Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

Syntaxin 有助于在胞吐作用期间催化膜融合。它还在质膜中形成簇，其中它的跨膜结构域和 SNARE 结构域都被认为是同源寡聚化的。为了研究活 PC12 细胞中的突触蛋白聚类，我们用神经肽-Y-mCherry 标记颗粒，用突触蛋白-1a 绿色荧光蛋白 (GFP) 标记突触蛋白簇。在全内反射 (TIRF) 照明下出现了丰富的簇，其中一些与颗粒相关（“颗粒上的簇”）。Syntaxin-1a-GFP 或其突变体以低水平表达，并与过量的内源性 syntaxin 竞争以纳入簇。已知在体外抑制与 Munc18-1 结合的突变减少了颗粒内含物。Munc18-1 的敲低揭示了 Munc18 依赖和独立的颗粒聚类。预计会破坏突触蛋白的 Hb 和 SNARE 结构域之间的盐桥的突变抑制了聚类，并通过预计恢复它们的额外突变来拯救。最有可能的是，突触蛋白在颗粒上聚集时处于闭合构象，其 SNARE 和 Hb 结构域接近原子距离。用丙氨酸成对替换 Munc18 接触残基只有适度的效果，除了这对R114A/I115A基本上取消了颗粒上的聚类。总之，颗粒上的簇源于颗粒和密集的突触蛋白-Munc18-1 复合物簇之间的特定相互作用。相比之下，即使是我们尝试过的最强突变，颗粒外簇也能抵抗，既不需要 Munc18-1，也不需要存在 SNARE 域。它们很可能通过与其他人在无细胞系统中观察到的膜脂的非化学计量相互作用形成。