIP, histidine-tagged CHIP was used. Purified human BAG-1M and BAG-1S (each 0.6 μg/10-μl reaction) were incubated in the presence of 0.1 μm rabbit E1, 4 μm UbcH5b, 2 μm His-CHIP, and 2.5 μg/μl ubiquitin in 20 mm MOPS, pH 7.2, 100 mm KCl, 5 mm MgCl2, 10 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride (buffer E) for 2 h at 30 °C. Samples were analyzed by SDS-PAGE and immunoblotting with anti-BAG-1 antibody. To analyze ubiquitylation of the 4K-R mutant of BAG-1M, a soluble extract from BL21 (DE3) cells expressing the recombinant protein was prepared by sonication in buffer A and centrifugation at 100,000 × g for 30 min. 30 μg of the bacterial extract was used in the ubiquitylation assay. For ubiquitylation reactions that involved formation of Hsc70·BAG-1·CHIP complexes untagged CHIP was used to avoid steric hindrance. Purified human BAG-1S (2 μm) was incubated in the presence of 0.1 μm wheat E1, 8 μmUbcH5b, 3 μm CHIP, and 1.25 μg/μl ubiquitin (Sigma) in buffer E for 2 h at 30 °C. When indicated, ubiquitin was replaced by K48R ubiquitin at a concentration of 1.25 μg/μl, and purified Hsc70 and Hsp40 were added at a concentration of 3 and 0.3 μm, respectively. To analyze the linkage of the BAG-1-attached ubiquitin chain in more detail, ubiquitylation was performed as described above in the presence of soluble bacterial extracts containing point mutants of ubiquitin. Before the experiment, the ubiquitin concentration within the extracts was analyzed by immunoblotting, and the amount of extract added to the ubiquitylation reactions was adjusted so that similar amounts of ubiquitin and mutant ubiquitin were present during the reaction (about 0.3 μg/μl final concentration). Ubiquitylation in the presence ofN-methylated ubiquitin (1.25 μg/μl final concentration) was performed at an increased concentration of conjugation components to counteract the reduced conjugation efficiency of the methylated protein, i.e. 0.3 μm wheat E1, 24 μm UbcH5b, and 6 μm CHIP were used. Samples were analyzed by SDS-PAGE and immunoblotting. Binding of ubiquitylated BAG-1M to the ATPase domain of Hsc70 was assayed by previously established ATPase domain affinity chromatography (15Lüders J. Demand J. Höhfeld J. J. Biol. Chem. 2000; 275: 4613-4617Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Prior to ATPase domain binding, in vitro ubiquitylation of BAG-1M was performed as described above in the presence of reticulocyte lysate and purified ubiquitin. After incubation for 2 h, a 5-fold volume of 20 mm MOPS, pH 7.2, 150 mm KCl, 5 mm EDTA (buffer F) was added, and samples were incubated in the presence of the ATPase domain affinity resin for 30 min at 4 °C. The affinity resins were washed four times with buffer F followed by boiling in SDS-PAGE sample buffer. COS-7 cells were transiently transfected with 1.5 μg of pcDNA3.1-hbag-1S and 2.5 μg of pcDNA3.1-chip or pcDNA3.1-myc/his-lacZ/35-mm dish. 24 h post-transfection cells were washed with phosphate-buffered saline and incubated for an additional hour at 37 °C in 1 ml of methionine-free medium. Metabolic labeling was performed for 1 h by the addition of 50 μCi/ml [35S]methionine. The labeled cells were washed twice with phosphate-buffered saline and incubated further in medium supplemented with 25 μg/ml cycloheximide and 1 mmunlabeled methionine. At the indicated time points the cells were harvested, frozen immediately in fluid N2, and stored at −80 °C. For immunoprecipitation, cells were thawed on ice and lysed in RIPA buffer supplemented with Complete protease inhibitor. Cell lysates were cleared by centrifugation at 30,000 × gat 4 °C for 20 min. Protein concentration of the lysates was determined, and equal amounts of lysate protein were incubated with 1 μg of anti-BAG-1 antibody at 4 °C for 1 h followed by the addition of protein G-Sepharose and an additional incubation period for 1 h at 4 °C. The Sepharose was collected by centrifugation and washed four times with RIPA buffer, prior to elution of bound proteins by boiling in