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Folding of the Glucocorticoid Receptor by the Heat Shock Protein (hsp) 90-based Chaperone Machinery

1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês

10.1074/jbc.272.34.21213

ISSN

1083-351X

Autores

Kurt D. Dittmar, Damon R. Demady, Louis F. Stancato, Priti Krishna, William B. Pratt,

Tópico(s)

thermodynamics and calorimetric analyses

Resumo

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor·hsp90 heterocomplexes. We have reconstituted a minimal receptor·hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR·hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997)J. Biol. Chem. 272, 13047–13054). In this work, we show that addition of p23 to native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR·hsp90 heterocomplexes prepared with the minimal (hsp90·p60·hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR·hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90·p60·hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR·hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90·p60·hsp70 forms a GR·hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex to inactivation and disassembly. In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor·hsp90 heterocomplexes. We have reconstituted a minimal receptor·hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR·hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997)J. Biol. Chem. 272, 13047–13054). In this work, we show that addition of p23 to native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR·hsp90 heterocomplexes prepared with the minimal (hsp90·p60·hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR·hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90·p60·hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR·hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90·p60·hsp70 forms a GR·hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex to inactivation and disassembly. Steroid receptors are recovered from hormone-free mammalian cells in multiprotein complexes that contain hsp90, 1The abbreviations used are: hsp, heat shock protein; GR, glucocorticoid receptor; HBD, hormone binding domain; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.1The abbreviations used are: hsp, heat shock protein; GR, glucocorticoid receptor; HBD, hormone binding domain; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. a 23-kDa protein (p23), one of several immunophilins, and, often, substoichiometric amounts of hsp70 (for review, see Refs. 1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1514) Google Scholar and 2Pratt W.B. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 297-326Crossref PubMed Google Scholar). Several protein kinases involved in signal transduction (e.g. Src, Raf) exist in similar cytosolic heterocomplexes with hsp90 (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1514) Google Scholar, 2Pratt W.B. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 297-326Crossref PubMed Google Scholar), and genetic experiments in yeast show that hsp90 is an integral component of both steroid receptor and protein kinase signaling pathways (see Nathan and Lindquist (3Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (368) Google Scholar) and references therein). Steroid receptor·hsp90 heterocomplexes can be created in vitro by incubating immunoadsorbed, hormone-free receptors with rabbit reticulocyte lysate (4Smith D.F. Schowalter D.B. Kost S.L. Toft D.O. Mol. Endocrinol. 1990; 4: 1704-1711Crossref PubMed Scopus (112) Google Scholar, 5Scherrer 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). This cell-free heterocomplex assembly system also forms heterocomplexes between hsp90 and protein kinases, such as Src, Raf, and Mek (6Hutchison 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, 7Stancato 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, 8Stancato L.F. Silverstein A.M. Owens-Grillo J.K. Chow Y.-H. Jove R. Pratt W.B. J. Biol. Chem. 1997; 272: 4013-4020Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In that concentrated lysates from mammalian, insect, and even plant cells are able to assemble the glucocorticoid receptor (GR) into a complex with hsp90 (9Stancato L.F. Hutchison K.A. Krishna P. Pratt W.B. Biochemistry. 1996; 35: 554-561Crossref PubMed Scopus (73) Google Scholar), it is likely the heterocomplex assembly system performs an ubiquitous and basic cellular function. The hormone binding domain (HBD) of the GR must be bound to hsp90 for it to bind steroid (10Bresnick E.H. Dalman F.C. Sanchez E.R. Pratt W.B. J. Biol. Chem. 1989; 264: 4992-4997Abstract Full Text PDF PubMed Google Scholar), and the heterocomplex assembly system converts the GR HBD from a non-steroid-binding to a steroid-binding form (5Scherrer 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,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). Physical studies suggest that the HBD of the hsp90-free GR is in a folded conformation in which the steroid-binding pocket is not accessible to ligand and that the hsp90-bound HBD may be partially unfolded, such