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, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present 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 denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.
Melki
R
R
Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France. melki@lebs.cnrs-gif.fr
Batelier
G
G
Soulié
S
S
Williams
R C
RC
D000199:Actins / Q000378:metabolism; D000818:Animals; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D003593:Cytoplasm / Q000737:chemistry* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000378:metabolism; D014461:Ultracentrifugation
D000199:Actins; D014404:Tubulin; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins