Stoichiometry, Abundance, and Functional Significance of the hsp90/hsp70-based Multiprotein Chaperone Machinery in Reticulocyte Lysate
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.m103773200
ISSN1083-351X
AutoresPatrick J. Murphy, Kimon C. Kanelakis, Mario D. Galigniana, Yoshihiro Morishima, William B. Pratt,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoRabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90·Hop·hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR·hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90·Hop·hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for ∼30% of the hsp90 and ∼9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR·hsp90 assembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90·hsp70 complexes with a stoichiometry of 2:1. Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the hormone binding domain of the receptor to its high affinity steroid binding state. This system has been resolved into five proteins, with hsp90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperones that optimize assembly. Hop binds independently to hsp70 and hsp90 to form an hsp90·Hop·hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rejected any role for Hop in GR·hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90·Hop·hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for ∼30% of the hsp90 and ∼9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR·hsp90 assembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90·hsp70 complexes with a stoichiometry of 2:1. heat shock protein glucocorticoid receptor ligand binding domain tetratricopeptide repeat hsp70/hsp90 organizing protein 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid Unliganded steroid receptors exist in cytosols in a heterocomplex with the ubiquitous protein chaperone hsp901 (for review, see Ref.1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar). Hsp90 binds to the ligand binding domain (LBD) of the receptors (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar), and the glucocorticoid receptor (GR) LBD must be bound to hsp90 for the receptor to have high affinity steroid binding activity (2Bresnick E.H. Dalman F.C. Sanchez E.R. Pratt W.B. J. Biol. Chem. 1989; 264: 4992-4997Abstract Full Text PDF PubMed Google Scholar, 3Hutchison K.A. Czar M.J. Scherrer L.C. Pratt W.B. J. Biol. Chem. 1992; 267: 14047-14053Abstract Full Text PDF PubMed Google Scholar). The receptor·hsp90 heterocomplexes are assembled by a multiprotein chaperone system that was first studied in reticulocyte lysate (4Smith D.F. Schowalter D.B. Kost S.L. Toft D.O. Mol. Endocrinol. 1990; 4: 1704-1711Crossref PubMed Scopus (113) 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). Both biochemical data (6Stancato 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 (36) Google Scholar) and data from GR mutants (7Xu M. Dittmar K.D. Giannoukos G. Pratt W.B. Simons Jr., S.S. J. Biol. Chem. 1998; 273: 13918-13924Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Giannoukos G. Silverstein A.M. Pratt W.B. Simons Jr., S.S. J. Biol. Chem. 1999; 274: 36527-36536Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) support a model (3Hutchison K.A. Czar M.J. Scherrer L.C. Pratt W.B. J. Biol. Chem. 1992; 267: 14047-14053Abstract Full Text PDF PubMed Google Scholar) in which the hydrophobic ligand binding cleft in the LBD is opened to access by steroid during heterocomplex assembly. The assembly system in reticulocyte lysate has been reconstituted (9Dittmar 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 a mixture of five purified proteins, hsp90, hsp70, 2In this report, we use the term “hsp70” collectively to refer to both the heat shock-induced hsp70 and the constitutively expressed heat shock cognate hsc70.2In this report, we use the term “hsp70” collectively to refer to both the heat shock-induced hsp70 and the constitutively expressed heat shock cognate hsc70. Hop, hsp40, and p23, is now used to achieve optimal receptor·hsp90 heterocomplex assembly (10Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Kosano H. Stensgard B. Charlesworth