Stoichiometry, Abundance, and Functional Significance of the hsp90/hsp70-based Multiprotein Chaperone Machinery in Reticulocyte Lysate

Abstract

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 hsp90⅐Hop⅐hsp70 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 GR⅐hsp90 heterocomplex assembly. Here, we counter that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90⅐Hop⅐hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for ϳ30% of the hsp90 and ϳ9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR⅐hsp90 assembly activity resides in the peak containing Hopbound 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 hsp90⅐hsp70 complexes with a stoichiometry of 2:1.Unliganded steroid receptors exist in cytosols in a heterocomplex with the ubiquitous protein chaperone hsp90 1 (for review, see Ref. 1). Hsp90 binds to the ligand binding domain (LBD) of the receptors (1), and the glucocorticoid receptor (GR) LBD must be bound to hsp90 for the receptor to have high affinity steroid binding activity (2, 3). The receptor⅐hsp90 heterocomplexes are assembled by a multiprotein chaperone system that was first studied in reticulocyte lysate (4, 5). Both biochemical data (6) and data from GR mutants (7, 8) support a model (3) 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 (9), and a mixture of five purified proteins, hsp90, hsp70, 2 Hop, hsp40, and p23, is now used to achieve optimal receptor⅐hsp90 heterocomplex assembly (10, 11).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 GR⅐hsp90 heterocomplex assembly (12). Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (13), and assembly proceeds faster when Hop is present to bring the two essential chaperones together (12). These complexes also contain small amounts of the hsp70 co-chaperone hsp40 (10), 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 (10,14). When mixed with immunoadsorbed GR, the immunoadso...

HOP is a monomer: Investigation of the oligomeric state of the co‐chaperone HOP

“…In the cell, if we assume HOP is monomeric, and we know that its endogenous levels are significantly lower than these of Hsp90, a model in which one Hsp90 dimer interacts with one monomeric HOP molecule seems the most reasonable. Indeed, in a study by the Murphy et al 17 using rabbit reticulocyate lysate demonstrated that all the HOP, 30% of Hsp90 and 9% of Hsp70, in the lysate is present in a Hsp70-HOPHsp90 complex with a stoichiometry of 1:1:2.…”

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

“…[6, H]Dexamethasone (40 Ci/mmol) and 125 Iconjugated goat anti-mouse and goat anti-rabbit IgGs were obtained from PerkinElmer Life Sciences. Protein A-Sepharose, goat anti-mouse, and goat anti-rabbit horseradish peroxidase conjugate, and monoclonal anti-GST antibody (clone GST-2) were from Sigma.…”

“…It is possible that only one molecule of hsp70 is bound directly to and interacting productively with the receptor, and that under these conditions of assembly with purified proteins the receptor-bound hsp70 can associate with other molecules of hsp70 to form dimers and trimers, much as purified hsp70 and its homologs have been shown to do in solution (32)(33)(34). It is perhaps important to note that analysis of both the native and assembled hsp90⅐Hop⅐hsp70 machinery has revealed a molar ratio of 1 hsp70 per hsp90 dimer (6,18). If the preassembled machinery can interact with the receptor to produce receptor⅐hsp90 heterocomplexes, then it is reasonable to predict that the initial binding to the receptor and the priming step is by one molecule of hsp70 interacting with one molecule of receptor.…”

“…Hsp90 binds to the ligand binding domain (LBD) of the GR, and the steroid binding activity of the GR is absolutely hsp90-dependent (3,4). When the GR is stripped of its associated hsp90, it immediately loses its ability to bind steroid, and steroid binding activity is regenerated when GR⅐hsp90 heterocomplexes are reformed by the hsp90/hsp70-based chaperone machinery (5,6). The steroids bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand, such that the LBD must change its conformation to allow entry of ligand (7).…”

Interactions of S100A2 and S100A6 with the Tetratricopeptide Repeat Proteins, Hsp90/Hsp70-organizing Protein and Kinesin Light Chain

“…because it contains both Hsp70 (ϳ0.4 g/2 mg of RRL) and Hsp90 (ϳ2.0 g/2 mg of RRL) abundantly, but the S100 proteins were undetectable (Ref. 31 and data not shown). The GST-Hop-WT was incubated with recombinant S100B, S100A2, and S100A6 in the presence of 1 mM CaCl 2 or EGTA, and then the resin was washed.…”