Ordering UNC-13 and UNC-18 function at the synapse

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DESCRIPTION (provided by applicant): UNC-18 and UNC-13 are presynaptic proteins involved in the synaptic vesicle exocytosis steps of docking, priming, and fusion. UNC-13 functions in priming, yet UNC-18 function is obscure. To determine if UNC-18 can function in either docking or fusion, the ordering of UNC-13 and UNC-18 interactions will be determined. Four specific aims will be pursued. 1) To implicate UNC-18 in docking, syntaxin-complexes will be purified from wild type and unc-13 mutant C. elegans. An increased UNC-18 in the unc-13 background will implicate UNC-18 in vesicle docking. 2) To test for a fusion role, the synaptobrevin and SNAP-25 content of a syntaxin/UNC-18 co-complex will be measured. If UNC-18 is present in complexes containing the requisite SNARE proteins, then a fusion role is implicated. 3) To detect UNC-18 binding to open syntaxin, constitutively open syntaxin mutants will be TAP tagged and screened for the presence of UNC-18, SNAP- 25 and synaptobrevin. The presence of UNC-18 will demonstrate UNC-18 interacts with the open conformation of syntaxin. If SNAP-25 and synaptobrevin are also present, then a fusion role for UNC-18 will be further supported.