Munc18-1:: Sequential interactions with the fusion machinery stimulate vesicle docking and priming
Munc18-1:: Sequential interactions with the fusion machinery stimulate vesicle docking and priming
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DOI：
10.1523/jneurosci.0658-07.2007
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发表时间：
2007-08-08
影响因子：
5.3
通讯作者：
Sorensen, Jakob B.
中科院分区：
文献类型：
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作者：
Gulyas-Kovacs, Attila;de Wit, Heidi;Sorensen, Jakob B.
分泌或突触囊泡的胞吐作用由包括SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白的机制执行。Munc 18 -1是这种融合机制的一部分，但它的作用是有争议的，因为它是融合不可缺少的，但也抑制了纯化的SNARE在体外的组装。这种抑制反映了Munc 18 -1与靶SNARE突触融合蛋白1的闭合构象的结合。如果结合到封闭的突触融合蛋白1被证明是刺激囊泡融合和/或额外的必要的相互作用被确定之间的Munc 18解剖的顺序步骤的胞吐级联，同时表达Munc 18变体在Munc 18 -1空背景下，争议将得到解决。在Munc 18 -1无效嗜铬细胞中，囊泡对接被取消，突触融合蛋白水平降低。一个突变，减少Munc 18结合突触融合蛋白1在体外衰减的小泡对接步骤，但抢救小泡启动超过对接。相反，表达Munc 18 -2同种型，其也显示与闭合的突触融合蛋白1结合，挽救了与Munc 18 -1相同的囊泡对接，但损害了更多下游囊泡引发步骤。所有的Munc 18变体都将突触融合蛋白1水平至少恢复到野生型水平，表明对接表型不是由突触融合蛋白1减少引起的. Munc 18变体均不影响囊泡融合动力学或融合孔持续时间。总之，结合Munc 18 -1封闭的突触融合蛋白1刺激囊泡对接和一个独特的相互作用模式调节连续的启动步骤。
Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target- SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild- type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.