Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach M F, Ravazzola M, Amherdt M, Schekman R
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720.
Cell. 1994 Jun 17;77(6):895-907. doi: 10.1016/0092-8674(94)90138-4.
In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.
利用洗涤过的膜和三种可溶性蛋白质（Sar1p、Sec13p复合物和Sec23p复合物）已在体外重建了内质网衍生运输小泡的合成过程。小泡形成需要GTP，但可由诸如GMP-PNP等不可水解类似物驱动。然而，GMP-PNP小泡无法靶向高尔基体复合物并与之融合，而GTP小泡具有功能。小泡形成所需的所有胞质蛋白都保留在GMP-PNP小泡上，而Sar1p则从GTP小泡上解离。纯化制剂的超薄切片电子显微镜显示出一群均匀的60 - 65纳米小泡，其具有10纳米厚的电子致密衣被。这种新型衣被复合物的亚基在分子水平上与参与高尔基体内运输的非网格蛋白衣被蛋白复合物的成分不同。由于由这两种衣被驱动的出芽总体循环在机制上似乎相似，我们建议将衣被结构称为COPI和COPII。
