Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity
Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity
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DOI：
10.1006/jmbi.2001.4889
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发表时间：
2001-08-24
影响因子：
5.6
通讯作者：
Reed, J
中科院分区：
文献类型：
--
作者：
Langosch, D;Crane, JM;Reed, J
SNARE蛋白对于不同类型的细胞膜融合是必不可少的。虽然它们的细胞质结构域之间的相互作用被认为是建立膜邻近的原因，但跨膜片段在融合过程中的作用目前尚不清楚。在这里，我们使用基于脂质混合和电子显微镜的体外方法来检查跨膜段的潜在融合活性。我们发现，在脂质膜中存在代表突触前可溶性n -乙基丙烯酰亚胺敏感因子附着蛋白受体（SNARES）突触brevin II（也称为VAMP II）或syntaxin 1A跨膜片段的合成肽，但不存在不相关的控制肽，驱动它们的融合。毫摩尔Ca2+浓度的脂质体聚集强烈增强了肽的作用；这表明在没有胞质SNARE结构域的情况下，双分子层的并置有利于它们的融合。肽驱动的融合让人想起天然膜融合，因为它被溶脂抑制，并涉及两层小叶。这表明在完全膜合并之后短暂存在半融合中间体。傅里叶变换红外光谱对脂质双分子层中多肽的结构进行了研究，发现其结构为a-螺旋和p -薄片的混合物。在各向同性溶液中，圆二色光谱显示肽以浓度依赖的a-螺旋和p -片结构平衡存在。有趣的是，一组变异肽的促聚变活性随着a-螺旋溶液结构稳定性的增加而降低。因此，跨膜片段的结构可塑性可能对SNARE蛋白在膜融合后期的功能起重要作用。(C) 2001学术出版社。
SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARES) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca2+ concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of a-helical and P-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of a-helical and P-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the a-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion. (C) 2001 Academic Press.