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

描述（由申请人提供）：UNC-18和UNC-13是突触前蛋白，参与突触囊泡胞吐的对接、启动和融合步骤。UNC-13在启动中起作用，而UNC-18的作用尚不清楚。为了确定UNC-18是否可以在对接或融合中发挥作用，将确定UNC-13和UNC-18相互作用的顺序。将追求四个具体目标。1)为了使UNC-18参与对接，将从野生型和unc-13突变型秀丽隐杆线虫中纯化syntaxin复合物。在unc-13背景下，UNC-18的增加将暗示UNC-18参与囊泡对接。2)为了测试融合作用，将测量syntaxin/UNC-18共络合物的synaptobrevin和SNAP-25含量。如果UNC-18存在于含有所需SNARE蛋白的复合物中，则涉及融合作用。3)为了检测UNC-18与open syntaxin的结合，我们将对组成型open syntaxin突变体进行TAP标记，并筛选UNC-18、SNAP- 25和synaptobrevin的存在。UNC-18的存在将证明UNC-18与syntaxin的开放构象相互作用。如果SNAP-25和synaptobrevin也存在，那么将进一步支持UNC-18的融合作用。

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.