The Role of DnaJ-like Proteins in Glucocorticoid Receptor·hsp90 Heterocomplex Assembly by the Reconstituted hsp90·p60·hsp70 Foldosome Complex
1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês
10.1074/jbc.273.13.7358
ISSN1083-351X
AutoresKurt D. Dittmar, Maria Banach, Mario D. Galigniana, William B. Pratt,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoThe glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR·hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor·hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90·p60·hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047–13054). The resulting GR·hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997)J. Biol. Chem. 272, 21213–21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR·hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90·p60·hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23·hsp90·p60·hsp70·hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR·hsp90 heterocomplex isolated from L cell cytosol, it is in the GR·hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR·hsp90 heterocomplex assembly. The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR·hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor·hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90·p60·hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047–13054). The resulting GR·hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997)J. Biol. Chem. 272, 21213–21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR·hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90·p60·hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23·hsp90·p60·hsp70·hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR·hsp90 heterocomplex isolated from L cell cytosol, it is in the GR·hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR·hsp90 heterocomplex assembly. Several members of the nuclear receptor family, the dioxin receptor, and several protein kinases (e.g. Src and Raf) exist in multiprotein cytosolic complexes with the abundant heat shock protein (hsp) 1The abbreviations used are: hsp, heat shock protein; GR, glucocorticoid receptor; HBD, hormone binding domain; TA, triamcinolone acetonide; TPR, tetratricopeptide repeat; Src, pp60v-src; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. hsp90 (for review, see Refs. 1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1559) Google Scholar and 2Pratt W.B. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 297-326Crossref PubMed Google Scholar). These complexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with rabbit reticulocyte lysate (3Smith D.F. Schowalter D.B. Kost S.L. Toft D.O. Mol. Endocrinol. 1990; 4: 1704-1711Crossref PubMed Scopus (113) Google Scholar, 4Scherrer L.C. Dalman F.C. Massa E. Meshinchi S. Pratt W.B. J. Biol. Chem. 1990; 265: 21397-21400Abstract Full Text PDF PubMed Google Scholar, 5Hutchison K.A. Brott B.K. De Leon J.H. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1992; 267: 2902-2908Abstract Full Text PDF PubMed Google Scholar, 6Stancato L.F. Chow Y.-H. Hutchison K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar) or with concentrated cytosols prepared from a variety of animal, insect, and plant cells (7Stancato L.F. Hutchison K.A. Krishna P. Pratt W.B. Biochemistry. 1996; 35: 554-561Crossref PubMed Scopus (73) Google Scholar). The formation of these protein·hsp90 heterocomplexes reflects a dynamic assembly/disassembly process (8Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar), and regardless of the protein being complexed with hsp90, there appears to be a common mechanism of heterocomplex assembly (9Nair S.C. Toran E.J. Rimerman R.A. Hjermstad S. Smithgall T.E. Smith D.F. Cell Stress Chaperones. 