The
sec-1/Munc-18 family of proteins (SM proteins) play a critical but uncertain role in regulating vesicle fusion during synaptic transmission, secretion, and intracellular membrane trafficking. Genetic disruption of neuronal SM function results in a severe reduction of exocytosis. For example, synaptic transmission in Munc18-1 null mutant embryos is completely abolished. SM proteins are well known to bind to the SNARE protein syntaxin, and also to interact with other proteins localized at sites of vesicle exocytosis. We are exploiting C. elegans genetics to identify proteins that interact with (or in parallel to) SM proteins to regulate exocytosis at synapses. The C. elegans
unc-18 gene, a founding member of the SM family, encodes a protein that is expressed in neurons and is present at presynaptic terminals.
unc-18 loss of function mutant animals are paralyzed, and have markedly reduced levels of evoked and endogenous neurotransmitter release at neuromuscular junctions. In three
unc-18 alleles, the magnitude of evoked post-synaptic currents is reduced to an average of 15% of that in wild-type animals, and the frequency of endogenous release events is reduced to an average of 12% of wild-type. We are using genetic supression of
unc-18 paralysis to identify proteins that interact with UNC-18 or that can compensate for the loss of
unc-18 function. First, we have isolated an extragenic suppressor of
unc-18 mutations that arose spontaneously during the characterization of
unc-18(
e234). Animals with improved motility were picked and outcrossed, after which the extragenic suppressor sup
(ox151) was isolated. sup
(ox151) animals are wild type in phenotype. At least three alleles of
unc-18 are suppressed by sup
(ox151). Suppressed animals exhibit a significant improvement of both evoked and spontaneous release. The magnitude of the evoked current in suppressed animals is elevated about 2.5 fold, to an average of 38% of that observed in wild-type. The frequency of endogenous release is increased about 4-fold, to an average of 51% of wild-type level. Second, to identify new
unc-18 suppressors, we have conducted pilot screens of EMS mutagenized
unc-18(
b403) populations for animals having improved motility. We have isolated one moderately strong and several weaker suppressors from 100 populations containing 200-500 mutagenized haploid genomes. The strongest suppressor, sup
(ja1), is extragenic, and elevates evoked release to about 35% that of wild-type. We are currently using SNP mapping to locate sup
(ox151) and sup
(ja1), and we are conducting extensive screens for more suppressors using multiple
unc-18 alleles.