Title: The Ubiquitin-related BAG-1 Provides a Link between the Molecular Chaperones Hsc70/Hsp70 and the Proteasome
Abstract: The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated. The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated. 70-kDa heat shock protein family minimal essential medium polyacrylamide gel electrophoresis phosphate-buffered saline Molecular chaperones of the 70-kDa heat shock protein family (Hsp70s)1 are involved in protein folding, protein translocation, and protein degradation (1.Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 31-52Google Scholar, 2.Hartl F.-U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3077) Google Scholar, 3.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar). To exert their function during such diverse cellular processes, Hsp70s cooperate with a multitude of chaperone cofactors. The cofactors either modulate the ATPase and peptide binding cycle of the chaperones or mediate their targeting to specific proteins and subcellular compartments (3.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar, 4.Cheetham M.E. Caplan A.J. Cell Stress Chap. 1998; 3: 28-36Crossref PubMed Scopus (480) Google Scholar, 5.Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 6.Höhfeld J. Biol. Chem. 1998; 379: 269-274PubMed Google Scholar). The BAG-1 protein is a recently identified cofactor of the constitutively expressed Hsc70 and the heat-inducible Hsp70 in the mammalian cytosol and nucleus (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 8.Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (433) Google Scholar, 9.Zeiner M. Gebauer M. Gehring U. EMBO J. 1997; 16: 5483-5490Crossref PubMed Scopus (148) Google Scholar, 10.Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (153) Google Scholar). In human HeLa cells at least three BAG-1 isoforms can be distinguished by the lengths of their amino termini: BAG-1 (termed BAG-1S throughout this study), BAG-1M (previously also termed Rap46/Hap46), and BAG-1L (11.Takayama 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) (see also Fig.1). The isoforms associate with Hsc70 and Hsp70 through binding to the ATPase domain of the chaperone proteins (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 8.Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (433) Google Scholar, 9.Zeiner M. Gebauer M. Gehring U. EMBO J. 1997; 16: 5483-5490Crossref PubMed Scopus (148) Google Scholar). Moreover, BAG-1M has been shown to stimulate the ATP hydrolytic activity of Hsc70 by accelerating ADP/ATP exchange (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 12.Gebauer M. Zeiner M. Gehring U. Mol. Cell. Biol. 1998; 18: 6238-6244Crossref PubMed Scopus (33) Google Scholar). BAG-1M thus fulfills a regulatory function opposite to that of another ATPase domain-binding protein, Hip, which stabilizes the ADP-bound state of Hsc70 (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 13.Höhfeld J. Minami Y. Hartl F.-U. Cell. 1995; 83: 589-598Abstract Full Text PDF PubMed Scopus (377) Google Scholar,14.Prapapanich V. Chen S. Nair S.C. Rimerman R.A. Smith D.F. Mol. Endocrinol. 1996; 10: 420-431PubMed Google Scholar). The antagonistic regulatory functions of BAG-1M and Hip severely affect the chaperone activity of Hsc70. While BAG-1M and also BAG-1S were shown to inhibit Hsc70-mediated protein folding in vitro, Hip promotes the folding capacity of the chaperone protein (8.Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (433) Google Scholar, 10.Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (153) Google Scholar, 15.Gebauer M. Zeiner M. Gehring U. FEBS Lett. 1997; 417: 109-113Crossref PubMed Scopus (87) Google Scholar, 16.Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar). It thus appears that balancing the intracellular levels of Hip and the BAG-1 isoforms might provide a central mechanism to influence the functional specificity of Hsc70/Hsp70. In this context it is important to note that overexpression of BAG-1 isoforms was frequently observed in tumor cells (11.Takayama 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, 17.Yang X. Chernenko G. Hao Y. Ding Z. Pater M.M. Pater A. Tang S.C. Oncogene. 