Regulated exocytosis of secretory vesicles is critically dependent on the assembly of SNARE complexes between the plasma membrane SNAREs, synatxin and SNAP-25 and vesicle-associated synaptobrevin, which render vesicles fusion competent. Tomosyn, a syntaxin-binding protein, forms a complex with syntaxin and SNAP-25 in competition with synaptobrevin predicted to limit fusogenic SNARE complex formation (Fujita et al, 1998). Consistent with these data, the highly conserved C. elegans tomosyn homolog, TOM-1, has been shown to negatively regulate both synaptic and dense-core vesicle release (Gracheva et al., 2006; Gracheva et al.,2007). TOM-1 appears to be associated with both synaptic vesicles (Takamori et al., 2006) and dense core vesicles (Gracheva et al., 2007) via an unknown mechanism. Consequently, the synaptic localization of TOM-1 is dependent on the anterograde vesicle kinesin motor (UNC-104) (McEwen et al., 2006). Given the importance of TOM-1 in the regulation of synaptic strength, we are interested in identifying the mechanism responsible for the synaptic localization of this protein. Neither syntaxin nor SNAP-25, the two established tomosyn-interacting proteins appear to be involved in TOM-1 transport to synapses (McEwen et al., 2006). In order to screen for TOM-1 trafficking defective mutants, we have generated an integrated transgene expressing TOM-1::GFP. We have verified that this protein is expressed at synapses and localizes in an UNC-104-dependent manner. We are currently screening known vesicle-associated candidate proteins by RNAi or by crossing the TOM-1::GFP transgene into reference alleles to examine their potential role in the regulation of tomosyn trafficking. Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, YokoyamaS, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH, Takai Y (1998) Tomosyn: asyntaxin-1-binding protein that forms a novel complex in the neurotransmitterrelease process. Neuron 20:905-915. Gracheva EO, Burdina AO, Touroutine D, Berthelot-Grosjean M, Parekh H,Richmond JE (2007) Tomosyn negatively regulates CAPS-dependent peptide releaseat Caenorhabditis elegans synapses. J Neurosci 27:10176-10184. Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD,Hadwiger G, Nonet ML, Weimer RM, Richmond JE (2006) Tomosyn inhibits synapticvesicle priming in Caenorhabditis elegans. PLoS Biol 4:
e261.McEwen JM, Madison JM, Dybbs M, Kaplan JM (2006) Antagonistic regulationof synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51:303-315. Takamori S et al. (2006) Molecular anatomy of a trafficking organelle.Cell 127:831-846.