Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.
Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.
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DOI：
10.1091/mbc.e15-07-0503
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发表时间：
2016-03-15
影响因子：
3.3
通讯作者：
Chapman ER
中科院分区：
文献类型：
--
作者：
Evans CS;He Z;Bai H;Lou X;Jeggle P;Sutton RB;Edwardson JM;Chapman ER
Synaptotagmin (syt)-1是一种Ca2+传感器，可触发快速突触囊泡胞吐。破坏syt-1串联Ca2+传感模块（C2结构域）之间物理相互作用的突变破坏了重建融合反应和神经元中受调节的膜融合。因此，这些区域之间的联系对功能很重要。C2结构域是广泛存在的基序，通常作为Ca2+结合模块；有些蛋白质有不止一个拷贝。一个悬而未决的问题是，当这些结构域在同一亲本蛋白中复制时，是否会相互作用以调节功能。在本研究中，我们讨论了突触塔蛋白(syt)-1串联C2结构域之间界面残基的功能意义，突触塔蛋白(syt)-1是神经元胞外分泌的Ca2+传感器。在结构域界面上替换四个残基YHRD，破坏了串联C2结构域之间的相互作用，改变了syt-1对Ca2+的内在亲和力，并改变了Ca2+对膜结合和驱动膜融合的依赖性。当在syt-1敲除神经元中表达时，与野生型蛋白相比，YHRD突变体产生突触传递减少。这些结果表明syt-1串联C2结构域之间的物理相互作用有助于兴奋-分泌耦合。
Synaptotagmin (syt)-1 is a Ca2+ sensor that triggers rapid synaptic vesicle exocytosis. Mutations that disrupt physical interactions between the tandem Ca2+-sensing modules (C2 domains) of syt-1 disrupt regulated membrane fusion in reconstituted fusion reactions and in neurons. Hence contacts between these domains are important for function. C2 domains are widespread motifs that often serve as Ca2+-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca2+ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca2+, and shifted the Ca2+ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.