Peter F, Plutner H, Zhu H, Kreis T E, Balch W E
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
J Cell Biol. 1993 Sep;122(6):1155-67. doi: 10.1083/jcb.122.6.1155.
Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin-coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.
利用一种新型体外测定法，该方法使我们能够区分囊泡出芽与随后的靶向和融合步骤，我们提供了首个生物学证据，即β-COP（一种被认为介导高尔基体内运输的非网格蛋白包被囊泡的组分）对于蛋白质从内质网运输到顺式高尔基区室至关重要。在β-COP特异性抗体和F(ab)片段存在下孵育可阻止水泡性口炎病毒糖蛋白（VSV-G）从内质网中排出。这些结果表明β-COP是介导囊泡出芽的包被复合物组装所必需的。大鼠肝细胞溶胶分级分离显示，β-COP的一种主要生物活性形式存在于一个与外被体不同的高分子量池中（>1000 kD），且该形式能促进内质网高效囊泡出芽。令人惊讶的是，rab1B可与该含β-COP的复合物定量共沉淀，且对其功能也必不可少。我们认为β-COP在囊泡形成的早期步骤中发挥作用，并且rab1B可能作为预包被复合物的一个组分被招募，该预包被复合物通过囊泡载体参与蛋白质从内质网的输出。