1998; 17: 981-989Crossref PubMed Scopus (126) Google Scholar). Furthermore, gene transfer-mediated elevations of the level of BAG-1 isoforms cause a variety of cellular phenotypes, possibly through a modulation of Hsc70/Hsp70 activity. This includes increased resistance to apoptosis, enhanced cell proliferation, and altered transcriptional activity of steroid hormone receptors (18.Takayama S. Sato T. Krajewski S. Kochel K. Irie S. Millan J.A. Reed J.C. Cell. 1995; 80: 279-284Abstract Full Text PDF PubMed Scopus (793) Google Scholar, 19.Bardelli A. Longati P. Albero D. Goruppi S. Schneider C. Ponzetto C. Comoglio P.M. EMBO J. 1996; 15: 6205-6212Crossref PubMed Scopus (301) Google Scholar, 20.Froesch B.A. Takayama S. Reed J.C. J. Biol. Chem. 1998; 273: 11660-11666Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 21.Kullmann M. Schneikert J. Moll J. Heck S. Zeiner M. Gehring U. Cato A.C.B. J. Biol. Chem. 1998; 273: 14620-14625Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22.Liu R. Takayama S. Zheng Y. Froesch B. Chen G. Zhang X. Reed J.C. Zhang X.K. J. Biol. Chem. 1998; 273: 16985-16992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 23.Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (183) Google Scholar). A remarkable structural feature of the BAG-1 isoforms is the presence of a ubiquitin-like domain close to their amino termini (6.Höhfeld J. Biol. Chem. 1998; 379: 269-274PubMed Google Scholar, 18.Takayama S. Sato T. Krajewski S. Kochel K. Irie S. Millan J.A. Reed J.C. Cell. 1995; 80: 279-284Abstract Full Text PDF PubMed Scopus (793) Google Scholar) (see also Fig. 1). Ubiquitin is a 76-residue protein found in all eukaryotic organisms. It can be covalently attached to other protein substrates through the action of a complex enzymatic machinery required for substrate recognition, ubiquitin activation, and ubiquitin conjugation (24.Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 25.Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 26.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (511) Google Scholar, 27.Koegl M. Hoppe T. Schlenker S. Ulrich H.D. Mayer T.U. Jentsch S. Cell. 1999; 96: 635-644Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar). Conjugation of a multiubiquitin chain plays a well established role in targeting substrates to a high molecular mass proteolytic complex, the proteasome, for their degradation. Notably, the ubiquitin-like domain of BAG-1 is not required for Hsc70 binding and regulation but is essential for BAG-1 to fulfill its anti-apoptotic function (8.Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (433) Google Scholar, 16.Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 19.Bardelli A. Longati P. Albero D. Goruppi S. Schneider C. Ponzetto C. Comoglio P.M. EMBO J. 1996; 15: 6205-6212Crossref PubMed Scopus (301) Google Scholar). The presence of the ubiquitin-like domain within the chaperone cofactor suggests that BAG-1 may function as a link between Hsc70/Hsp70 and the ubiquitin/proteasome system. Here we provide evidence for this notion. We show that BAG-1 associates with the proteasome in an ATP-dependent manner and promotes binding of Hsc70 and Hsp70 to the proteolytic complex. BAG-1 can apparently act as a coupling factor between the Hsp70 chaperone system and the protein degradation machinery. Human BAG-1M and BAG-C were expressed in Sf9 insect cells using recombinant baculoviruses and were purified as described previously (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 16.Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar). The monoclonal anti-Hsc70/Hsp70 and monoclonal anti-Hsp90 antibodies were obtained from Stressgen; polyclonal anti-BAG-1/C-16 (recognizing the carboxyl-terminal 16 residues of human and mouse BAG-1) was from Santa Cruz Biotechnology. The antibody against Hip was raised in chicken using a recombinant deletion fragment of rat Hip (amino acids 1–257) as the source of antigene. For gene transfer-mediated expression of human BAG-1S, the corresponding cDNA was amplified by polymerase chain reaction using plasmid pVL1392-bag-1M (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar) as the template, and was subcloned into pcDNA3.1 (Invitrogen). As a control plasmid, pcDNA3.1B-myc/his-lacZ (Invitrogen) was used. Transfection of human HeLa cells was performed using the CalPhos