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.

A C. elegans Hetero-Oligomeric Chaperonin Related to Hsp60/GroEL Contains CCT-1, a Member of a Family of Related Proteins.

Michel R. Leroux, E. Peter M. Candido

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
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