1996; 1: 237-250Crossref PubMed Scopus (201) Google Scholar). Receptor·hsp90 heterocomplex assembly by reticulocyte lysate requires ATP/Mg2+, a monovalent cation, such as K+ (10Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar, 11Hutchison K.A. Czar M.J. Scherrer L.C. Pratt W.B. J. Biol. Chem. 1992; 267: 14047-14053Abstract Full Text PDF PubMed Google Scholar), and at least three proteins, hsp70 (10Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar, 12Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar), p60 (13Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 14Chen S. Prapapanich V. Rimerman R.A. Honoré B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar), and p23 (15Johnson J.L. Toft D.O. J. Biol. Chem. 1994; 269: 24989-24993Abstract Full Text PDF PubMed Google Scholar, 16Hutchison K.A. Stancato L.F. Owens-Grillo J.K. Johnson J.L. Krishna P. Toft D.O. Pratt W.B. J. Biol. Chem. 1995; 270: 18841-18847Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently, we have reconstituted the heterocomplex assembly system of reticulocyte lysate (13Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). We have also developed a minimal heterocomplex assembly system in which incubation of a GR immune pellet with a mixture containing purified rabbit hsp90 and hsp70, purified human p23, and bacterial lysate containing human p60 yields GR·hsp90 heterocomplex assembly and restoration of the steroid binding conformation of the HBD (13Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). hsp90, hsp70, and p60 are present in a common complex in reticulocyte lysate (17Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (252) Google Scholar), and Chen et al.(14Chen S. Prapapanich V. Rimerman R.A. Honoré B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar) have suggested that p60 binds to hsp70 via an N-terminal TPR (tetratricopeptide repeat) region and to hsp90 via a central TPR region. Mixture of the three proteins results in spontaneous formation of an hsp90·p60·hsp70 complex that can be adsorbed with an anti-p60 antibody, and the resulting immune complex converts the GR HBD to a steroid binding state in an ATP-dependent and K+-dependent manner (18Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Although hsp90, hsp70, and p60 are sufficient to alter the folding state of the GR HBD to produce a steroid binding site, the GR·hsp90 complexes that are formed rapidly disassemble unless p23 is present to stabilize them (19Dittmar K.D. Demady D.R. Stancato L.F. Krishna P. Pratt W.B. J. Biol. Chem. 1997; 272: 21213-21220Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). There is an ATP binding site in the N-terminal domain of hsp90 (20Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar), and Sullivan et al. (21Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) have shown that hsp90 assumes two conformations depending upon whether or not this site is occupied by ATP. When hsp90 is bound by ADP, it has a high affinity for a hydrophobic resin, and when bound by ATP, it has a low affinity for the hydrophobic resin (21Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In direct experiments, Sullivan et al. (21Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) have shown that p23 binds to the ATP-dependent state of hsp90 and stabilizes it in the conformation with low affinity for hydrophobic resin (21Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). This ATP-dependent conformation of hsp90 appears to be required for the GR HBD to have a steroid binding site, and binding of p23 to that state of GR-bound hsp90 stabilizes the GR·hsp90 heterocomplex to disassembly and loss of the binding site (19Dittmar K.D. Demady D.R. Stancato L.F. Krishna P. Pratt W.B. J. Biol. Chem. 1997; 272: 21213-21220Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Mammalian homologs of the bacterial DnaJ protein are often contaminants of purified hsp70 preparations, and DnaJ-like proteins are possible components of the reconstituted assembly system. In Escherichia coli, DnaJ interacts with DnaK, the bacterial hsp70 homolog, to stimulate its ATPase activity (22Liberek K. Marszalek J. Ang D. Georgopolous C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (711) Google Scholar). The resulting transition of DnaK to the ADP-bound state stabilizes the interaction of DnaK with protein substrates (23Langer T. Lu C. Echols H.J. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (839) Google Scholar). A variety of DnaJ-like proteins in eukaryotes also stimulate the ATPase activity of various members of the hsp70 protein family (for review, see Ref. 24Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (407) Google Scholar). One of the DnaJ-like proteins cloned from humans is hsp40 (25Ohtsuka K. Biochem. Biophys. Res. Commun. 