that a hydrophobic binding pocket is accessible to the ligand (12Stancato L.F. Silverstein A.M. Gitler C. Groner B. Pratt W.B. J. Biol. Chem. 1996; 271: 8831-8836Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Using formation of steroid binding sites as a rapid assay of “folding” 2In this report, we will use the word “folding” to encompass a change in the folding state of the receptor HBD, which may be toward either a more folded state or a partially unfolded state, depending upon the absence or presence of hsp90.2In this report, we will use the word “folding” to encompass a change in the folding state of the receptor HBD, which may be toward either a more folded state or a partially unfolded state, depending upon the absence or presence of hsp90. as well as direct assay of receptor· hsp90 complex assembly, several details of the assembly mechanism have been established. Assembly of receptor·hsp90 complexes requires ATP/Mg2+, a monovalent cation, such as K+ (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, 13Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar), and at least three proteins, hsp70 (13Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar, 14Hutchison 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 (15Dittmar 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 (145) Google Scholar), and p23 (16Johnson J.L. Toft D.O. J. Biol. Chem. 1994; 269: 24989-24993Abstract Full Text PDF PubMed Google Scholar, 17Hutchison 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). Although the mammalian DnaJ homolog, hsp40, has not been demonstrated in receptor heterocomplexes, it is possible that it also is required for heterocomplex assembly. The p60 component of the assembly system was first identified by Smithet al. (13Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar) in progesterone receptor heterocomplexes formed in reticulocyte lysate when ATP was limiting. The rabbit p60 (18Smith 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 (246) Google Scholar) was then shown to be the homolog of a human protein that had been cloned by Honoré et al. (19Honoré B. Leffers H. Madsen P. Rasmussen H.H. Vanderkerckhove J. Celis J.E. J. Biol. Chem. 1992; 267: 8485-8491Abstract Full Text PDF PubMed Google Scholar) and a homolog of the nonessential yeast heat shock protein Sti1 (20Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (201) Google Scholar). Smith (21Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar) showed that p60 associates with progesterone receptors incubated with reticulocyte lysate early in the heterocomplex assembly process and then exits the complex. Chen et al. (22Chen S. Prapapanich V. Rimerman R.A. Honoré B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar) have shown that p60 binds to hsp70 via an N-terminal tetratricopeptide repeat region, and it binds to hsp90 via a central tetratricopeptide repeat region to form an hsp90·p60·hsp70 complex. Recently, we have demonstrated that the mixture of purified rabbit hsp90 and hsp70 with bacterial lysate containing human p60 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 (23Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In this minimal reconstituted system, conversion of the GR HBD to the steroid binding state was detected by incubating the GR with the chaperones in the presence of [3H]triamcinolone acetonide, which binds to the receptor as soon as GR·hsp90 complexes are formed. Thus, despite the fact that the heterocomplexes formed by this minimal system undergo rapid inactivation and/or disassembly, evidence of proper folding of the GR HBD to the steroid binding conformation could be preserved. To demonstrate maximum formation of GR·hsp90 heterocomplexes directly by immunoblotting, p23 must be present in the heterocomplex assembly mixture along with hsp90, hsp70, and p60 (23Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). p23 is a 23-kDa acidic protein that was first coimmunoadsorbed from cell lysates with the chicken progesterone receptor (24Smith D.F. Faber L.E. Toft D.O. J. Biol. Chem. 1990; 265: 3996-4003Abstract Full Text PDF PubMed Google Scholar) and the murine GR (25Bresnick E.H. Dalman F.C. Pratt W.B. Biochemistry. 1990; 29: 520-527Crossref PubMed Scopus (56) Google Scholar). Toft and his co-workers prepared the human cDNA and showed that p23 was a unique and ubiquitous, 160- amino acid protein with an aspartic acid-rich COOH-terminal domain (26Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (179) Google Scholar). p23 binds directly to hsp90 by a process that requires ATP (27Johnson J.L. Toft D.O. Mol. Endocrinol. 1995; 9: 670-678Crossref PubMed Scopus (211) Google Scholar, 28Johnson J. Corbisier R. Stensgard B. Toft D.O. J. Steroid Biochem. Mol. Biol. 1996; 56: 31-37Crossref PubMed Scopus (64) Google Scholar). Interestingly, when human p23 is added to wheat germ extract, it binds to plant hsp90 in an ATP-dependent manner (29Owens-Grillo J.K. Stancato L.F. Hoffman K. Pratt W.B. Krishna P. Biochemistry. 