Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis
Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis
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DOI：
10.1242/jcs.126862
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发表时间：
2013-06-01
影响因子：
4
通讯作者：
Sugita, Shuzo
中科院分区：
文献类型：
--
作者：
Han, Gayoung Anna;Bin, Na-Ryum;Sugita, Shuzo
Munc18-1 除了陪伴其同源突触融合蛋白外，还被认为通过与 SNARE 复合物结合来引发或刺激 SNARE 介导的膜融合/胞吐作用。然而，尚未发现选择性丧失启动功能同时保留突触蛋白伴侣活性的 Munc18-1 突变体。因此，介导 Munc18-1 依赖性启动的机制仍不清楚。在分析 Munc18-1 结构域 3a 中各种点突变的功能结果的过程中，我们发现了插入突变体（插入 5 或 39 个残基的 K332E/K333E）。这些突变体完全失去了拯救分泌的能力，但它们有效地恢复了质膜上的 Syntaxin-1 表达以及 Munc18-1 和 Munc18-2 双敲低 PC12 细胞中的致密核心囊泡对接。突变体可以化学计量方式结合突触蛋白-1A。然而，与野生型或疏水口袋突变体 (F115E) 相比，与 SNARE 复合物的结合受到损害。我们的结果表明，Munc18-1 的结构域 3a 在胞吐作用的启动中起着至关重要的作用，其独立于其 Syntaxin-1 陪伴活性，并且位于致密核心囊泡对接的下游。我们还认为 Munc18-1 的启动机制涉及其与 SNARE 复合体的结构域 3a 依赖性相互作用。
Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.