M.C. McMahon N. Toft D. J. Biol. Chem. 1998; 273: 32973-32979Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar).The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR·hsp90 heterocomplex assembly (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Hop binds independently to hsp90 and hsp70 to form an hsp90·Hop·hsp70 complex (13Chen S. Prapapanich V. Rimerman R.A. Honoré B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar), and assembly proceeds faster when Hop is present to bring the two essential chaperones together (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These complexes also contain small amounts of the hsp70 co-chaperone hsp40 (10Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), and together they form the hsp90/hsp70-based chaperone “machinery.” The chaperone machinery can be prepared simply by mixing purified components, or it can be immunoadsorbed from reticulocyte lysate with a monoclonal antibody against Hop (10Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). When mixed with immunoadsorbed GR, the immunoadsorbed chaperone machinery converts the GR to its steroid binding form in an ATP-dependent manner (10Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Once the machinery has assembled the GR·hsp90 heterocomplex, p23 binds dynamically (15Dittmar 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 (234) Google Scholar) to the ATP-dependent conformation of hsp90 (16Sullivan 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 (225) Google Scholar) and stabilizes its association with the receptor.The Hop (p60) of rabbit 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 (247) Google Scholar) is the homolog of a human protein cloned by Honoré et al. (18Honoré B. Leffers H. Madsen P. Rasmussen H.H. Vandekerckhove J. Celis J.E. J. Biol. Chem. 1992; 267: 8485-8491Abstract Full Text PDF PubMed Google Scholar) and the non-essential yeast heat shock protein Sti1 (19Nicolet C.M. Craig E.A. Mol. Cell. Biol. 1989; 9: 3638-3646Crossref PubMed Scopus (203) Google Scholar). Unlike hsp70 and hsp90, Hop alone does not possess any chaperone activity in protein refolding assays (20Bose S. Weikl T. Bugl H. Buchner J. Science. 1996; 274: 1715-1717Crossref PubMed Scopus (316) Google Scholar, 21Freeman B.C. Toft D.O. Morimoto R.I. Science. 1996; 274: 1718-1720Crossref PubMed Scopus (288) Google Scholar). Hop contains multiple tetratricopeptide repeats (TPR), with separate TPR domains determining its binding to hsp70 and hsp90. The N-terminal TPR1 domain binds to the C terminus of hsp70, and the central TPR2 domain binds to a TPR acceptor site in the C terminus of hsp90 (13Chen S. Prapapanich V. Rimerman R.A. Honoré B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar, 22Lassle M. Blatch G.L. Kundra V. Takatori T. Zetter B.R. J. Biol. Chem. 1997; 272: 1874-1876Abstract Full Text Full Text PDF Scopus (142) Google Scholar, 23Demand J. Luders J. Hohfeld J. Mol. Cell. Biol. 1998; 18: 2023-2028Crossref PubMed Scopus (230) Google Scholar, 24Young J.C. Oberman W.M.J. Hartl F.U. J. Biol. Chem. 1998; 273: 18007-18010Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 25Carello A. Ingley E. Minchin R.F. Tsai S. Ratajczak T. J. Biol. Chem. 1999; 274: 2682-2689Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 26Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1000) Google Scholar). In addition to bringing hsp70 and hsp90 together into the machinery for opening the steroid binding cleft in the GR LBD (14Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), studies of purified Hop·hsp90 interaction show that Hop inhibits hsp90 ATPase activity (27Prodromou 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 (1106) Google Scholar, 28Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (349) Google Scholar). Inasmuch as hsp90 ATPase activity is required to generate steroid binding activity (29Grenert J.P. Johnson B.D. Toft D.O. J. Biol. Chem. 1999; 274: 17525-17533Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and the presence of Hop in the purified five-protein system accelerates the rate at which GR steroid binding activity is generated (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), inhibition of hsp90 ATPase activity may not be a critical component of Hop function in the activation of steroid binding sites by the hsp90·Hop·hsp70 machinery.In 1992, we purified (by ammonium sulfate precipitation and molecular sieve chromatography) a high molecular mass complex from rabbit reticulocyte lysate that contained hsp90 and hsp70 and had a low ability to assemble GR·hsp90 heterocomplexes with steroid binding activity (30Scherrer L.C. Hutchison K.A. Sanchez E.R. Randall S.K. Pratt W.B. Biochemistry. 