THE 23-KDA ACIDIC PROTEIN IN RETICULOCYTE LYSATE IS THE WEAKLY-BOUND COMPONENT OF THE HSP FOLDOSOME THAT IS REQUIRED FOR ASSEMBLY OF THE GLUCOCORTICOID RECEPTOR INTO A FUNCTIONAL HETEROCOMPLEX WITH HSP90

被引:107
作者
HUTCHISON, KA
STANCATO, LF
OWENSGRILLO, JK
JOHNSON, JL
KRISHNA, P
TOFT, DO
PRATT, WB
机构
[1] UNIV MICHIGAN,SCH MED,DEPT PHARMACOL,ANN ARBOR,MI 48109
[2] MAYO GRAD SCH,DEPT BIOCHEM & MOLEC BIOL,ROCHESTER,MN 55905
[3] UNIV WESTERN ONTARIO,DEPT PLANT SCI,LONDON,ON N6A 5B7,CANADA
关键词
D O I
10.1074/jbc.270.32.18841
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified ''foldosome'' directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K. A., Dittmar, K. D., and Pratt, W. B. (1994) J. Biol, Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte ly sate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directed folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography, Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.
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页码:18841 / 18847
页数:7
相关论文
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