1993; 197: 235-240Crossref PubMed Scopus (92) Google Scholar), and we use the term hsp40 here to refer to mammalian members of the DnaJ-like protein family. To date, hsp40 has not been identified either in native receptor·hsp90 heterocomplexes recovered from mammalian cells or in heterocomplexes assembled in reticulocyte lysate. However, the yeast DnaJ homolog YDJ-1 has been recovered in GR·hsp90 (26Chang H.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (194) Google Scholar) and v-Src·hsp90 heterocomplexes (27Dey B. Caplan A.J. Boschelli F. Mol. Biol. Cell. 1996; 7: 91-100Crossref PubMed Scopus (59) Google Scholar) isolated from yeast lysates. Both genetic and biochemical observations suggest that hsp40 may play a role in heterocomplex assembly by the hsp90-based chaperone system. Studies in yeast involving the genetic manipulation of chaperone activity have shown that hsp90 plays a critical role in determining the ligand responsiveness of steroid and dioxin receptors (Refs. 28Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Crossref PubMed Scopus (693) Google Scholar, 29Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428Crossref PubMed Scopus (193) Google Scholar, 30Xu Y. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (386) Google Scholar, 31Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (376) Google Scholar, 32Carver L.A. Jackiw V. Bradfield C.A. J. Biol. Chem. 1994; 269: 30109-30112Abstract Full Text PDF PubMed Google Scholar, 33Fang Y. Fliss A.E. Robins D.M. Caplan A.J. J. Biol. Chem. 1997; 271: 28697-28702Abstract Full Text Full Text PDF Scopus (190) Google Scholar and see Ref. 34Picard D. Freedman L.P. The Molecular Biology of Steroid and Nuclear Hormone Receptors. Birkhäuser Boston, Inc., Cambridge, MA1998: 1-18Google Scholar for review) and also growth arrest caused by v-Src (30Xu Y. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (386) Google Scholar,31Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (376) Google Scholar). Deletion of STI1, the yeast homolog of the p60 component of the hsp90-based chaperone system, reduces the in vivo activity of both the GR and v-Src (26Chang H.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (194) Google Scholar). Inasmuch as mutations in the gene for YDJ-1 also affect both steroid receptor and v-Src function in yeast (27Dey B. Caplan A.J. Boschelli F. Mol. Biol. Cell. 1996; 7: 91-100Crossref PubMed Scopus (59) Google Scholar, 35Kimura Y. Yahara I. Lindquist S. Science. 1995; 268: 1362-1365Crossref PubMed Scopus (220) Google Scholar, 36Caplan A.J. Langley E. Wilson E.M. Vidal J. J. Biol. Chem. 1995; 270: 5251-5257Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), it is possible that hsp40 is a component of the hsp90 heterocomplex assembly system. In biochemical studies, reticulocyte lysate has been utilized to promote chaperone-mediated refolding of denatured proteins. For example, Schumacher et al. (37Schumacher R.J. Hurst R. Sullivan W.P. McMahon N.J. Toft D.O. Matts R.L. J. Biol. Chem. 1994; 269: 9493-9499Abstract Full Text PDF PubMed Google Scholar) examined ATP-dependent refolding of thermally denatured firefly luciferase and found that reactivation of the enzyme activity correlated with the concentration of both hsp70 and hsp90. Additionally, both hsp70 and hsp90 immune pellets, as well as a mixture of purified hsp90 and hsp70, yielded partial luciferase reactivation (37Schumacher R.J. Hurst R. Sullivan W.P. McMahon N.J. Toft D.O. Matts R.L. J. Biol. Chem. 1994; 269: 9493-9499Abstract Full Text PDF PubMed Google Scholar). Subsequently, it was found that the purified preparations of hsp70 and hsp90 were contaminated with low levels of hsp40, and when hsp70 and hsp90 that were free of hsp40 were used to facilitate luciferase activation, the system had to be supplemented with a DnaJ-like protein (YDJ-1) to obtain renaturation activity (38Schumacher R.J. Hansen W.J. Freeman B.C. Alnemri E. Litwack G. Toft D.O. Biochemistry. 