Abstract
The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.
Highlights
More than 100 substrates or client proteins for Hsp90 have been identified (2– 4). These proteins include a diverse family of kinases, transcription factors, and cell cycle regulators, many of which are involved in cancer (5–7)
The primary amino acid sequence of the steroid binding domain (SBD) is highly conserved throughout the steroid receptor family and a crystal structure of this domain of human progesterone receptor (PR) bound to hormone is available (11)
The glycine-phenylalanine (G/F)rich region is required for the proper function of these proteins (35), while the significance of the glycine-methionine (G/M) region, unique to the type II Hsp40 proteins, is unclear (38)
Summary
HeLa Cell Lysate Pull-downs—HeLa cells stably expressing PR-B (28) were grown to 70% confluency in MEM medium enriched with 5% fetal bovine serum (HyClone Laboratories, Logan, UT), 6 ng/l insulin (Invitrogen), nonessential amino acids, penicillin, and streptomycin at 37 °C. PR Binding Assays—PR resin pellets (20 l) were suspended with 200 l of cold reaction buffer (20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 0.01% Nonidet P-40, and 2 mM dithiothreitol, pH 7.5) containing the specified amount of wild type or mutant Ydj, DjA1, or DjB1. Reactions that assess the stimulation of Hsp binding to PR include wild type or mutant Hsp plus 2 mM ATP These reactions proceeded at 30 °C for 20 min; the samples were chilled on ice for 2 min, washed four times with 1 ml of reaction buffer. Progesterone Receptor Reconstitution—PR resin (20 l) was suspended with 200 l of cold reaction buffer containing 20 g of Hsp70, 5 g of Ydj, DjA1, or DjB1, 5 g of Hop, 20 g of Hsp90, 5 g of p23, and 5 mM ATP unless otherwise noted. Analysis of complex formation after reconstitution assays were performed in 10% acrylamide gels, while analysis of Hsp and Hsp binding to PR used 7.5% acrylamide gels to get better resolution in the 50 – 40 kDa range
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