The assembly of progesterone receptor-hsp90 complexes using purified proteins.
The progesterone receptor can be reconstituted into hsp90-containing complexes in vitro, and the resulting complexes are needed to maintain hormone binding activity. This process requires ATP/Mg2+, K+, and several axillary proteins. We have developed a defined system for the assembly of progesterone receptor complexes using purified proteins. Five proteins are needed to form complexes that are capable of maintaining hormone binding activity. These include hsp70 and its co-chaperone, hsp40, the hsp70/hsp90-binding protein, Hop, hsp90, and the hsp90-binding protein, p23. The proteins Hip and FKBP52 were not required for this in vitro process even though they have been observed in receptor complexes. Each of the five proteins showed a characteristic concentration dependence. Similar concentrations of hsp70, hsp90, and p23 were needed for optimal assembly, but hsp40 and Hop were effective at about 1/10 the concentration of the other proteins, suggesting that these two proteins act catalytically or are needed at levels similar to the receptor concentration. ATP was required for the functioning of both hsp70 and hsp90. The binding of hsp70 to the receptor requires hsp40 and about 10 microM ATP; however, hsp90 binding appears to occur subsequent to hsp70 binding and is optimal with 1 mM ATP. A three-step model is presented to describe the assembly process.
Kosano
H
H
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan.
Stensgard
B
B
Charlesworth
M C
MC
McMahon
N
N
Toft
D
D
D000818:Animals; D002460:Cell Line; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae