Reconstitution of the Steroid Receptor·hsp90 Heterocomplex Assembly System of Rabbit Reticulocyte Lysate

Dittmar, Kurt D.; Hutchison, Kevin A.; Owens-Grillo, Janet K.; Pratt, William B.

doi:10.1074/jbc.271.22.12833

Cited by 155 publications

(152 citation statements)

References 32 publications

(20 reference statements)

Supporting

Mentioning

148

Contrasting

Order By: Relevance

“…Furthermore, previous studies have implicated cytosolic Hsp90 in the targeting of preproteins to the TOC translocon (30), and the use of urea-denatured recombinant pre-SSU-3xFLAG eliminated the possibility that the effect on import was due to inhibition of cytosolic Hsp90 present in the reticulocyte lysate of in vitro translated preproteins (31). Three general stages of import have previously been defined (2,3,(21)(22)(23)(24)(25): energy-independent binding to the TOC receptors, early import intermediates spanning both TOC and TIC translocons formed in the presence of 0.1 mM ATP/GTP, and late import intermediates captured in the presence of high concentrations of ATP (>1 mM) that are fully translocated across the TOC translocon and remain engaged with the TIC complex.…”

Section: Dsp (mentioning

confidence: 99%

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

Inoue

Schnell

2013

Proc. Natl. Acad. Sci. U.S.A.

108

View full text Add to dashboard Cite

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.

show abstract

Section: Dsp (mentioning

confidence: 99%

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

Inoue

Schnell

2013

Proc. Natl. Acad. Sci. U.S.A.

108

View full text Add to dashboard Cite

show abstract

“…Samples were then washed twice with 1 ml of TEGM and counted by liquid scintillation spectrometry as described previously (13). The steroid binding is expressed as counts/minute of [ 3 H]TA bound/anti-GST immune pellet prepared from 100 l of cytosol.…”

Section: Methodsmentioning

confidence: 99%

“…Hsp90 is an ubiquitous cellular protein that is part of a multiprotein complex with chaperone activity (12). When hsp90 dissociates from GRs, steroid binding is lost but can be regenerated by incubating immobilized GRs with reticulocyte lysate or with a five-protein, minimal chaperone system consisting of hsp90, hsp70, Hop, hsp40, and p23 (11)(12)(13). Several sequences of the GR have been implicated in the binding of hsp90, but most are near the middle of the GR LBD (amino acids 568 -671 of the rat GR (14 -16)).…”

mentioning

confidence: 99%

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Giannoukos¹,

Silverstein²,

Pratt³

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the aminoterminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in fulllength GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/ L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg 651 . The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550 -554 acting as a hydrophobic clasp.

show abstract

“…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).…”

mentioning

confidence: 99%

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

Murphy

Morishima

Chen

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR)⅐hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR⅐hsp70 complex that primes the receptor for subsequent ATPdependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR⅐hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size~1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR⅐hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/ 554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/ 554C, and it is the subsequent assembly step with hsp90 that is defective.Glucocorticoid receptors (GR) 1 are recovered from hormonefree cells as large multiprotein complexes containing a dimer of hsp90 (for review see Refs. 1 and 2). 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). The hsp90/hsp70-dependent chaperone machinery carries out the ATP-dependent opening of the binding cleft in the GR LBD such that it can be accessed by steroid. In addition to opening the steroid binding cleft, formation of a complex with hsp90 is accompanied by conformational changes that increase the sensitivity of the GR LBD to attack by thiol-derivatizing agents and trypsin (8 -10).The multiprotein chaperone machinery that forms receptor⅐hsp90 heterocomplexes was originally identified in reticulocyte lysate (11,12). 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 b...

show abstract

Reconstitution of the Steroid Receptor·hsp90 Heterocomplex Assembly System of Rabbit Reticulocyte Lysate

Cited by 155 publications

References 32 publications

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

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

Contact Info

Product

Resources

About