Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate.
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 approximately 30% of the hsp90 and approximately 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.
Murphy
P J
PJ
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. pjmurphy@umich.edu
Kanelakis
K C
KC
Galigniana
M D
MD
Morishima
Y
Y
Pratt
W B
WB
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002847:Chromatography, Agarose; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005938:Glucocorticoids / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D015151:Immunoblotting; D007313:Insecta; D051379:Mice; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry*; D013256:Steroids / Q000378:metabolism
D003432:Cross-Linking Reagents; D005938:Glucocorticoids; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D013256:Steroids