1996; 35: 14889-14898Crossref PubMed Scopus (144) Google Scholar). As DnaJ-like proteins have been found to operate in conjunction with eukaryotic hsp70s in several in vitro refolding systems (e.g. Refs. 39Freeman B.C. Myers M.P. Schumacher R. Morimoto R.I. EMBO J. 1995; 14: 2281-2292Crossref PubMed Scopus (385) Google Scholar, 40Levy E.J. McCarty J. Bukau B. Chirico W.J. FEBS Lett. 1995; 368: 435-440Crossref PubMed Scopus (68) Google Scholar, 41Freeman B.C. Morimoto R.I. EMBO J. 1996; 15: 2969-2979Crossref PubMed Scopus (385) Google Scholar), it is reasonable to ask whether they play a role in GR·hsp90 heterocomplex assembly by the minimal (hsp90, hsp70, p60, and p23) assembly system. In this work, we use a new commercial antibody against hsp40 to detect contamination of our purified rabbit hsp70 and hsp90 preparations with small amounts of hsp40. We also find that hsp40 is a component of the hsp90·p60·hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, and molybdate) a five-protein p23·hsp90·p60·hsp70·hsp40 heterocomplex is formed in reticulocyte lysate or from purified proteins. Elimination of rabbit hsp40 reduces the GR·hsp90 assembly activity of the purified system, and activity is restored by addition of the purified yeast homolog YDJ-1. We show that GR·hsp90 heterocomplexes assembled in reticulocyte lysate contain hsp40, but hsp40 is not a component of native GR·hsp90 heterocomplexes isolated from L cell cytosol. [6,7-3H]Triamcinolone acetonide (42.8 Ci/mmol) and125I-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from NEN Life Science Products. Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI). Protein A-Sepharose and goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates were from Sigma. The BuGR2 monoclonal IgG antibody against the GR was from Affinity Bioreagents (Golden, CO). The AC88 monoclonal IgG against hsp90, the anti-hsp40 rabbit polyclonal antibody, the N27F3-4 anti-72/73-kDa hsp monoclonal IgG (anti-hsp70), and purified DnaJ fromE. coli were from StressGen (Victoria, British Columbia, Canada). The JJ3 monoclonal IgG against p23, E. coliexpressing human p23, and purified YDJ-1 were gifts from Dr. David Toft (The Mayo Clinic). The DS14F5 monoclonal IgG against p60 and E. coli expressing p60 were kindly provided by Dr. David Smith (University of Nebraska Medical School). Actigel-ALD (activated aldehyde agarose) affinity support for protein immobilization was from Sterogene Biochemicals (San Gabriel, CA). Hybridoma cells producing FiGR monoclonal IgG against the GR were generously provided by Dr. Jack Bodwell (Dartmouth Medical School). L929 mouse fibroblasts (L cells) were grown in monolayer in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum. Cells were harvested by scraping into Earle's balanced saline, suspended in 1.5 volumes of HE buffer (10 mm Hepes, 1 mm EDTA, pH 7.4), and ruptured by Dounce homogenization. Homogenates were centrifuged for 1 h at 100,000 × g, and the supernatant is referred to as “cytosol.” Receptors were immunoadsorbed from 100-μl aliquots of L cell cytosol by rotation for 2 h at 4 °C with 8 μl of Actigel-ALD precoupled to 80 μl of FiGR ascites suspended in 300 μl of TEG (10 mm TES, pH 7.6, 50 mm NaCl, 4 mm EDTA, 10% glycerol). Prior to incubation with reticulocyte lysate or with other additions as noted, immunoadsorbed receptors were stripped of associated hsp90 by incubating the immunopellet an additional 2 h at 4 °C with 0.5m NaCl followed by one wash with 1 ml of TEG and a second wash with 1 ml of Hepes buffer (10 mm Hepes, pH 7.4). For immunoadsorption of p60, 400-μl aliquots of reticulocyte lysate or various mixtures of purified proteins as noted were immunoadsorbed to 8 μl of protein A-agarose prebound with DS14F5 antibody against p60 (5%) or nonimmune mouse IgG (5%). The samples were rotated at 4 °C for 2 h, and immunopellets were washed twice with 1 ml of Hepes buffer. FiGR immunopellets containing GR stripped of hsp90 were incubated with 50 μl of rabbit reticulocyte lysate or with combinations of proteins (12 μg of purified hsp90, 20 μg of purified hsp70, 3 μl of lysate from bacteria expressing p60, 4 μg of purified p23, 1.0 