1996; 35: 15249-15255Crossref PubMed Scopus (73) Google Scholar), and it has the same effect on GR heterocomplex assembly by plant chaperones as it does when added to a heterocomplex reconstitution mixture containing purified mammalian hsp90·p60· hsp70 (17Hutchison 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). This suggests that the binding of p23 to hsp90 is a highly conserved protein interaction in eukaryotic cells. A recent study of hsp90 binding to phenyl-Sepharose suggests that hsp90 can exist in two functional states; in one state, hsp90 is bound by ADP and has a high affinity for hydrophobic resin, and in the other state, hsp90 is bound by ATP and has a low affinity for hydrophobic resin (30Sullivan 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 (223) Google Scholar). In direct experiments utilizing the purified proteins, it was shown that p23 binds to the ATP-dependent state of hsp90 and stabilizes it in the conformation with low affinity for hydrophobic resin (30Sullivan 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 (223) Google Scholar). Although p23 is required for the reconstituted assembly system to form stable GR·hsp90 heterocomplexes (23Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), it is not known whether p23 accelerates the rate of heterocomplex assembly or stabilizes assembled heterocomplexes, or both. It is also not known at what stage in the assembly process p23 can enter receptor heterocomplex. In this work, we show that p23 engages in a dynamic association with hsp90, not only when hsp90 is free as shown by Toft and his co-workers (27Johnson J.L. Toft D.O. Mol. Endocrinol. 1995; 9: 670-678Crossref PubMed Scopus (211) Google Scholar, 30Sullivan 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 (223) Google Scholar) but to hsp90 in GR·hsp90·p60·hsp70 complexes formed by the minimal assembly system and also to native GR·hsp90 complexes formed in intact cells. In contrast to the ATP dependence of p23 association with free hsp90, the association of p23 with hsp90 that is complexed with the GR does not require ATP. p23 stabilizes GR·hsp90 heterocomplexes once they are formed and does not appear to accelerate their rate of formation. Molybdate has the same effect as p23 in the reconstituted heterocomplex assembly system except when purified rabbit hsp90 is replaced with a purified, recombinant plant hsp90, which has equivalent heterocomplex forming activity and is stabilized by p23 but not by molybdate. Considering our observations together with Toft laboratory's observation that p23 binds an ATP-dependent state of hsp90, we would propose that the ATP-dependent conformation of hsp90 is required for the HBD to have a high affinity steroid binding site and that binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex against inactivation and disassembly. [6,7-3H]Triamcinolone acetonide (42.8 Ci/mmol) and125I-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from DuPont NEN. Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI), and wheat germ extract was from Promega. 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 and the N27F3-4 anti-72/73-kDa hsp monoclonal IgG (anti-hsp70) were from StressGen (Victoria, British Columbia). The JJ3 monoclonal IgG against p23 and Escherichia coli expressing human p23 were gifts from Dr. David Toft (The Mayo Clinic). The DS14F5 monoclonal IgG against p60 and E. coliexpressing 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 a mixture 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, 12.5 μg of purified p23, 3 μl of lysate from bacteria expressing p60) and adjusted to 50 μl with HKD buffer (10 mm Hepes, 100 mm KCl, 5 mm dithiothreitol, pH 7.35). 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 its associated proteins. 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 mmcreatine 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 mmsodium 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 5 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 [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 60,000–80,000 cpm [3H]triamcinolone acetonide binding capacity and we immunoadsorb about 50% of the GR. Thus, reactivation of 100% of receptors to the steroid binding form represents 30,000–40,000 cpm of binding activity. 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 7% 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, or 0.1% JJ3 mouse ascites for p23. The immunoblots were then incubated a second time with the appropriate125I-conjugated counterantibody to visualize the immunoreactive bands. The bacterial expression of human p23 and its purification have been described by Johnson and Toft (16Johnson 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 mmKCl, and 10% glycerol, pH 7.4, and stored at −70 °C. Rabbit hsp70 and hsp90 were purified from brain cytosol as described previously (14Hutchison 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, reticulocyte lysate was chromatographed on a DE52 column exactly as described by Dittmar et al. (15Dittmar 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 (145) 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. (14Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar). The purified hsp70 and hsp90 were dialyzed against HKD buffer, flash frozen, and stored at −70 °C. For expression and purification of plant hsp90, the Brassica napus hsp90-1 cDNA clone (31Krishna P. Sacco M. Cherutti J.F. Hill S. Plant Physiol. 