1992; 31: 7325-7329Crossref PubMed Scopus (40) Google Scholar). A factor that was separated from the complex during the ammonium sulfate step was required for efficient heterocomplex reconstitution (30Scherrer L.C. Hutchison K.A. Sanchez E.R. Randall S.K. Pratt W.B. Biochemistry. 1992; 31: 7325-7329Crossref PubMed Scopus (40) Google Scholar), and this factor was identified as p23 (31Hutchison 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). Subsequently, heterocomplexes containing hsp90, Hop, and hsp70 were immunoadsorbed from reticulocyte lysate with monoclonal antibodies against hsp90 (32Hutchison K.A. Dittmar K.D. Pratt W.B. J. Biol. Chem. 1994; 269: 27894-27899Abstract Full Text PDF PubMed Google Scholar) or Hop (14Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) and shown to assemble GR·hsp90 heterocomplexes with steroid binding activity. This multiprotein chaperone complex, originally called a “foldosome” (32Hutchison K.A. Dittmar K.D. Pratt W.B. J. Biol. Chem. 1994; 269: 27894-27899Abstract Full Text PDF PubMed Google Scholar), is now known as the hsp90/hsp70-based chaperone machinery. Yeast (Saccharomyces cerevisiae) contains similar hsp90(hsp82)·Hop(Sti1)·hsp70(Ssa) complexes (33Chang H.C.J. Lindquist S. J. Biol. Chem. 1994; 269: 24983-24988Abstract Full Text PDF PubMed Google Scholar), and mutation ofsti1 results in decreased GR activation of a reporter genein vivo (34Chang H.C.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (192) Google Scholar).These observations suggest that the hsp90·Hop·hsp70 chaperone machinery plays a role in GR·hsp90 heterocomplex assembly under cell-free conditions by reticulocyte lysate and by yeast in vivo, but Hop is not essential in either case. Immune depletion of Hop from reticulocyte lysate, for example, reduces its ability to generate GR steroid binding activity by 50% (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and Sti1mutant yeast still have one-third the GR activity of wild-type yeast at high hormone concentration (34Chang H.C.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (192) Google Scholar). Recently, studies utilizing purified proteins for GR·hsp90 assembly have caused some investigators to totally repudiate the notion of the hsp90/hsp70-based chaperone machinery (35Rajapandi T. Greene L.E. Eisenberg E. J. Biol. Chem. 2000; 275: 22597-22604Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In a purified protein assembly system, GR steroid binding activity can be generated with a combination of the core chaperones hsp90 and hsp70 without Hop or hsp40 (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 35Rajapandi T. Greene L.E. Eisenberg E. J. Biol. Chem. 2000; 275: 22597-22604Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Also, GR·hsp90 heterocomplexes can be assembled by purified proteins in a two-step procedure that does not involve prior formation of the hsp90·Hop·hsp70 assembly machinery (36Morishima Y. Murphy P.J.M. Li D.P. Sanchez E.R. Pratt W.B. J. Biol. Chem. 2000; 275: 18054-18060Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 37Morishima Y. Kanelakis K.C. Murphy P.J.M. Shewach D.S. Pratt W.B. Biochemistry. 