mammalian transfection kit (CLONTECH). Transfection efficiency was monitored by cotransfection of a vector expressing green fluorescent protein using 1/10 of the amount of the bag-1carrying constructs. Efficiency was usually between 50% and 70%. For the preparation of mammalian cell extracts, HeLa cells were grown in minimal essential medium (MEM) (Life Technologies, Inc.) supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. At about 90% confluence, attached cells were washed two times with phosphate-buffered saline (PBS) and collected in PBS. HeLa cells were lysed in 25 mm Tris, pH 7.5, 100 mm KCl, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin (buffer A), containing 0.5% Tween 20. When indicated buffer A contained 2 mm MgCl2 and 1 mm ATP, or 5 mm EDTA. Lysates were centrifuged for 20 min at 30,000 × g, and the obtained supernatants were used as soluble cell extracts. To the cell extracts, antibodies were added as indicated. Rabbit anti-chicken IgGs were used as unrelated control antibody. Samples were incubated for 1 h at 4 °C with constant mixing, followed by addition of protein G-Sepharose and further incubation for 1 h at 4 °C. The immobilized immunocomplexes were collected by centrifugation, washed four times with buffer A containing 0.5% Tween 20, and washed once with buffer A lacking detergent. The immunocomplexes were subsequently incubated for 15 min at 30 °C in 500 μl of buffer A containing 2 mm MgCl2, 1 mm ATP, and protease inhibitors. The protein G-Sepharose was collected by centrifugation, and ATP-eluted proteins were precipitated from the supernatant fraction by addition of trichloroacetic acid. The Sepharose beads were washed again using buffer A, followed by elution of immunocomplexes through boiling in SDS-PAGE loading buffer. To analyze the effect of BAG-1M on the association of Hsc70/Hsp70 with the proteasome, HeLa cell extracts were prepared in buffer A containing 0.5% Tween 20, 2 mm MgCl2, and 1 mm ATP. When indicated, purified BAG-1M (3 μg of purified protein/1 mg of cell extract) or an equimolar amount of BAG-C were added and samples were incubated for 2 h at 4 °C. EDTA was added to a final concentration of 10 mm, and immunoprecipitations were performed as outlined above. HeLa cells were grown to about 80% confluence, and attached cells were washed with PBS and incubated in methionine- and cysteine-free MEM containing 20 μCi/ml35S-Redivue Pro-Mix (Amersham Pharmacia Biotech), 10% dialyzed fetal calf serum, penicillin, and streptomycin. Following labeling for 24 h under standard growth conditions, cells were collected and lysed as described above. In chase experiments, attached cells were washed once with prewarmed complete MEM, 10% fetal calf serum, and 2 mm methionine, followed by growth in the same medium for the indicated times. The BAG-1 isoforms expressed in human HeLa cells share a common ubiquitin-like domain within their amino-terminal portions (Fig. 1). Conceivably, the ubiquitin-like domain might serve as a signal to induce the rapid degradation of the BAG-1 isoforms by the proteasome. Therefore, the half-life of BAG-1 isoforms in HeLa cells was investigated. For this purpose, we first established immunoprecipitation conditions. HeLa cells were radiolabeled for 24 h to detect significant amounts of BAG-1L and BAG-1M in anti-BAG-1 immunoprecipitates (Fig.2 A). The radiolabeled isoforms were not precipitated when the antibody was blocked with purified BAG-1M (Fig. 2 A, lane 3). In this situation, an increased amount of Hsc70 and Hsp70 was coprecipitated due to an association of the chaperones with purified BAG-1M (arrowhead). Moreover, proteins of high molecular mass (>90 kDa) were coprecipitated, which we could identify as multiubiquitinated proteins that associate with the BAG-1 isoforms. 