Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
Department of Chemical Physiology and Biochemistry, School of Medicine, Oregon Health and Science University, Portland, OR 97239.
Proc Natl Acad Sci U S A. 2021 Dec 7;118(49). doi: 10.1073/pnas.2025748118.
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 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 有助于促进胞吐作用中的膜融合。它还在质膜中形成簇，其跨膜和 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 syntaxin 簇集，我们用神经肽-Y-mCherry 标记颗粒，用 syntaxin-1a 绿色荧光蛋白（GFP）标记 syntaxin 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（“在颗粒上的簇”）。Syntaxin-1a-GFP 或其突变体以低水平表达，并与过量的内源性 syntaxin 竞争包含在簇中。已知抑制与 Munc18-1 在体外结合的突变会减少与颗粒相关的包含。Munc18-1 的敲低揭示了依赖于 Munc18 和独立于 Munc18 的在颗粒上的簇集。预期会破坏 syntaxin 的 Hb 和 SNARE 结构域之间盐桥的突变会抑制簇集，并通过预期恢复它们的额外突变来挽救。最有可能的是，当 syntaxin 在颗粒上聚集时，它处于封闭构象，其 SNARE 和 Hb 结构域接近原子距离。用丙氨酸替代 Munc18 接触残基的成对替换只有适度的影响，除了一对基本消除了在颗粒上的簇集。总之，一个在颗粒上的簇集是由颗粒和密集的 syntaxin-Munc18-1 复合物簇之间的特异性相互作用产生的。相比之下，离线簇即使是我们尝试过的最强突变也具有抗性，既不需要 Munc18-1 也不需要 SNARE 结构域的存在。它们很可能通过与膜脂质的非化学计量相互作用形成，其他人在无细胞系统中观察到了这种相互作用。
