The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting
The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting
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DOI：
10.1006/meth.2000.0955
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发表时间：
2000-04-01
期刊：
影响因子：
--
通讯作者：
Schekman, R
中科院分区：
文献类型：
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作者：
Matsuoka, K;Schekman, R
我们已经建立了系统，重建与脂质体制成的纯脂质和纯化的外壳蛋白包被的运输囊泡的生物发生。脂质体中的脂质组合物的优化允许外壳蛋白I和外壳蛋白II（COPII）外壳亚基两者的有效结合。通过离心分析和电子显微镜检测并表征了从生物膜产生的大约大小的包被囊泡。这种出芽反应的一种变体使我们能够测量v-SNARE蛋白到合成COPII囊泡中的分选。我们开发了一种新的系统，拴谷胱甘肽S-转移酶（GST）的杂交蛋白的脂质体表面与谷胱甘肽衍生磷脂。该系统使我们能够检测包装成COPII囊泡的两个v-SNARE蛋白质的胞质结构域的积极作用。因此，包被囊泡的产生和蛋白质分选到囊泡中可以用脂质体和纯化的蛋白质再现。(C)北京大学出版社.
We have established systems that reconstitute the biogenesis of coated transport vesicles with liposomes made of pure lipids and purified coat proteins. Optimization of the lipid composition in the liposomes allowed the efficient binding of both coat protein I and coat protein II (COPII) coat subunits. Coated vesicles of approximately the size generated from biomembranes were detected and characterized by centrifugation analysis and electron microscopy. A variation of this budding reaction allowed us to measure the sorting of v-SNARE proteins into synthetic COPII vesicles. We developed a novel system to tether glutathione S-transferase (GST)-hybrid proteins to the surface of liposomes formulated with a glutathione-derivatized phospholipid. This system allowed us to detect the positive role of cytoplasmic domains of two v-SNARE proteins that are packaged into COPII vesicles. Therefore, both generation of coated vesicles and protein sorting into the vesicles can be reproduced with liposomes and purified proteins. (C) 2000 Academic Press.