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Characterization of two C. elegans molecular chaperone families, CCT (chaperonin containing TCP-1) and the small heat shock proteins

University of British Columbia

Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partly or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated into an organelle, or damaged by cellular stress. The work presented in this thesis describes the first studies aimed at characterizing some of the structural and functional properties of two molecular chaperone families of the nematode Caenorhabditis elegans, CCT (chaperonin containing TCP-1) and the small heat shock proteins (smHSPs). CCT is involved in folding newly synthesized actin and tubulin, and may play a more general role in protein folding within the eukaryotic cytosol. C. elegans CCT is an ATP-binding complex of about 900 kDa. Sucrose gradient fractionation and ATP-agarose chromatography were used to purify the CCT complex from embryos. Over 7 subunits ranging between 52- 65 kDa were detectable, of which two were shown to be CCT-1 and CCT-5 by immunoblotting. Native gel electrophoresis of the CCT revealed three distinct species: one contains CCT-1 and CCT-5 and hence represents the CCT complex, another contains HSP60, which has the highest affinity for chemically-denatured actin, and the last species remains unidentified. A 3.7 kb PstI genomic fragment encoding the 59 kDa CCT-1 protein was cloned and sequenced. The cct-1 transcript undergoes both c/s-splicing of its four introns and trans-splicing to SL1. Three additional cct cDNAs were sequenced (cct-2, cct-4, and cct-5), and the sequence of a fifth cct gene (cct-6) was obtained from the C. elegans sequencing consortium. The C. elegans cct multigene family displays 23-35% sequence identity between members and about 65% identity to the corresponding murine cct homologues. Northern blot analyses show that the five C. elegans cct genes are expressed in all life stages. Transgenic lines carrying a cct-1-promoter-lacZ construct revealed that cct-1 is expressed in various tissues, including muscles and the nervous system. The smHSP and α-crystallin genes encode a family of proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the α-crystallin domain. Studies on wild-type HSP16-2 and five derivatives demonstrate that multimerization and chaperone activity depend on the full-length nonconserved N-terminal region and are not affected by removal of most of the C-terminal extension which follows the α-crystallin domain. The N-terminal region of HSP16-2 is buried within an oligomeric complex which can accomodate an additional 4 kDa of heterologous protein sequence. It was found that HSP16-2 has an equally high affinity for unfolded actin and tubulin intermediates which form early on the renaturation or aggregation pathway. The structure-function data on HSP16-2 are complemented by studies on HSP12.6, the smallest smHSP to be characterized. In contrast to other smHSPs, the stage-specific HSP12.6 does not multimerize and lacks chaperone activity.

Thesis/Dissertation

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2009-04-02

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http://hdl.handle.net/2429/6733

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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