2001; 40: 1109-1116Crossref PubMed Scopus (36) Google Scholar). However, we have shown that the rate of GR·hsp90 heterocomplex assembly is accelerated markedly when Hop is added to a mixture of purified hsp90, hsp70, YDJ-1, and p23 (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Because Hop binds to hsp70 and hsp90, it co-purifies with both, and fastidious purification procedures are required to assure Hop-free preparations of these chaperones (12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). It is likely that Hop contamination of the purified chaperones accounts for the difference between those who have concluded that Hop has no effect (35Rajapandi T. Greene L.E. Eisenberg E. J. Biol. Chem. 2000; 275: 22597-22604Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and both ourselves (9Dittmar 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, 12Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 14Dittmar K.D. Pratt W.B. J. Biol. Chem. 1997; 272: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) and the Toft laboratory (11Kosano H. Stensgard B. Charlesworth M.C. McMahon N. Toft D. J. Biol. Chem. 1998; 273: 32973-32979Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) who have shown that the presence of Hop in a purified five-protein system optimizes receptor·hsp90 heterocomplex assembly.Although assembly with the purified chaperones is useful for studying the mechanism by which GR·hsp90 heterocomplexes are formed and how steroid binding sites are created, the purified system does not replicate the conditions of reticulocyte lysate. The concentrations of hsp90 and hsp70 in the purified system, for example, are several times those of reticulocyte lysate. Here, we revisit the reticulocyte lysate system to determine the relative abundance of hsp90, hsp70, and Hop and the amount of each protein that is present in the chaperone machinery. We find that all of the Hop, ∼30% of the hsp90, and ∼9% of the hsp70 in rabbit reticulocyte lysate exist in hsp90·Hop·hsp70 heterocomplexes. When the reticulocyte lysate is separated into two pooled fractions by molecular sieve chromatography, 92% of the ability to activate GR steroid binding activity resides in the largeM r peak of hsp90, which contains 92% of the Hop. A second pool of fractions containing the unbound hsp90 (38%) and hsp70 (53%) and 8% of the Hop has a low GR activating activity. This suggests that in reticulocyte lysate the Hop-containing chaperone machinery plays the major role in GR·hsp90 heterocomplex assembly, with free hsp90 and hsp70 playing a considerably lesser role. We have determined by both native gel electrophoresis and cross-linking that the chaperone machinery in reticulocyte lysate possesses an hsp90:Hop:hsp70 stoichiometry of 2:1:1. We also show by cross-linking that purified hsp90 and hsp70 bind weakly to each other in the absence of Hop, producing an hsp90·hsp70 complex with a stoichiometry of 2:1. This is consistent with the notion (37Morishima Y. Kanelakis K.C. Murphy P.J.M. Shewach D.S. Pratt W.B. Biochemistry. 2001; 40: 1109-1116Crossref PubMed Scopus (36) Google Scholar) that hsp90 and hsp70 interact directly with each other while the steroid binding cleft is being opened by either the chaperone machinery or by the purified chaperones without Hop.EXPERIMENTAL PROCEDURESMaterialsUntreated rabbit reticulocyte lysate was purchased from Green Hectares (Oregon, WI). [6,7-3H]Triamcinolone acetonide (38 Ci/mmol) and 125I-conjugated goat anti-mouse IgG were obtained from PerkinElmer Life Sciences. Sephacryl S-300 was fromAmersham Pharmacia Biotech. Protein A-Sepharose, goat anti-mouse horseradish peroxidase conjugate, and molecular weight markers used for non-denaturing gels were from Sigma Chemical Co. The BuGR2 monoclonal IgG antibody against the GR and the 3G3 monoclonal IgM against hsp90 were 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, BC, Canada).Escherichia coli expressing YDJ-1 was a gift from Dr. Avrom Caplan (Mount Sinai School of Medicine). The DS14F5 monoclonal IgG against Hop and E. coli expressing Hop were kindly provided by Dr. David Smith (Mayo Clinic, Scottsdale, AZ). Hybridoma cells producing the FiGR monoclonal IgG against the GR were generously provided by Dr. Jack Bodwell (Dartmouth Medical School).MethodsImmunoadsorption of GRMouse GR was expressed in Sf9 cells, and cytosol was prepared as previously described (36Morishima Y. Murphy P.J.M. Li D.P. Sanchez