Mutations in Munc18, an essential protein for several stages of synaptic vesicle exocytosis, have been linked to early infantile epileptic encephalopathy. Null mutations in Munc18 and its homologs in flies (Rop) and yeast (SEC1) are lethal, but null mutations in the C. elegans homolog (
unc-18) are not. Consequently C. elegans provide a suitable model to investigate the effects of loss-of-function. We conducted an ethylmethanesulfonate (EMS) mutagenesis suppressor screen of the
unc-18 e81 allele resulting in a novel strain, RESCUE, in which the paralysed phenotype of
unc-18 null mutants was rescued. Two novel mutations found in the rescue strain, present in the
dgk-1 and
sorf-2 genes were hypothesised to facilitate bypass of
unc-18 function. Diacylglycerol kinase (
dgk-1) catalyses the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), thus functioning in synaptic transmission. Sorf-2 encodes the C. elegans homolog of WDR81, predicted to function in organelle fusion and endosomal transport. We confirmed that RESCUE worms were significantly better at locomotion compared to
unc-18 null mutants and statistically indistinguishable from wild-type worms. Introduction of wild-type
dgk-1 into RESCUE worms reversed the improvement in locomotion to
unc-18 null levels suggesting necessity of the
dgk-1 mutation for the rescued phenotype. Elevating DAG levels in
unc-18 null mutants failed to improve locomotion, both genetically and pharmacologically, suggesting the
dgk-1 mutation alone is not sufficient for phenotypic rescue. Sorf-2 RNAi in
unc-18 null mutants improved locomotion following treatment with the DGK inhibitor II, R59949. Further investigation into the
sorf-2 mutation will lead to a better understanding of the mechanisms through which the two mutations bypass the
unc-18 null mutation. This will further allow research into WDR81 function, little of which is known as of yet.