Subtle Interplay between synaptotagmin and complexin binding to the SNARE complex.
Subtle Interplay between synaptotagmin and complexin binding to the SNARE complex.
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DOI：
10.1016/j.jmb.2013.07.001
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发表时间：
2013-09-23
影响因子：
5.6
通讯作者：
Rizo, Josep
中科院分区：
文献类型：
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作者：
Xu, Junjie;Brewer, Kyle D.;Perez-Castillejos, Raquel;Rizo, Josep
关键词：
Ca2+触发的神经递质释放依赖于将突触囊泡和质膜结合在一起的SNARE复合物的形成，依赖于Ca2+传感器synaptotagmin-1，以及起积极和抑制作用的络合素。通过synaptotagmin-1结合SNARE复合体释放络合蛋白抑制活性，引起络合蛋白位移，被认为是触发胞吐的原因。然而，该模型的有效性受到质疑，因为观察到复杂素-1和含有synaptotagmin-1 C2结构域（C2AB）的片段同时结合到膜锚定的SNARE复合物上。通过使用多种生物物理技术，我们发现C2AB和络合蛋白i并不相互结合，但在溶液中确实可以同时与SNARE复合物结合。因此，SNARE复合体包含两种蛋白质的单独结合位点。然而，全内反射荧光显微镜实验表明，在平衡条件下，C2AB可以从膜锚定的SNARE复合物中取代含有其中心SNARE结合螺旋和抑制螺旋（Cpx26-83）的络合蛋白- 1片段。有趣的是，与Cpx26-83相比，全长络合蛋白i与膜锚定的SNARE复合物结合更紧密，并且不会被C2AB取代。这些结果表明，复合物i的N端和/或c端序列与SNARE复合物和/或磷脂的相互作用增加了复合物i对SNARE复合物的亲和力，阻碍了C2AB诱导的解离。我们提出了一种模型，即synaptotagmin-1与SNARE复合体的结合直接或间接导致复合物-1抑制螺旋的重排，而不诱导复合物-1解离，从而减轻抑制活性，并使synaptotagmin-1与复合物-1合作触发释放。
Ca2+-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca2+ sensor synaptotagmin-1, and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C2 domains (C2AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C2AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C2AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26–83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26–83, and is not displaced by C2AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C2AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release.
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