2J. Höhfeld, unpublished observations. The BAG-1S protein (apparent molecular mass of 36 kDa) was not detectable after immunoprecipitation. Possibly, BAG-1S resides in a distinct protein complex or displays an altered conformation in which the carboxyl terminus of the protein is not accessible to the anti-BAG-1 antibody. In pulse-chase experiments, we therefore focused on the readily detected BAG-1L and BAG-1M isoforms. During a 3-h chase, the amount of both isoforms decreased by about 20%, revealing a half-life of the two isoforms of about 7.5 h (Fig. 2 B). Apparently, BAG-1L and BAG-1M are rather stable proteins despite the presence of a ubiquitin-like domain. The data suggest that the domain may fulfill degradation-independent targeting functions. The BAG-1 isoforms resemble the Rad23 protein, which possesses a similar ubiquitin-like domain at its amino terminus, and utilizes this domain to bind to the proteasome in a stable manner (28.Watkins J.J. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar, 29.Schauber C. Chen L. Tongaonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (402) Google Scholar, 30.Russel S.J. Reed S.H. Huang W. Friedberg E.C. Johnston S.A. Mol. Cell. 1999; 3: 687-695Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). We therefore investigated whether BAG-1 forms stable complexes with the proteasome. Following immunoprecipitation of BAG-1 isoforms from HeLa cells, the C-8 subunit, a component of the 20 S catalytic core of the proteolytic complex, and the S-1 subunit of the regulatory particle of the proteasome were found in association with the chaperone cofactor (Fig.3). This reveals the existence of BAG-1/proteasome complexes in HeLa cells. The association of BAG-1 with the proteasome was destabilized in the presence of ATP (Fig. 3). Under the same conditions, Hsc70 and Hsp70 were also released form immunoprecipitated BAG-1. This raised the possibility that the cofactor's association with the proteolytic complex is mediated by the chaperone proteins. Alternatively, binding of BAG-1 to Hsc70/Hsp70 and to the proteasome, respectively, may represent independent events that are both regulated by ATP. To distinguish between these possibilities, we investigated the nucleotide requirements for the formation of the BAG-1/proteasome complex. For this purpose the antibody used for immunoprecipitations was saturated with purified BAG-1M protein. In this situation, endogenous BAG-1/proteasome complexes were no longer efficiently precipitated and binding of the proteasome to the purified, antibody-bound BAG-1M could be analyzed. In the presence of EDTA, proteasomes were not detectable in association with purified BAG-1M (Fig.4, lane 3), whereas endogenous BAG-1/proteasome complexes were stabilized (Fig. 4,lane 2). In contrast, when lysates were prepared and incubations were done in the presence of ATP and MgCl2the proteasome associated with purified BAG-1M (Fig. 4, lane 6). Thus, complex formation apparently requires ATP hydrolysis. Since ATP was also found to destabilize isolated BAG-1/proteasome complexes (see above), an equilibrium between free and complexed forms appears to exist in the presence of the nucleotide. Complexes that form transiently in this situation can be stabilized during isolation by washing immunopellets with nucleotide-free buffer and can finally be dissociated by ATP treatment. The requirement for ATP hydrolysis distinguishes the interaction of BAG-1 with the proteasome from the interaction of BAG-1 with Hsc70/Hsp70. While binding of the proteasome to added BAG-1M was blocked in the presence of EDTA, the chaperones efficiently associated with the cofactor in this situation (Fig. 4). The different nucleotide requirements for Hsc70 and proteasome binding to BAG-1 thus indicate two distinct binding events. Since BAG-1 associates with Hsc70/Hsp70 and the proteasome, the chaperone cofactor may act as a coupling factor between the chaperone machinery and the degradation system. To verify this notion the effect of BAG-1 on the association of Hsc70/Hsp70 with the proteasome was analyzed. Immunoprecipitation experiments from HeLa cell extracts were performed in the presence and absence of purified BAG-1M, respectively, using a monoclonal anti-Hsc70/Hsp70 antibody. To allow for ATP-dependent interactions of endogenous proteins,i.e. the proteasome, with added BAG-1M the extracts were prepared and initially incubated in the presence of ATP and MgCl2, followed by addition of an excess of EDTA to stabilize the formed complexes prior to immunoprecipitation. Association of BAG-1M with Hsc70/Hsp70 severely reduced the amount of the cofactor Hip bound to the chaperone proteins (Fig.5 A). This is in agreement with previous findings identifying Hip and BAG-1 as antagonistic regulators that compete with each other for an interaction with Hsc70 and Hsp70 (7.Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (335) Google Scholar, 31.Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Remarkably, the association of Hsc70/Hsp70 with the proteasome was significantly increased in the presence of BAG-1M, while a similar effect was not observed for binding of the chaperone proteins to Hsp90 (Fig. 5 A). Apparently, BAG-1 specifically promotes an interaction of Hsc70/Hsp70 with the proteasome. Notably, an induced association was observed using antibodies against the C-8 catalytic subunit as well as against the S-1 regulatory subunit of the proteasome (Fig. 5 B). We conclude that BAG-1 provides a link between Hsc70/Hsp70 and the apparently intact 26 S proteasome. Consistent with the in vitro observations, gene-transfer mediated elevation of the level of BAG-1S in HeLa cells resulted in an increased amount of proteasomes associated with Hsc70/Hsp70 (Fig.6 A). Similar findings were made when BAG-1M was overexpressed (data not shown). Apparently, the intracellular levels of BAG-1 isoforms significantly affect the amount of Hsc70/Hsp70 tethered to the proteasome. An induced association of Hsc70/Hsp70 with the proteasome was not observed in in vitro experiments using the carboxyl-terminal fragment BAG-C (Fig. 5 B). Although BAG-C is sufficient for Hsc70 binding and regulation, the fragment does not support targeting of Hsc70 to the proteasome. Apparently, the amino terminus of BAG-1, which includes the ubiquitin-like domain, is required for the targeting function of BAG-1. In this study an interaction of the chaperone cofactor BAG-1 with the proteasome is demonstrated. BAG-1 thus resembles Rad23, a protein involved in nucleotide excision repair in yeast and man that associates with the proteasome in a rather stable manner (28.Watkins J.J. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (215) Google Scholar, 29.Schauber C. Chen L. Tongaonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (402) Google Scholar, 30.Russel S.J. Reed S.H. Huang W. Friedberg E.C. Johnston S.A. Mol. Cell. 1999; 3: 687-695Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Both BAG-1 and Rad23 possess non-cleavable and non-transferable ubiquitin-like domains at their amino termini. The ubiquitin-like domain of Rad23 is in fact required for its association with the proteasome (29.Schauber C. Chen L. Tongaonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (402) Google Scholar). Similarly, we observed that the amino terminus of BAG-1, which includes the ubiquitin-like domain, is necessary for targeting of the chaperone cofactor to the proteasome (Fig. 5 B). We cannot exclude, however, that a hexapeptide repeat region preceding the ubiquitin-like domain (see Fig. 1, TRSEEX repeats) contributes to the interaction of BAG-1 with the proteasome. Deletion of the repeat region rendered the remaining fragment highly unstable and thus abolished attempts to demonstrate an exclusive role of the ubiquitin-like domain in targeting of BAG-1 to the proteasome. 3J. Demand, and J. Höhfeld, unpublished observations. The fact that the BAG-1S isoform contains only an incomplete repeat region (see Fig.1), but is still able to trigger proteasome association (Fig.6 A), points to the targeting function of the ubiquitin-like domain. Interaction of BAG-1 with the proteasome apparently includes the regulatory particle of the proteolytic complex. This particle comprises several ATPases of the AAA type that have been implicated in the recognition and unfolding of ubiquitinated substrates prior to their degradation (32.Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1295) Google Scholar, 33.Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell. 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The chaperones have well established roles in stabilizing non-native polypeptides (1.Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegelhoffer T. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 31-52Google Scholar, 2.Hartl F.-U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3077) Google Scholar, 3.