Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex
Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex
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DOI：
10.1093/emboj/17.21.6200
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发表时间：
1998-11-02
期刊：
影响因子：
11.4
通讯作者：
Brennwald, P
中科院分区：
文献类型：
--
作者：
Katz, L;Hanson, PI;Brennwald, P
在酵母SNAP-25同系物Sec9的温度敏感突变的筛查中，我们已经确定了酵母Synaptobrevin同系物Snc2的功能获得突变，这种抑制的遗传特性指向SNARE复合体中Sec9的C末端和Snc2之间的特定相互作用。对野生型和突变蛋白之间相互作用的生化分析证实了这一预测，证明了这些突变对SNARE之间相互作用的特定影响，突变的位置表明Sec9的C端H2螺旋结构域很可能与SNARE复合体中的Snc2平行排列。为了验证这一预测，我们用深蚀刻电子显微镜检查了酵母胞外陷阱复合体的结构，就像神经元陷阱复合体一样，它是一个类似于14纳米长的杆状结构。利用表位标签、抗体和麦芽糖结合蛋白标记，我们发现SSO、SNC和Sec9的两半螺旋结构域在SNARE复合体中平行排列，表明酵母胞外SNARE复合体由一个平行的四螺旋束组成。最后，我们发现SNAP-25在神经元SNAR复合体中也有类似的排列。这为胞外SNARE复合体是一个高度保守的结构提供了强有力的证据，该结构由四个平行的螺旋结构域组成，它们的C末端必须聚合才能实现膜融合。
In a screen fur suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9,,ve have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2, The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs, The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy, Like the neuronal SNARE complex, it is a rod similar to 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned, in parallel within the SNARE complex, suggesting that tbe yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.