Partial occlusion of both cavities of the eukaryotic chaperonin with antibody has no effect upon the rates of beta-actin or alpha-tubulin folding

The eukaryotic chaperonin containing T-complex polypeptide 1 (CCT) is requi red in vivo for the production of native actin and tubulin. It is a 900-kDa oligomer formed from two back-to back rings, each containing eight differe nt subunits surrounding a central cavity in which interactions with substra tes are thought to occur, Here, we show that a monoclonal antibody recogniz ing the C terminus of the CCT alpha subunit can bind inside, and partially occlude, both cavities of apo-CCT, Rabbit reticulocyte lysate was programme d to synthesize beta-actin and alpha-tubulin in the presence and absence of anti-CCT alpha antibody. The binding of the antibody inside the cavity and its occupancy of a large part of it does not prevent the folding of beta-a ctin and alpha-tubulin by CCT, despite the fact that all the CCT in the in vitro translation reactions was continuously bound by two antibody molecule s. Furthermore, no differences in the protease susceptibility of actin boun d to CCT in the presence and absence of the monoclonal antibody were detect ed, indicating that the antibody molecules do not perturb the conformation of actin folding intermediates substantially. These data indicate that comp lete sequestration of substrate by CCT may not be required for productive f olding, suggesting that there are differences in its folding mechanism comp ared with the Group I chaperonins.