Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
EMBO J. 2013 Jan 9;32(1):159-71. doi: 10.1038/emboj.2012.307. Epub 2012 Nov 27.
Among SNARE proteins mediating synaptic vesicle fusion, syntaxin-1 uniquely includes an N-terminal peptide ('N-peptide') that binds to Munc18-1, and a large, conserved H(abc)-domain that also binds to Munc18-1. Previous in vitro studies suggested that the syntaxin-1 N-peptide is functionally important, whereas the syntaxin-1 H(abc)-domain is not, but limited information is available about the in vivo functions of these syntaxin-1 domains. Using rescue experiments in cultured syntaxin-deficient neurons, we now show that the N-peptide and the H(abc)-domain of syntaxin-1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the H(abc)-domain regulates this fusion, in part by forming the closed syntaxin-1 conformation. Moreover, we observed that deletion of the H(abc)-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the H(abc)-domain stabilizes Munc18-1. Thus, the N-terminal syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes.
在介导突触囊泡融合的 SNARE 蛋白中，突触融合蛋白-1 独特地包含一个 N 端肽（“N 肽”），该肽与 Munc18-1 结合，以及一个大的、保守的 H(abc)-结构域，该结构域也与 Munc18-1 结合。先前的体外研究表明，突触融合蛋白-1 的 N 肽在功能上很重要，而 H(abc)-结构域则不重要，但关于这些突触融合蛋白-1 结构域的体内功能的信息有限。通过在培养的突触融合蛋白缺陷神经元中进行挽救实验，我们现在表明，突触融合蛋白-1 的 N 肽和 H(abc)-结构域在突触囊泡融合中发挥不同且独立的作用。具体来说，我们发现 N 肽本身对于囊泡融合是必需的，而 H(abc)-结构域调节这种融合，部分是通过形成封闭的突触融合蛋白-1 构象。此外，我们观察到 H(abc)-结构域的缺失而不是 N 肽的缺失导致 Munc18-1 的丢失，这导致突触处可释放囊泡的易释放池减少，表明 Munc18 与 H(abc)-结构域的结合稳定了 Munc18-1。因此，N 端突触融合蛋白-1 结构域在突触囊泡融合中介导不同的功能，可能通过形成不同的 Munc18/SNARE 蛋白复合物。