SDS-PAGE loading buffer. Samples were separated on an SDS-polyacrylamide gel and analyzed using a PhosphorImager. Quantification of bands was carried out with OptiQuant software version 3.0 (Packard Instrument). To analyze the effects of BAG-1 ubiquitylation on the association of the cofactor with the proteasome, 30-μl ubiquitylation reactions were performed in the presence of 6 μm BAG-1S, 6 μm C130, 0.1 μmwheat E1, 8 μm UbcH5b, 6 μm CHIP, 0.6 μm Hsp40, 6 μm Hsc70, and 2.5 μg/μl ubiquitin as indicated in buffer E for 2 h at 30 °C. Samples were cooled on ice followed by the addition of purified human 26 S proteasomes (16 μg/sample) and incubation for 1 h at 4 °C. Samples were diluted in 400 μl of 20 mm MOPS, pH 7.2, 100 mm KCl, 3 mm MgCl2, 2 mm ATP, 1 mm phenylmethylsulfonyl fluoride, and split into two equal aliquots. One aliquot received 5 μg of anti-BAG-1 antibody, the other one the same amount of control IgG. Aliquots were incubated at 4 °C for 1 h, protein G-Sepharose was added, and incubation was continued at 4 °C for an additional hour. The Sepharose was collected by centrifugation and washed four times with buffer G (20 mm MOPS, pH 7.2, 100 mmKCl, 0.5% Tween 20) and once with buffer G lacking detergent. Bound proteasomes were dissociated from the immunocomplexes by the addition of detergent-free buffer G containing 2 mmMgCl2, 1 mm ATP, and 1 mmphenylmethylsulfonyl fluoride and incubation at 30 °C for 20 min. The protein G-Sepharose was sedimented by centrifugation, and ATP-eluted proteins were precipitated from the supernatant fraction by the addition of trichloroacetic acid. Sepharose beads were washed again using buffer G followed by elution of BAG-1 through boiling in SDS-PAGE loading buffer. Samples were analyzed by SDS-PAGE and immunoblotting with specific antibodies. BAG-1 exists as multiple isoforms in human HeLa cells (25Takayama S. Krajewski S. Krajewska M. Kitada S. Zapata J.M. Kochel K. Knee D. Scudiero D. Tudor G. Miller G.J. Miyashita T. Yamada M. Reed J.C. Cancer Res. 1998; 58: 3116-3131PubMed Google Scholar). The most abundant isoforms are the nuclear BAG-1L (∼55 kDa apparent molecular mass) and the cytosolic BAG-1M (∼46 kDa) and BAG-1S (∼36 kDa) (Fig.1 A). However, after transient expression of BAG-1M and BAG-1S in human HeLa cells, the anti-BAG-1 antibody specifically detected additional polypeptides that possibly arose from covalent modification of the isoforms (Fig. 1 A,arrowheads). Modification led to an increase of the apparent molecular mass by about 10 kDa. It thus appeared conceivable that the isoforms were modified by attachment of a ubiquitin moiety. To verify this hypothesis a tagged form of ubiquitin carrying a hemagglutinin (HA) epitope was coexpressed with BAG-1S. Coexpression led to the appearance of a second modified form of BAG-1S, consistent with the attachment of both HA-tagged and endogenous ubiquitin (Fig.1 B). Apparently, the chaperone cofactor can be covalently modified by attachment of ubiquitin. To exclude the possibility that BAG-1 ubiquitylation was only a consequence of transient overexpression, endogenous BAG-1 was immunoprecipitated from human HEK 293 cells and analyzed for the presence of ubiquitylated isoforms by subsequent immunoblotting using an anti-ubiquitin antibody. The immunoprecipitation was performed under denaturing conditions to remove BAG-1-associated ubiquitylated proteins (9Demand J. Alberti S. Patterson C. Höhfeld J. Curr. Biol. 2001; 11: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). In this situation high molecular mass ubiquitin conjugates were still detectable in BAG-1 immunoprecipitates (Fig.2). The data reveal modification of endogenous BAG-1 isoforms by multiple ubiquitin attachment in vivo. We sought to reconstitute BAG-1 ubiquitylation in vitro. As a first step toward this goal rabbit reticulocyte lysate was used as a source for the ubiquitin-activating enzyme E1, ubiquitin-conjugating enzymes, and ubiquitin ligases. In t
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