CYTOPLASMIC CHAPERONIN CONTAINING TCP-1 - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additio nal protein cofactors. It has been previously demonstrated that nucleo tide exchange and ATP hydrolysis act to switch CCT between conformatio ns that interact either strongly or weakly with unfolded substrates [M elki, R., & Cowan, N.J. (1994) Mel. Cell. Biol. 14, 2895-2904]. The pr esent study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatu red labeled beta-actin and beta-tubulin as model substrates for bindin g to CCT, we demonstrate that the CCT particle can contain two substra te protein chains simultaneously. Third, by electron microscopy, sedim entation velocity, and intrinsic fluorescence measurements, we documen t the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate pr otein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target protein s.