The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly

Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion.

细胞内货物的囊泡运输需要精准的靶向膜融合，以及能够将相互贴近的待融合膜拉近的SNARE蛋白复合体（SNARE protein complex）的形成。胰岛素调控的葡萄糖转运蛋白4（glucose transporter-4, GLUT4）储存囊泡在细胞表面的递送与膜融合，依赖于两种关键蛋白：作为SNARE整合膜蛋白的突触融合蛋白4（Syntaxin4, Sx4），以及可溶性调控蛋白Munc18c。既往诸多针对Munc18c与Sx4相互作用及SNARE复合体形成的体外研究，均采用了缺失天然跨膜结构域的可溶性Sx4构建体。正因如此，Sx4 C端锚定区域的重要性至今仍未得到充分阐明。
本研究发现，相较于C端跨膜结构域被T4溶菌酶融合蛋白替代的Sx4，可溶性C端截短的Sx4与Munc18c的解离速率更快。我们还观察到：当使用可溶性C端截短的Sx4时，Munc18c似乎会抑制SNARE复合体的形成；但当Sx4带有C端锚定结构时（如结合层析树脂的C端组氨酸标签（His-tag）、C端T4溶菌酶融合结构，或是去污剂胶束中的天然C端跨膜结构域），Munc18c并不会抑制SNARE复合体的形成。
基于上述结果，我们得出结论：Sx4的C端对于其与Munc18c的相互作用至关重要，而此前报道的Munc18c的抑制作用可能是实验设计带来的人为假象。本研究结果支持这一观点：Munc18c的主要功能是促进SNARE复合体的形成与膜融合。