DISASSEMBLY OF THE RECONSTITUTED SYNAPTIC VESICLE MEMBRANE-FUSION COMPLEX IN-VITRO
DISASSEMBLY OF THE RECONSTITUTED SYNAPTIC VESICLE MEMBRANE-FUSION COMPLEX IN-VITRO
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DOI：
10.1002/j.1460-2075.1995.tb07226.x
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发表时间：
1995-05-15
期刊：
影响因子：
11.4
通讯作者：
NIEMANN, H
中科院分区：
文献类型：
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作者：
HAYASHI, T;YAMASAKI, S;NIEMANN, H
突触前膜蛋白SNAP-25和syntaxin与突触囊泡蛋白synaptobrevin（VAMP）的相互作用在调节神经递质的胞吐中起关键作用。梭菌神经毒素，蛋白水解这些多肽，是神经传递的有效抑制剂。三种膜蛋白的胞质结构域连接成紧密的SDS抗性复合物（Hayashi艾德等人，1994年）。在这里，我们表明，这种重建的复合物，以及异源二聚体组成的syntaxin和SNAP-25，可以被拆解的协调行动的N-乙基马来酰亚胺敏感因子，NSF和可溶性NSF附着蛋白，α-SNAP，α-SNAP结合预测的α-螺旋卷曲螺旋区域的syntaxin和SNAP-25，以前被证明是从事他们的直接相互作用。小突触泡蛋白虽然不能单独结合α-SNAP，但当与突触融合蛋白和SNAP-25结合成异源三聚体时，诱导第三个α-SNAP结合位点。NSF从全长的突触融合蛋白中释放预先结合的α-SNAP，但不从N-末端截短的突触融合蛋白衍生物中释放。含有这种突触融合蛋白突变体的复合物的构象受损，表明N-末端结构域在α-SNAP/NSF介导的解离过程中起关键作用。含有C-末端缺失的SNAP-25衍生物的复合物，如由A型和E型肉毒毒素产生的，更有效地解离。相比之下，N-末端片段产生的小突触素肉毒毒素F型产生的SDS敏感的复合物，解离差。
The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi ed al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the n-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP, alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.