E.R. Pratt W.B. J. Biol. Chem. 2000; 275: 18054-18060Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Receptors were immunoadsorbed from 50-µl aliquots of Sf9 cytosol by rotation for 2 h at 4 °C with 14 µl of protein A-Sepharose precoupled to 7 µl of FiGR ascites suspended in 200 µl of TEG (10 mm TES, pH 7.6, 50 mm NaCl, 4 mmEDTA, 10% glycerol). Prior to incubation with reticulocyte lysate or lysate subfractions, immunoadsorbed receptors were stripped of associated hsp90 by incubating the immunopellet for an additional 2 h at 4 °C with 350 µl of 0.5 m NaCl in TEG. The pellets were then washed once with 1 ml of TEG followed by a second wash with 1 ml of Hepes buffer (10 mm Hepes, pH 7.4).Glucocorticoid Receptor Heterocomplex ReconstitutionFor assembly of GR·hsp90 heterocomplexes, FiGR immunopellets containing GR stripped of chaperones were incubated with 40 µl of reticulocyte lysate or with 40 µl of a lysate subfraction from Sephacryl S-300 chromatography plus 6 µg of purified p23 and 0.4 µg of purified YDJ-1. Incubation volumes were adjusted to 50 µl with HKD buffer (10 mm Hepes, 100 mm KCl, 5 mmdithiothreitol, pH 7.35) containing 20 mm sodium molybdate and 5 µl of an ATP-regenerating system (50 mm ATP, 250 mm creatine phosphate, 20 mm magnesium acetate, and 100 units/ml creatine phosphokinase). The assay mixtures were incubated for 20 min at 30 °C with suspension of the pellets by shaking the tubes every 2 min. At the end of the incubation, the pellets were washed twice with 1 ml of ice-cold TEGM buffer (TEG with 20 mm sodium molybdate) and assayed for steroid binding capacity.Assay of Steroid Binding CapacityImmune pellets to be assayed for steroid binding were incubated overnight at 4 °C in 50 µl of HEM buffer (10 mm Hepes, pH 7.35, 1 mmEDTA, 20 mm molybdate) plus 50 nm[3H]triamcinolone acetonide. Samples were then washed three times with 1 ml of TEGM and counted by liquid scintillation spectrometry. The steroid binding is expressed as counts per minute of [3H]triamcinolone acetonide bound/FiGR immunopellet prepared from 50 µl of Sf9 cytosol.Gel Electrophoresis and Western BlottingImmune pellets were resolved on 12% SDS-polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were probed with 0.25 µg/ml BuGR for GR or 1 µg/ml AC88 for hsp90. The immunoblots were then incubated a second time with the appropriate125I-conjugated or horseradish peroxidase-conjugated counterantibody to visualize the immunoreactive bands. For electrophoresis under non-denaturing conditions, 10 µl of reticulocyte lysate was mixed with 50 µl of detergent-free buffer (312 mm Tris-HCl, pH 6.8, 50% glycerol, 0.05% bromphenol blue), and proteins were resolved on a 7.5% polyacrylamide gel, followed by Western blotting. The immunoblots were probed with AC88 for hsp90, 1 µg/ml N27F3-4 for hsp70, or 0.1% DS14F5 mouse ascites for Hop. Molecular weight markers for non-denaturing gels were bovine serum albumin, monomer (66,000) and dimer (132,000), and Jack bean urease, trimer (272,000) and hexamer (545,000).Glutaraldehyde Cross-linking of Purified ProteinsFor cross-linking of purified hsp90 or hsp70, 15 µg of purified protein was incubated for 1 h at room temperature with 0.8 mmglutaraldehyde in a final volume of 50 µl adjusted with HKD buffer. The cross-linking was terminated by adding 12 µl of 0.5 mTris, pH 8.0, and continuing incubation for 30 min at room temperature. Proteins were resolved by SDS-polyacrylamide gel electrophoresis on 6% gels followed by Western blotting.Relative Abundance of Proteins in Heterocomplexes and Reticulocyte LysateFor determining stoichiometry of hsp90·Hop·hsp70 heterocomplexes, 50-µl aliquots of reticulocyte lysate were immunoadsorbed to 18 µl of protein A-Sepharose prebound with 0.5 µl (∼7 µg) of DS14F5 antibody against Hop or non-immune mouse IgG. The samples were rotated at 4 °C for 2 h, and immunopellets were washed three times with 1 ml of TEG buffer. Relative amounts of hsp90 and hsp70 in immunoadsorbed Hop complexes were estimated by resolving the immune pellet proteins on 12% SDS-polyacrylamide gels and staining with Coomassie Blue. Ratios