Sitarska Ewa, Xu Junjie, Park Seungmee, Liu Xiaoxia, Quade Bradley, Stepien Karolina, Sugita Kyoko, Brautigam Chad A, Sugita Shuzo, Rizo Josep
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States.
Elife. 2017 May 6;6:e24278. doi: 10.7554/eLife.24278.
Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the 'furled conformation' of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.
Munc18-1与Munc13-1共同协调SNARE复合体组装，以介导神经递质释放。Munc18-1与突触小泡蛋白结合，但其相互作用的相关性及其与Munc13功能的关系尚不清楚。核磁共振实验表明，Munc18-1与突触小泡蛋白特异性和非特异性结合。Munc18-1中的L348R突变抑制特异性结合，而设计用于破坏Munc18-1环“卷曲构象”的D326K突变增强特异性结合。相应地，在需要Munc18-1和Munc13-1进行膜融合的重组实验中，Munc18-1的活性受到D326K突变的刺激和L348R突变的抑制。此外，D326K突变允许不依赖Munc13-1的融合，并在空突变体的拯救实验中导致功能获得。与之前的研究一起，我们的数据支持一个模型，即Munc18-1作为SNARE复合体组装的模板，突触小泡蛋白结合的自抑制有助于Munc13-1对神经递质释放的调节。
