A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly
A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly
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DOI：
10.1074/jbc.m312064200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Margittai, M
中科院分区：
文献类型：
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作者：
Fasshauer, D;Margittai, M
SNARE蛋白syntaxin， SNAP-25和synaptobrevin在神经末端Ca2+依赖性胞吐过程中发挥核心作用。syntaxin和SNAP-25位于质膜上，而synaptobrevin则位于突触囊泡的膜上。据认为，这些蛋白质逐渐组装成膜桥接三元SNARE复合物最终导致膜融合。根据该模型，syntaxin和SNAP-25构成synaptobrevin的受体复合体。然而，在体外，syntaxin和SNAP-25形成一个稳定的复合物，包含两个syntaxin分子，其中一个占据并可能阻碍synaptobrevin的结合位点。为了阐明突触SNAREs的组装途径，我们现在应用了荧光和CD光谱的组合。我们发现SNARE组装始于syntaxin和SNAP-25缓慢且限速的相互作用。它们的相互作用被n端阻止，而不是被c端截断，这表明为了有效组装，所有三个参与的螺旋必须同时聚集在一起。这表明一个复杂的成核过程可能是观察到的缓慢组装速率的原因。SNAP-25和syntaxin的N端截断也阻止了三元配合物的形成，而N端和c端缩短的突触缩短蛋白螺旋都没有失去相互作用的能力。这表明synaptobrevin的结合发生在syntaxin-SNAP-25相互作用建立之后。此外，synaptobrevin的结合被过量的syntaxin抑制，这表明syntaxin和SNAP-25的1:1相互作用是通路上SNARE组装的中间体。
The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca2+-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1: 1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.