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 required 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 different subunits surrounding a central cavity in which interactions with substrates are thought to occur. Here, we show that a monoclonal antibody recognizing the C terminus of the CCTalpha subunit can bind inside, and partially occlude, both cavities of apo-CCT. Rabbit reticulocyte lysate was programmed to synthesize beta-actin and alpha-tubulin in the presence and absence of anti-CCTalpha 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-actin and alpha-tubulin by CCT, despite the fact that all the CCT in the in vitro translation reactions was continuously bound by two antibody molecules. Furthermore, no differences in the protease susceptibility of actin bound to CCT in the presence and absence of the monoclonal antibody were detected, indicating that the antibody molecules do not perturb the conformation of actin folding intermediates substantially. These data indicate that complete sequestration of substrate by CCT may not be required for productive folding, suggesting that there are differences in its folding mechanism compared with the Group I chaperonins.
Grantham
J
J
Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom.
Llorca
O
O
Valpuesta
J M
JM
Willison
K R
KR
D000199:Actins / Q000378:metabolism*; D000818:Animals; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008854:Microscopy, Electron; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D014404:Tubulin; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins