Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States.
Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.
Elife. 2017 Sep 12;6:e28409. doi: 10.7554/eLife.28409.
The synaptic vesicle Ca sensor Synaptotagmin binds Ca through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca-binding mutants in . This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca activation of the synaptic vesicle fusion machinery.
突触囊泡 Ca 传感器突触融合蛋白通过其两个 C2 结构域与 Ca 结合，从而触发膜相互作用。除了 C2 结构域介导的膜插入之外，突触融合蛋白活性的其他要求仍在阐明中。为了确定突触融合蛋白中对囊泡循环所必需的关键残基，我们利用了这样的观察结果，即 C2B 结构域 Ca 结合口袋中的突变主要破坏了从无脊椎动物到人释放的现象。我们在. 中进行了一种基因内筛选，以寻找表达突触融合蛋白 C2B Ca 结合突变体时诱导的致死性的抑制子。该筛选揭示了突触融合蛋白中必需的残基，这些残基为融合所必需的几种活性提供了结构基础，包括涉及 SNARE 复合物相互作用的 C2B 表面，这对于囊泡释放的快速同步和 Ca 协同作用是必需的。通过对这些突变体进行电生理学、形态学和计算特性的研究，我们提出了由突触融合蛋白介导的促进 Ca 激活突触囊泡融合机制的一系列分子相互作用。
