SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles
SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles
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DOI：
10.1074/jbc.m407020200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Bacia, K;Schuette, CG;Schwille, P
膜结构域作为蛋白质信号传递和运输的潜在平台受到了极大的关注。由于木筏被认为是由协作性脂质相互作用形成的，但在体内不能直接接触到，人工相分离脂质双层是有用的模型系统。巨大的单层囊泡(GUV)提供了大的独立双层，但合适的结合蛋白质的方法仍然很少。在这里，我们报告了两个不溶于水的SNARE蛋白在没有融合性添加剂的情况下重组成GUV的过程。重组后，通过共聚焦成像、荧光自相关光谱和互相关光谱分析蛋白质的功能。掺入含有相分离脂质的GUV表明，在没有其他细胞因素的情况下，这两种蛋白质都表现出对液体无序相的内在偏好。尽管对整个细胞的洗涤剂抗性分析的结果是模糊的，但通过这种新的方法将胞外机制的组件重组到GUV中，应该可以深入了解蛋白质复合体与假想的液体有序相微域的结合动力学，耐洗涤剂膜和液体有序相之间的对应关系，以及SNARE介导膜融合的机制。
Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.