of hsp90 to hsp70 were determined by scanning multiple stained bands. For cross-linking, Hop immune pellets were suspended in 50 µl of HKD buffer and incubated with 0.4 mm glutaraldehyde as described above.To determine the concentration of Hop, hsp90 and hsp70 in reticulocyte lysate, aliquots of lysate were electrophoresed on SDS-polyacrylamide gels that also contained various amounts of purified hsp90, hsp70, and Hop to provide a standard curve for each protein. Immunoblots were prepared and probed with monoclonal IgGs against each protein, followed by incubation with 125I-labeled anti-IgG counterantibody. Samples and purified standards were then excised and counted to permit calculation of the concentration of each protein.Protein Purificationhsp90 and hsp70 were purified from rabbit reticulocyte lysate by sequential chromatography on DE52, hydroxyapatite, and ATP-agarose as described previously (38Hutchison K.A. Dittmar K.D. Czar M.J. Pratt W.B. J. Biol. Chem. 1994; 269: 5043-5049Abstract Full Text PDF PubMed Google Scholar). Human p23 was purified from 10 ml of bacterial lysate by chromatography on DE52 followed by hydroxyapatite chromatography as described previously (39Johnson J.L. Toft D.O. J. Biol. Chem. 1994; 269: 24989-24993Abstract Full Text PDF PubMed Google Scholar). For purification of YDJ-1, bacterial sonicates were cleared by centrifugation, and YDJ-1 was purified by sequential chromatography on DE52 and hydroxyapatite as described previously (10Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The bacterial expression of YDJ-1 has been described (40Caplan A.J. Tsai J. Casey P.J. Douglas M.G. J. Biol. Chem. 1992; 267: 18890-18895Abstract Full Text PDF PubMed Google Scholar) as has the expression of human Hop (9Dittmar 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). Purification of human Hop was carried out in a similar manner by sequential chromatography on DE52 and hydroxyapatite. In all cases the protein-containing fractions were identified by immunoblotting, and fractions from the final purification step were pooled, concentrated by Amicon filtration, dialyzed against HKD buffer, flash frozen and stored at −70 °C.Fractionation of Reticulocyte LysateReticulocyte lysate (500 µl) was applied to a column (1.5 × 113 cm) of Sephacryl S-300, and the column was eluted with HKD buffer. Aliquots (100 µl) of each 2.5-ml fraction were assayed for hsp90, hsp70, and Hop by SDS-polyacrylamide gel electrophoresis and immunoblotting, and total protein was assayed by the Bradford method. The indicated fractions were pooled and contracted to a volume of 300 µl by Amicon filtration. Aliquots (40 µl) of each contracted pool were assayed for GR·hsp90 heterocomplex reconstitution as described above. Unliganded steroid receptors exist in cytosols in a heterocomplex with the ubiquitous protein chaperone hsp901 (for review, see Ref.1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar). Hsp90 binds to the ligand binding domain (LBD) of the receptors (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar), and the glucocorticoid receptor (GR) LBD must be bound to hsp90 for the receptor to have high affinity steroid binding activity (2Bresnick E.H. Dalman F.C. Sanchez E.R. Pratt W.B. J. Biol. Chem. 1989; 264: 4992-4997Abstract Full Text PDF PubMed Google Scholar, 3Hutchison K.A. Czar M.J. Scherrer L.C. Pratt W.B. J. Biol. Chem. 1992; 267: 14047-14053Abstract Full Text PDF PubMed Google Scholar). The receptor·hsp90 heterocomplexes are assembled by a multiprotein chaperone system that was first studied in reticulocyte lysate (4Smith D.F. Schowalter D.B. Kost S.L. Toft D.O. Mol. Endocrinol. 1990; 4: 1704-1711Crossref PubMed Scopus (113) 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). Both biochemical data (6Stancato 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 (36) Google Scholar) and data from GR mutants (7Xu M. Dittmar K.D. Giannoukos G. Pratt W.B. Simons Jr., S.S. J. Biol. Chem. 1998; 273: 13918-13924Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Giannoukos G. Si
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