μg of purified YDJ-1, 1.0 μg of purified HDJ-1, or 0.5 μg of purified HDJ-2) and adjusted to 50 μl with HKD buffer (10 mmHepes, 100 mm KCl, 5 mm dithiothreitol, pH 7.35). In some experiments p23 was substituted with 20 mmsodium molybdate, which has the same effect on GR·hsp90 heterocomplex assembly (19Dittmar K.D. Demady D.R. Stancato L.F. Krishna P. Pratt W.B. J. Biol. Chem. 1997; 272: 21213-21220Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). For reconstitution of GR by the immunoadsorbed p60 heterocomplex, stripped receptors were suspended in 50 μl of an assay mix consisting of HKD buffer and then the whole GR immunopellet suspension was pipetted onto the DS14F5 immunopellet containing the immunoadsorbed p60 and the associated protein complex. Dithiothreitol (1 μl) was added to each incubation to a final concentration of 5 mm, and 5 μl of an ATP-regenerating system (50 mm ATP, 250 mm creatine phosphate, 20 mm MgOAc, and 100 units/ml creatine phosphokinase) were added to all assays to yield a final assay volume of 56 μl. The assay mixtures were incubated for 20 min at 30 °C with suspension of the pellets by shaking the tubes every 5 min for soluble protein conditions or every minute for the immunoadsorbed p60 condition. At the end of the incubation, the pellets were washed twice with 1 ml of ice-cold TEGM buffer (TEG buffer with 20 mm sodium molybdate) and assayed for steroid binding capacity and, in some experiments, receptor-associated proteins. To conserve the purified components of the reconstitution system, each experimental condition represents a single sample. The experimental observations have been replicated, and in most cases, the key observation from an experiment appears again as one of the conditions presented in another panel in the same figure or in one of the subsequent figures. Immune pellets to be assayed for steroid binding were incubated overnight in 100 μl of TEGM buffer plus 4 mm dithiothreitol and 50 nm[3H]triamcinolone acetonide. Samples were then washed twice with 1 ml of TEGM and counted by liquid scintillation spectrometry as described previously. The steroid binding is expressed as counts/min of [3H]triamcinolone acetonide-bound/FiGR immunopellet prepared from 100 μl of cytosol. As noted previously (11Hutchison K.A. Czar M.J. Scherrer L.C. Pratt W.B. J. Biol. Chem. 1992; 267: 14047-14053Abstract Full Text PDF PubMed Google Scholar), 100 μl of L cell cytosol contains 60,000 cpm of [3H]triamcinolone acetonide binding capacity, and we immunoadsorb about 50% of the GR. Thus ∼30,000 cpm represents 100% of receptors reactivated to the steroid binding form. For assay of GR and associated proteins or p60 and associated proteins, immune pellets were boiled in SDS sample buffer with 10% β-mercaptoethanol, and proteins were resolved on 10% SDS-polyacrylamide gels (12% for resolving p23). Proteins were then transferred to Immobilon-P membranes and probed with 2 μg/ml BuGR monoclonal antibody for the GR, 1 μg/ml AC88 for hsp90, 1 μg/ml N27F3-4 for hsp70, 0.1% DS14F5 anti-p60 mouse ascites for p60, 0.5% anti-hsp40 for hsp40, or 0.1% JJ3 mouse ascites for p23. The immunoblots were then incubated a second time with the appropriate 125I-conjugated or horseradish peroxidase-conjugated counterantibody to visualize the immunoreactive bands. It is important to note that hsp40 is very sticky and that immunoadsorption of p60 from reticulocyte lysate or from a mixture of purified proteins yields substantial amounts of hsp40 in nonimmune as well as immune pellets. Thus, 0.02% Nonidet P-40 was added to lysates and protein solutions during immunoadsorption, and the resulting protein A pellets were washed once with 1 ml of detergent-containing (0.02% Nonidet P-40) buffer prior to two washes with detergent-free buffer. This markedly reduces or eliminates nonspecific hsp40 and YDJ-1 binding. The bacterial expression of human p23 and its purification have been described (15Johnson J.L. Toft D.O. J. Biol. Chem. 1994; 269: 24989-24993Abstract Full Text PDF PubMed Google Scholar). Briefly, p23 is soluble in bacterial lysates, and its abundance and high affinity for DEAE-cellulose allowed purification to 90% purity by chromatography on DEAE-cellulose. The protein was concentrated by precipitation with ammonium sulfate at 80% of saturation. It was dissolved and dialyzed into 10 mm Tris, 100 mm KCl, and 10% glycerol, pH 7.4, and stored at −70 °C. hsp70 and hsp90 were routinely purified from rabbit brain cytosol as described previously (12Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar). Briefly, 20 ml of brain cytosol were chromatographed on a DE52 column exactly as described by Dittmaret al. (13Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Fractions containing hsp70 were chromatographed on an ATP-agarose column and eluted with ATP followed by ammonium sulfate precipitation, and DE52 fractions containing hsp90 were chromatographed on hydroxylapatite followed by chromatography over ATP-agarose exactly as described by Hutchison et al. (12Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar). To prepare hsp40-free hsp70, the fractions eluting prior to hsp90 from the DE52 column (fraction A of Dittmar et al. (13Dittmar K.D. Hutchison K.A. Owens-Grillo J.K. Pratt W.B. J. Biol. Chem. 1996; 271: 12833-12839Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) were chromatographed on hydroxylapatite before chromatography over ATP-agarose. To prepare hsp40-free hsp90, the routine three-step (DE52, hydroxylapatite, and ATP-agarose) procedure was followed, but at each step a sample from every fraction in the hsp90-containing region of the DE52 and hydroxylapatite eluates was immunoblotted for both hsp90 and hsp40. With very conservative pooling of peak hsp90 fractions containing the least hsp40, hsp40-free hsp90 is obtained. This procedure increases the purification time by ∼50%, and we have noted that the activity of hsp90 in the purified reconstitution system declines as the time of purification is extended. Thus, it is important to purify hsp90 in as few days as possible. In that we do not see cleavage products by immunoblot, it is unlikely that the inactivation is due to proteolysis. The purified hsp70 and hsp90 were dialyzed against HKD buffer, flash frozen, and stored at −70 °C. The bacterial expression and purification of YDJ-1 (42Caplan A.J. Tsai J. Casey P.J. Douglas M.G. J. Biol. Chem. 1992; 267: 18890-18895Abstract Full Text PDF PubMed Google Scholar) has been described (38Schumacher R.J. Hansen W.J. Freeman B.C. Alnemri E. Litwack G. Toft D.O. Biochemistry. 1996; 35: 14889-14898Crossref PubMed Scopus (144) Google Scholar). Briefly, bacterial sonicates were cleared by centrifugation, and YDJ-1 was purified by sequential chromatography on DEAE-cellulose and hydroxylapatite. The pooled YDJ-1 fractions (identified by gel electophoresis) were concentrated by vacuum dialysis against 10 mm Tris, pH 7.5, 10 mmdithiothreitol, 50 mm KCl, 10% glycerol, and aliquots were stored at −70 °C. The final preparation is about 80% pure. The bacterial expression of p60 has been described previously (4Scherrer L.C. Dalman F.C. Massa E. Meshinchi S. Pratt W.B. J. Biol. Chem. 1990; 265: 21397-21400Abstract Full Text PDF PubMed Google Scholar). Control E. coli and bacteria expressing p60 were grown to an A600 of 0.6, induced with isopropyl-1-thio-β-d-galactopyranoside for 3 h at 25 °C, and harvested. Bacterial lysates were prepared by sonication in phosphate-buffered saline, and aliquots were flash frozen and stored at −70 °C. In the experiment of Fig. 1 A, GR immune pellets that were stripped free of receptor-associated proteins with salt were incubated with the purified assembly system (hsp90, hsp70, p60, and p23) and various concentrations of the yeast DnaJ-like protein YDJ-1. Addition of the purified YDJ-1 produces a small increase in the level of steroid binding activity that is generated by the purified assembly system (Fig. 1 A). The increase in steroid binding activity is accompanied by a small increase in the amount of GR·hsp90 heterocomplexes that are assembled (Fig. 1 B). The YDJ-1 preparation used here is very active in promoting the activation of thermally denatured luciferase by hsp70 (38Schumacher R.J. Hansen W.J. Freeman B.C. Alnem
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