In vitro formation of estrogen receptor-heat shock protein 90 complexes.

We previously showed that the 9 S estrogen receptor can be reconstituted from purified vero ER (estradiol binding subunit) and purified hsp 90 (heat shock protein 90) in vitro [Inano, K. et al. (1990) FEBS Lett. 267, 157-159]. In this study, we further characterized our reconstitution system to investigate the mechanism underlying the formation of 9 S ER. When a vero ER preparation stored at 4 degrees C for more than 20 h after affinity chromatography was used for the reconstitution of 9 S ER, 0.5 M NaSCN was essential, but not Na2MoO4 or other reagents. When, however, vero ER was used within 3 h after dissociation from an affinity resin, 9 S ER could be reconstituted in a relatively high yield without NaSCN. Moreover, if such a fresh vero ER preparation was used, 9 S ER could be reconstituted in the absence of NaSCN from not only unoccupied vero ER but also the occupied form. From these results it was suggested that the conformation of purified vero ER tends to change quickly in a time dependent manner, and so a chemical perturbant, NaSCN, is generally necessary for the reconstitution of 9 S ER from purified vero ER and purified hsp 90. The concentration of hsp 90 required for the reconstitution was only about 1.0 microM, which was lower than its physiological concentration. Based on these results, the mechanism underlying the formation of 9 S ER was discussed.