Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, Oregon, United States.
Sci Rep. 2018 Jan 9;8(1):193. doi: 10.1038/s41598-017-18597-z.
SNAREs and SNARE-binding accessory proteins are believed to be central molecular components of neurotransmitter release, although the precise sequence of molecular events corresponding to distinct physiological states is unclear. The mechanism of docking of vesicles to the plasma membrane remains elusive, as the anchoring protein residing on vesicles is unknown. Here I show that targeting small amounts of syntaxin to granules by transmembrane domain alteration leads to a substantial enhancement of syntaxin clustering beneath granules, as well as of morphological granule docking. The effect was abolished without munc18 and strongly reduced by removal of the N-terminal peptide in the syntaxin mutant. Thus, in contrast to the current paradigm, I demonstrate that syntaxin acts from the vesicular membrane, strongly facilitating docking of vesicles, likely via interaction of its N-peptide with munc18. Docking was assayed by quantifying the syntaxin clusters beneath granules, using two-color Total Internal Reflectance Fluorescence microscopy in live PC-12 cells and confirmed by electron microscopy. Hereby, I propose a new model of vesicle docking, wherein munc18 bridges the few syntaxin molecules residing on granules to the syntaxin cluster on the plasma membrane, suggesting that the number of syntaxins on vesicles determines docking and conceivably fusion probability.
SNAP 受体和与其结合的辅助蛋白被认为是神经递质释放的核心分子成分，尽管与不同生理状态相对应的分子事件的确切顺序尚不清楚。尽管囊泡上的锚定蛋白尚不清楚，但囊泡与质膜对接的机制仍难以捉摸。在这里，我发现通过跨膜结构域改变将少量突触融合蛋白靶向颗粒会导致颗粒下突触融合蛋白簇的大量增强，以及形态学颗粒对接的增强。该效应在没有 munc18 的情况下被消除，并且在突触融合蛋白突变体中去除 N 端肽后强烈降低。因此，与当前的范例相反，我证明了突触融合蛋白从囊泡膜上发挥作用，强烈促进囊泡的对接，可能是通过其 N 肽与 munc18 的相互作用。通过在活 PC-12 细胞中使用双色全内反射荧光显微镜对颗粒下的突触融合蛋白簇进行定量来检测对接，并通过电子显微镜进行确认。因此，我提出了一种新的囊泡对接模型，其中 munc18 将位于颗粒上的少数几个突触融合蛋白与质膜上的突触融合蛋白簇连接起来，这表明囊泡上的突触融合蛋白数量决定了对接，可能还决定了融合的概率。
