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.

The C. elegans CCT chaperonin complex contains 8-9 subunits and includes CCT-1 and CCT-5

Michel R. Leroux, E. Peter M. Candido

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