The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

Yoshihiro Morishima,Kimon C Kanelakis,Adam M Silverstein,Kurt D Dittmar,Lourdes Estrada,William B Pratt

doi:10.1074/jbc.275.10.6894

Yoshihiro Morishima, Kimon C Kanelakis + Show 4 more

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https://doi.org/10.1074/jbc.275.10.6894

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Abstract

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR).Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90.Hop.Hsp70.Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR.Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358-7366). Hop has been considered essential for the formation of receptor.Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR.Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

Highlights

The steroid receptors are recovered from cells as multiprotein heterocomplexes containing a dimer of Hsp90, substochiometric amounts of Hsp70,1 an acidic 23-kDa protein, p23, and a tetratricopeptide repeat domain protein, such as immunophilin or protein phosphatase 5
The ADP-bound conformation of Hsp70 has a high affinity for hydrophobic substrates, and Hsp40 potentiates GR1⁄7Hsp90 heterocomplex assembly when it is present in the purified system at about one-twentieth the concentration of Hsp70 [15]
In our studies with the purified five-protein system, we have considered Hop to be obligatory for assembly of GR1⁄7Hsp90 heterocomplexes and glucocorticoid receptor (GR) folding to the steroid binding state [12]

Summary

Introduction

The steroid receptors are recovered from cells as multiprotein heterocomplexes containing a dimer of Hsp, substochiometric amounts of Hsp an acidic 23-kDa protein, p23, and a tetratricopeptide repeat domain protein, such as immunophilin or protein phosphatase 5 (for review see Refs. 1 and 2). The ADP-bound conformation of Hsp has a high affinity for hydrophobic substrates, and Hsp (provided as the purified yeast homolog, YDJ-1) potentiates GR1⁄7Hsp heterocomplex assembly when it is present in the purified system at about one-twentieth the concentration of Hsp70 [15] It has not been clear whether Hsp is essential for GR folding by the five-protein system or whether it enhances a process that occurs, albeit less effectively, when it is not present [15]. The final player in the five-protein assembly system was identified as a 60-kDa rabbit protein intermediate in receptor1⁄7Hsp heterocomplex assembly [26] that was found to

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