Worm Breeder's Gazette 14(1): 84 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Dept. of Biochemistry and Molecular Biology, University of B.C., Vancouver, B.C. Canada V6T 1Z3
Chaperonins form a ubiquitous class of protein complexes involved in protein folding. Two chaperonin families exist, based on amino acid sequence similarity, quaternary structure, and evolutionary origin. The first family consists of eubacterial GroEL, mitochondrial Hsp60, and chloroplast RuBP (Rubisco-subunit binding protein). The members of the second family are the cytosolic eukaryotic CCT and archaebacterial TF55 chaperonins. GroEL, Hsp60, and RuBP (the so-called classical chaperonins) form double-torus structures with 7-fold rotational symmetry, and contain one or two types of subunits. CCT and TF55 also form double-toruses but have greater (8- or 9-fold) rotational symmetry. TF55 contains two types of subunits, but CCT is more complex, having 7-9 types (in mouse and bovine species) of subunits encoded by a multigene family. The classical and TF55 chaperonins appear to fold a large subset of proteins within their respective cellular environements, whereas the only in vivo targets detected so far for the cytosolic chaperonin have been tubulins and actins; however, it is possible that CCT has a more general role in protein folding, as it has been shown to be required for the proper maturation of in vitro-translated firefly luciferase. We have characterized five C. elegans genes, cct-1, cct-2, cct-4, cct-5, and cct-6. The multigene family encodes ~60 kDa proteins which share 23-35% amino acid sequence identity between members, and 31-35% identity to TF55. The C. elegans genes display 63-68% deduced amino acid sequence identity to mouse and yeast orthologues, indicating that the family is highly conserved across species and that each gene encodes a protein with a specific function. The genes map to chromosomes II and III, and are not closely associated. Each gene is expressed at a similar level in embryos, larvae, and adults, suggesting that the genes perform a necessary function throughout nematode development. Unlike the classical chaperonin genes and other molecular chaperones (many of which are heat-shock proteins), cct-1 expression is not up-regulated during heat stress. Our analysis of the predicted secondary structures of chaperonins has provided some insight into CCT/TF55 protein structure. Despite their divergent amino acid sequence (~30% identity), the C. elegans and mouse CCT/TF55 chaperonins have nearly identical predicted secondary structures. The GroEL/Hsp60/RuBP and CCT/TF55 chaperonin families possess highly conserved ATPase domains, but display some differences in their polypeptide binding domains. This latter difference may be functionally significant since CCT does not require a co-chaperonin, which is required by the classical chaperonins, and associates with the polypeptide binding domain. Also, the different polypeptide binding domain and the large number of subunits of CCT may account for its seemingly limited substrate specificity relative to the GroEL/Hsp60/RuBP chaperonins. We are now characterizing C. elegans CCT. The chaperonin was purified using a combination of sucrose gradient fractionation, ion-exchange chromatography, and ATP-agarose chromatography. The complex contains 8-9 polypeptides ranging from 52-65 kDa, and the distribution of proteins viewed on a single dimension SDS-polyacrylamide gel is similar to mouse and bovine CCT. Western blotting of the purified complex with two antibodies specific for C. elegans CCT-1 and CCT-5 reveals that both of these proteins are present within the complex. We will now assess the ability of C. elegans CCT to bind to various denatured substrate proteins, and assay for ATPase activity. This research is supported by the Medical Research Council of Canada.