Heidelberg University Biochemistry Center, Heidelberg University, 69120 Heidelberg, Germany.
J Cell Biol. 2011 Sep 5;194(5):765-77. doi: 10.1083/jcb.201011027.
Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle. Using a reconstituted system for COPI vesicle formation from purified components, we find that a dimerization-deficient Arf1 mutant, which does not display the ability to modulate membrane curvature in vitro or to drive formation of coated vesicles, is able to recruit coatomer to allow formation of COPI-coated buds but does not support scission. Chemical cross-linking of this Arf1 mutant restores vesicle release. These experiments show that initial curvature of the bud is defined primarily by coatomer, whereas the membrane curvature modulating activity of dimeric Arf1 is required for membrane scission.
形成有被膜的小泡需要对脂质双层进行两次显著的操作。首先，诱导膜曲率以驱动芽的形成。其次，在芽颈处发生的断裂反应释放出小泡。利用从纯化组分中形成 COPI 小泡的重组系统，我们发现一个二聚缺陷的 Arf1 突变体，它在体外不显示调节膜曲率的能力，也不能驱动有被小泡的形成，但是能够募集衣被蛋白复合物来允许 COPI 被膜小泡芽的形成，但不支持断裂。该 Arf1 突变体的化学交联恢复了小泡的释放。这些实验表明，芽的初始曲率主要由衣被蛋白复合物决定，而二聚的 Arf1 的膜曲率调节活性则是膜断裂所必需的。
