Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro
Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro
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DOI：
10.1074/jbc.m803478200
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发表时间：
2008-07-25
影响因子：
4.8
通讯作者：
Rothman, James E.
中科院分区：
文献类型：
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作者：
Giraudo, Claudio G.;Garcia-Diaz, Alejandro;Rothman, James E.
在受调节的胞吐作用中，核心膜融合机器蛋白（SNARE蛋白）由一组调节因子辅助，以将膜融合与细胞内钙离子（Ca 2（+））浓度的增加偶联。复合蛋白-I和突触结合蛋白-I已被证明是这种严格调控过程的关键要素。许多研究表明，复合蛋白-I可以阻止融合反应，突触结合蛋白-I可以以钙依赖性方式释放复合蛋白-I阻断。虽然它们发挥功能的实际分子机制仍然未知，但最近的体内实验假设复合蛋白-I的结构域对神经递质释放产生不同的影响。在此，通过使用体外翻转SNARE细胞融合试验，我们已经确定并表征了将钙和突触结合蛋白-I偶联到膜融合所需的复合蛋白-I的最小功能结构域。此外，我们提供的证据表明，其他亚型的复合蛋白，复合蛋白-II，-III，和-IV，也可以在功能上耦合到突触结合蛋白-I和钙。这些密切对应于体内实验的结果，提供了翻转SNARE系统的生理相关性的进一步验证。
In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca2(+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.