Worm Breeder's Gazette 13(5): 73 (February 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
The process of protein folding within cells is known to be assisted by a diverse class of proteins termed molecular chaperones. Chaperonins form an extensively studied family of related molecular chaperones represented largely by Hsp60, GroEL, and Rubisco subunit binding protein. The function of these chaperonins is to facilitate the folding of proteins within their respective cellular compartments (mitochondria, bacterial cytoplasm, and chloroplast). Only recently has a eukaryotic cytosolic protein complex with structural and functional similarities to the chaperonin family been discovered. This chaperonin, refered to as "Chaperonin containing TCP-I" (CCT) differs from Hsp60/GroEL in that it is composed of multiple related subunits encoded by a cct multigene family containing at least seven members in both mouse and bovine species. TCP-l (CCT-l) was the first subunit from the chaperonin to be described. We have characterized C elegans cct-l (tcp-l), a gene encoding a 549 a.a protein (59 kDa) with 66percent aa. identity to murine TCP-1. The primary cct-1 transcript undergoes both cis splicing of its four introns and trans-splicing to SL1. C. elegans cct-l is expressed constitutively throughout development, and contrary to heat-shock protein genes (many of which encode molecular chaperones), it is not heat-inducible. Five cDNAs isolated during the C. elegans cDNA sequencing project are significantly homologous to eukaryotic cct-l (rcp-l). We have named these cDNAs according to the C elegans nomenclature, based on the Greek letter assignment for the mouse genes. So far, three gene sequences are known (cct-l on chr. II, cct5 and cct-6 on chr. III), all genes have been mapped, and we are currently sequencing the two cct cDNAs for which no sequence is available (cct-2 and cct-4). The amino acid sequence identity between homologous eukaryote CCT subunits is greater than 60percent, and the identity between the different CCT subunits is 25-35percent. Little information is known of the tissue distribution of CCT in higher eukaryotes, apart from the observed high level expression in mouse and bovine testis. In C elegans, the five known cct genes are expressed constitutively at similar (relative) levels during development. In order to ascertain any tissue specificity of some of these genes in the nematode, we are currently injecting promoter-LacZ constructs, and are preparing antibodies for in situ immuno-localization. We have also begun to characterize the chaperonin protein complex. C elegans CCT was isolated using sucrose gradient fractionation and ATP-agarose chromatography. Using antibodies raised against C elegans CCT-1, the native chaperonin complex containing CCT-l was identified on the sucrose gradient as a particle of Mr greater than the protein standard thyroglobulin (669 kDa). Sucrose fractions containing the chaperonin were further purified on an ATP-agarose column. Western blot analysis of an aliquot eluted from the column with ATP reveals the presence of a 57-kDa protein corresponding to CCT-l. In brief, C elegans CCT-1 is a 57-kDa protein present within a high-Mr complex which can bind ATP. Additionally, preliminary results suggest that C. elegans CCT contains six or more protein subunits with Mr distributions similar to bovine and mouse CCT.