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar). Moreover, Hsp70 family members were shown to participate in protein degradation (38.Straus D.B. Walter W.A. Gross C.A. Genes Dev. 1988; 2: 1851-1858Crossref PubMed Scopus (136) Google Scholar, 39.Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (207) Google Scholar, 40.Savel'ev A.S. Novikova L.A. Kovaleva I.E. Luzikov V.N. Neupert W. Langer T. J. Biol. 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However, BAG-1 does not appear to act as a general accelerator of protein degradation, and known target proteins of BAG-1 and Hsc70/Hsp70 are differently affected by the regulatory function of the chaperone cofactor. BAG-1 isoforms were shown to enhance the functions of Bcl-2, the Raf-1 protein kinase, androgen receptor, and the hepatocyte growth factor and platelet-derived growth factor receptors, while inhibiting retinoic acid receptor, glucocorticoid receptor, and Siah-1 (18.Takayama S. Sato T. Krajewski S. Kochel K. Irie S. Millan J.A. Reed J.C. Cell. 1995; 80: 279-284Abstract Full Text PDF PubMed Scopus (793) Google Scholar, 19.Bardelli A. Longati P. Albero D. Goruppi S. Schneider C. Ponzetto C. Comoglio P.M. EMBO J. 1996; 15: 6205-6212Crossref PubMed Scopus (301) Google Scholar, 20.Froesch B.A. Takayama S. Reed J.C. J. Biol. Chem. 1998; 273: 11660-11666Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 21.Kullmann M. Schneikert J. Moll J. Heck S. Zeiner M. Gehring U. Cato A.C.B. J. Biol. Chem. 1998; 273: 14620-14625Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22.Liu R. Takayama S. Zheng Y. Froesch B. Chen G. Zhang X. Reed J.C. Zhang X.K. J. Biol. Chem. 1998; 273: 16985-16992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 23.Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (183) Google Scholar). The interaction of BAG-1 with the proteasome may thus affect only a certain subset of substrates, whereas in other cases an Hsc70-regulating activity independent of the proteasome may be prevalent. Alternatively, BAG-1-mediated association of Hsc70 and Hsp70 with the proteasome may not result in the degradation of all substrates. The association of Rad23 with the regulatory particle of the proteasome may in fact have functions independent of protein degradation during nucleotide excision repair (30.Russel S.J. Reed S.H. Huang W. Friedberg E.C. Johnston S.A. Mol. Cell. 1999; 3: 687-695Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and a chaperone function of the regulatory particle of the proteasome was recently demonstrated (43.Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.-M. Finley D. Schmidt M. Nat. Cell Biol. 1999; 1: 221-226Crossref PubMed Scopus (386) Google Scholar). Further experimental approaches will be necessary to distinguish between these alternative functional concepts. BAG-1 might conceivably stimulate an association of Hsc70 and Hsp70 with the proteasome by inducing an association-competent state of the molecular chaperones. However, several lines of evidence argue against this notion. BAG-1 does not generally induce an association of Hsc70/Hsp70 with partner proteins, but specifically promotes a chaperone/proteasome interaction (Fig. 5 A). Moreover, the carboxyl-terminal domain of BAG-1, which is sufficient for Hsc70 binding and regulation (16.Lüders J. 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Prapapanich V. Rimerman R.A. Honore B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar, 45.Lassle M. Blatch G.L. Kundra V. Takatori T. Zetter B.R. J. Biol. Chem. 1997; 272: 1876-1884Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In this way, Hop facilitates the cooperation of Hsc70 and Hsp90 during the regulation of signal transduction pathways (46.Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1513) Google Scholar, 47.Caplan A. Trends Cell Biol. 1999; 9: 262-268Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Coupling factors apparently help to organize chaperone networks in the cellular environment. The BAG-1 isoforms seem to act as coupling factors at the interface between protein folding and protein degradation. We thank Stefan Jentsch for generous support and helpful discussions. We acknowledge Helle Ulrich for critical reading of the manuscript, and Erika Seemüller and Wolfgang Baumeister for the gift of anti-S1 antibody.