Moore M S, Mahaffey D T, Brodsky F M, Anderson R G
Science. 1987 May 1;236(4801):558-63. doi: 10.1126/science.2883727.
During receptor-mediated endocytosis, coated pits invaginate to form coated vesicles, clathrin and associated proteins dissociate from the vesicle membrane, and these proteins form new coated pits at the cell surface. As a means of elucidating molecular mechanisms that govern the function of coated pits, the assembly phase of this cycle was reconstituted by incubating purified membranes that were treated to remove endogenous coated pits with cytoplasm extracted from cultured cells. The in vitro assembly of coated pits on these membranes satisfactorily mimics many features of coated pit formation in the intact cell. These studies indicate that: the membranes contain a limited number of coated pit assembly sites that bind clathrin with high affinity; the half-time for assembly is 5 minutes both at 4 degrees C and 37 degrees C; during assembly, proteins with molecular sizes of 180, 110, and 36 kilodaltons are recruited to the plasma membrane; and assembly is not dependent on adenosine triphosphate, but this nucleotide triggers a temperature-dependent loss of coated pits that are assembled in the absence of adenosine triphosphate.
在受体介导的内吞作用过程中，有被小窝内陷形成有被小泡，网格蛋白及相关蛋白从泡膜上解离，这些蛋白在细胞表面形成新的有被小窝。作为阐明调控有被小窝功能分子机制的一种手段，通过将去除内源性有被小窝的纯化膜与从培养细胞中提取的细胞质一起孵育，重建了该循环的组装阶段。这些膜上有被小窝的体外组装令人满意地模拟了完整细胞中有被小窝形成的许多特征。这些研究表明：这些膜含有数量有限的与网格蛋白高亲和力结合的有被小窝组装位点；在4℃和37℃时组装的半衰期均为5分钟；在组装过程中，分子大小为180、110和36千道尔顿的蛋白质被募集到质膜；组装不依赖于三磷酸腺苷，但这种核苷酸会引发在没有三磷酸腺苷的情况下组装的有被小窝的温度依赖性丢失。
