Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism
Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism
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DOI：
10.1074/jbc.m906729199
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Chapman, ER
中科院分区：
文献类型：
--
作者：
Bai, JH;Earles, CA;Chapman, ER
突触囊泡蛋白synaptotagmin I已被提出作为快速胞吐的Ca 2+传感器，Synaptotagmin跨越囊泡膜一次，并具有主要由两个C2结构域组成的胞质结构域，命名为C2 A和C2B。我们已经确定了Ca 2 + C2 A的Ca 2+结合环渗透到脂质双层的深度，并报告了突触结合蛋白的突变，该突变可以从Ca 2+触发的与SNARE复合物的相互作用中解偶联膜渗透。为了确定C2 A是否渗透到囊泡（“顺式”）或质膜（“反式”），我们重建了一个片段的突触结合蛋白，其中包括跨膜和C2 A结构域（C2 A-TMR）到脂蛋白体。动力学实验表明，顺式相互作用是快速的（小于或等于500 μ s）。以反式模式结合的特点是反式靶囊泡的缓慢扩散。观察到两种结合模式，表明膜锚和C2 A结构域之间的接头起柔性系链的作用。C2 A-TMR通过一个新的N-末端寡聚化结构域组装成寡聚体，表明突触结合蛋白可以在突触囊泡表面形成簇。这种新的聚类模式可以允许通过膜远端C2B结构域的蛋白质的快速Ca 2+触发的寡聚化。
The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis, Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+ C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (less than or equal to 500 mu s). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles, Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca2+- triggered oligomerization of the protein via the membrane distal C2B domain.