James, Claerwen Laura; (1998) Analysis of components of the Caenorhabditis elegans cell death apparatus in a heterologous system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The nematode worm Caenorhabditis elegans has proved to be an illuminating model for programmed cell death. Three principal genes are involved in nematode PCD, ced-3, ced-4 and ced-9: each has proved to have vertebrate homologues that are critical regulators of vertebrate apoptosis. Until recently, the molecular functions of CED-4 and CED-9 were largely obscure. I have used the yeast Schizosaccharomyces pombe as a naive model system in which to assay the function of the pro-apoptotic protein CED-4. Expression of wild-type ced-4 is toxic to S. pombe, while expression of the point mutant I258N, which gives rise to a ced-4-null phenotype in the worm, has no effect, suggesting that the toxicity is the result of a bona fide activity of CED-4. Mutation of the putative nucleotide-binding P-loop motif of CED-4 also eliminates the lethal phenotype, demonstrating for the first time the importance of this domain for CED-4 function. The anti-apoptotic protein CED-9 is able to rescue S. pombe from CED-4- induced lethality: the most parsimonious explanation for this observation is that CED-9 directly binds and inhibits CED-4. This is confirmed by immunogold labelling of CED-4 visualised by electron microscopy: CED-4 expressed alone is nuclear, but when co-expressed with CED-9 it is found on mitochondrial and ER membranes, the presumptive location of CED-9. The physical interaction between CED-4 and CED-9 is further confirmed by yeast two-hybrid analysis. In addition, the cloning and characterisation of two C. elegans homologues of baculovirus Inhibitor of Apoptosis Proteins (IAPs) is described. Cellular IAP homologues are found in Drosophila and humans and influence apoptosis in both organisms. The C. elegans IAP homologues do not inhibit the activity of CED-3 or CED-4 in S. pombe. Knockout of their activity in vivo by means of RNA-mediated inhibition reveals no obvious cell death-related phenotype.

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