Though 131 somatic calls and many more germ cells undergo apoptosis during C. elegans development, the identity and function of the protein interactions involved in this process are poorly understood. Through a combination of forward and reverse genetics, 21 genes have been shown to be involved in C. elegans apoptosis, with 13 additional genes predicted by homology to have a role in apoptosis. We generated a protein-protein interaction map using each one of these 34 known or suspected apoptosis gene products, and combined the results with our previously published worm interactome map version 5 (WI5) (Li et al Science, 2004). Our apoptosis interaction map consists of 218 potential interactions involving 20 known or suspected apoptosis proteins and 167 new potential components. We are currently analyzing the phenotypic consequences of disrupting individual protein-protein interactions within the apoptosis interactome. This approach complements a null allele approach, whereby the phenotype results from the absence of an entire protein and all the interactions it makes. The consequences of removing all interactions simultaneously may be to completely disrupt the network that the protein acted in, making it difficult to define the function of any individual interaction. By contrast, removing individual interactions from a network may allow us to preserve the integrity of the network, allowing us to observe more subtle and specific phenotypes. To analyze interaction specific phenotypes, we focused on four proteins found to interact with CED-9, an essential signal integrator in the apoptotic network. We disrupted each of these interactions by generating interaction defective alleles (IDA) with reverse yeast two-hybrid (Vidal et al PNAS 1996). This R-Y2H approach generated alleles that disrupt single interactions, while leaving the other interacting proteins largely intact. Thus far we have generated transgenic worms with IDAs that individually disrupt each of the four CED-9 interactions as assessed in yeast and in a co-affinity purification interaction assay. To assess the in vivo consequences of interaction disruption on the apoptotic protein interaction network, these transgenic animals are being crossed into
ced-9(lf) and other genetic backgrounds.