EFFECT OF GELDANAMYCIN ON THE KINETICS OF CHAPERONE-MEDIATED RENATURATION OF FIREFLY LUCIFERASE IN RABBIT RETICULOCYTE LYSATE

Renaturation of thermally denatured firefly luciferase in rabbit retic ulocyte lysate (RRL) requires hsp90, hsc70, and other as yet unidentif ied RRL components [Schumacher, R. J., et al. (1994) J. Biol. Chem. 26 9, 9493-9499]. Benzoquinonoid ansamycins (BAs) have recently been show n to specifically bind hsp90 and inhibit its function. In this report, we present data that indicate BAs are specific inhibitors of hsp90 fu nction. The effects of the BA geldanamycin (GA) on the kinetics of the luciferase renaturation in RRL were examined to gain insight into the mechanism by which GA inhibits the function of the hsp90 chaperone ma chinery. Chaperone-mediated renaturation of luciferase obeyed Michaeli s-Menten kinetics. The GA inhibited luciferase renaturation uncompetit ively with respect to ATP concentration and noncompetitively with resp ect to luciferase concentration, indicating that GA binds after the bi nding of ATP and that it binds to both the hsp90 chaperone machine/ATP complex and the hsp90 chaperone machine/ATP/luciferase complex. GA ma rkedly decreased the K-app, of the hsp90 chaperone machine for ATP, su ggesting that GA increases the binding affinity of the hsp90 chaperone machinery for ATP or it slows the rate of ATP hydrolysis. Consistent with the notion that GA specifically binds hsp90 and inhibits its func tion, addition of hsp90, but not hsc70, p60, or p23, reversed GA-induc ed inhibition of luciferase renaturation in RRL. Hsp90, hsc70, and the hsp cohorts p60, P48, and p23 were coimmunoprecipitated with lucifera se from RRL. GA increased the steady-state levels of luciferase associ ated with hsp90/hsp70 chaperone machine complexes that contain p60 and blocked the association of the hsp90 cohort p23 with chaperone-bound luciferase. The data suggest that the function of the hsp90 chaperone machinery is not specific to its previously described interaction with steroid hormone receptors, and that it carries out some more generali zed function in vivo.