1995; 107: 915-923Crossref PubMed Scopus (118) Google Scholar) was modified to add six histidines at the COOH terminus and expressed in Sf9 insect cells to prepare a recombinant, tagged hsp90. 3P. Mahnhoon, C. Y. Kang, and P. Krishna, submitted for publication. The recombinant protein was purified over a Ni2+-nitrilotriacetic acid-agarose column and its identity confirmed with the plant hsp90-specific rabbit R2 antiserum (31Krishna P. Sacco M. Cherutti J.F. Hill S. Plant Physiol. 1995; 107: 915-923Crossref PubMed Scopus (118) Google Scholar). The bacterial expression of p60 has been described previously by Johnson et al. (26Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (179) Google Scholar). ControlE. coli and bacteria expressing p60 were grown to anA600 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, p60 was immunoadsorbed from rabbit reticulocyte lysate, and the immune pellet was washed and incubated with GR immune pellets that had been stripped of associated proteins by washing them with salt. Fig. 1 A shows the composition of the washed p60 immune pellet. This native p60 heterocomplex contains hsp90 and hsp70, but not p23. In Fig. 1 B it can be seen that the immune pellet containing p60 and its coadsorbed proteins generates only a modest number of steroid binding sites (lane 4) and that their formation is ATP-dependent (cf. lanes 3and 4). In this experiment, steroid binding activity was assayed in the usual manner by incubating the immunoadsorbed GR with [3H]triamcinolone acetonide after heterocomplex assembly at 30 °C. When molybdate is present to stabilize the GR·hsp90 complexes as they are formed, then there are more steroid binding sites at the end of the incubation (cf. lane 6 with lane 4). In the presence of p23 (lane 8), GR reactivation is nearly to the level achieved with reticulocyte lysate (lane 2). It is interesting that addition of p23 to reticulocyte lysate increases the extent of GR reactivation by 20–50% (data not shown), suggesting that p23 may be limiting in the lysate. To examine the relationship between p23 potentiation of steroid binding activity and hsp90 association with the receptor, stripped GR immune pellets were incubated with the minimal assembly system consisting of purified rabbit hsp90 and hsp70 and bacterial lysate containing expressed human p60. As shown in Fig. 2, when stripped receptors (lane 1) are incubated with rabbit reticulocyte lysate (lane 2), they become associated with hsp90 and hsp70 and they are activated to the steroid binding state (it will be shown later (Fig. 7 A) that GR·hsp90 complexes assembled in reticulocyte lysate also contain p23). Incubation with the minimal assembly system produces a GR·hsp90·p60·hsp70 complex (lane 3) with low steroid binding activity. It is not known why p60 is present in GR·hsp90 heterocomplexes formed with purified hsp90·p60· hsp70 and not in complexes formed by reticulocyte lysate, but we have previously suggested that reticulocyte lysate must contain an as yet unidentified activity that facilitates the exit of p60 from the receptor heterocomplex and is not present in the reconstituted system (23Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).Figure 7Binding of p23 to hsp90 in different states. A, presence of p23 in reconstituted GR·hsp90 heterocomplexes. Stripped nonimmune or GR immune pellets were incubated for 20 min at 30 °C with rabbit reticulocyte lysate or with an assembly mixture containing purified hsp90, hsp70, p60, and p23. GR and associated proteins were assayed by Western blotting. Lanes 1 and 2, nonimmune and GR immune pellets, respectively, incubated with reticulocyte lysate; lanes 3 and4, nonimmune and GR immune pellets, respectively, incubated with hsp90, hsp70, p60, and p23. B, p23 does not bind to the hsp90·p60·hsp70 complex. Purified hsp90, hsp70, p60, and p23 were incubated at 30 °C with an ATP-generating system under the same conditions as for GR·hsp90 heterocomplex reconstitution except that no GR immune pellet was present. In the samples of lanes 3and 4, 0.02% Nonidet P-40 was also present during the incubation. Samples were immunoadsorbed with nonimmune IgG or with the JJ3 monoclonal IgG against p23. Lanes 1 and 2, nonimmune and p23 immune pellets, respectively, from mixtures incubated without Nonidet P-40; lanes 3 and 4, nonimmune and p23 immune pellets, respectively, from mixtures incubated with 0.02% Nonidet P-40. C, binding of purified p23 to purified hsp90. Purified hsp90 and p23 were incubated at 30 °C with an ATP-generating system, p23 was immunoadsorbed, and the two proteins were assayed by Western blotting. Lanes 1 and2, nonimmune and p23 immune pellets from mixtures incubated without Nonidet P-40; lanes 3 and 4, nonimmune and p23 immune pelle

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