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

Abstract

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)⅐Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90⅐Hop⅐Hsp70⅐Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR⅐Hsp90 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 receptor⅐Hsp90 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 GR⅐Hsp90 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.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 (for review see Refs. 1 and 2). The steroid receptor⅐Hsp90 heterocomplexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with reticulocyte lysate (3, 4). Inasmuch as the glucocorticoid receptor (GR) 2 must be associated with Hsp90 for it to have steroid binding activity (5), incubation of Hsp90-free GR with reticulocyte lysate results in generation of steroid binding activity in direct proportion to the number of GR⅐Hsp90 heterocomplexes that are assembled (6). Hsp90 binds directly to the ligand-binding domain (LBD) of the GR (1), and complexing with Hsp90 also opens up both thiol moieties (7) and trypsin cleavage sites (8, 9) in the LBD to attack by a thiol-derivatizing agent and the protease. These biochemical observations, coupled with data derived from GR mutants (10), support the idea (6, 11) that the Hsp90 heterocomplex assembly system in reticulocyte lysate directs an ATP-dependent partial unfolding of the GR LBD, thus opening the hydrophobic steroid-binding cleft to access by steroid.The heterocomplex assembly system has been reconstituted (12-16), and five proteins, including Hsp90, Hsp70, Hop (60-kDa Hsp organizer protein), Hsp40, and p23, participate in the ATP/Mg 2ϩ -dependent and K ϩ -dependent assembly process (for review of heterocomplex assembly see Refs. 17 and 18). Besides Hsp90 itself, the only component of this Hsp90-based chaperone system proven to be essential for both GR⅐Hsp90 heterocomplex...

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

“…It is clear that both GR (16) and PR (15) heterocomplexes can be assembled with purified proteins in the complete absence of YDJ-1/hsp40. Thus, there is not an absolute requirement for YDJ-1 co-chaperone function or for it to target hsp70 to either receptor.…”

“…This machinery has been reconstituted (13), and a mixture of five purified proteins (hsp90, hsp70, 2 Hop, hsp40, and p23) is now used to achieve efficient receptor⅐hsp90 heterocomplex assembly (14,15). 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 (16). Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (17).…”

“…Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (17). Although Hop is not essential for GR⅐hsp90 heterocomplex assembly, when Hop is present to form a machinery, assembly is much faster (16). Hop does not act solely as a tie rod to bring the essential chaperones together; it also influences the conformational state and function of each protein (18).…”

Glucocorticoid receptor alpha, beta and gamma expression vs in vitro glucocorticod resistance in childhood leukemia

“…[9][10][11][12][13][14][15][16][17] In the absence of hormone, the GRa predominantly resides in the cytoplasm as a multiprotein complex consisting of the GR itself, two molecules of hsp90 and several other proteins. [18][19][20] Upon binding of a GC, the receptor complex dissociates and translocates to the nucleus. In the nucleus, the GR binds as a homodimer to GC-responsive elements, which are usually found in the promotor regions of GC-responsive genes.…”

Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase

“…It is unclear whether folding occurs while the receptor is in association with Hsp90 or after its release. Whether the cochaperones are required for this reaction is not clear, because purified Hsp70 and Hsp90 can together facilitate folding of GR in vitro, whereas cochaperones such as Hop stimulate the reaction (Morishima et al, 2000;Rajapandi et al, 2000). Furthermore, deletion of yeast Hop (STI1), or p23 (SBA1) does not result in a slow growth phenotype except under stress conditions (Nicolet and Craig, 1989;Chang et al, 1997;Bohen, 1998;Fang et al, 1998).…”