Drake M T, Zhu Y, Kornfeld S
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Biol Cell. 2000 Nov;11(11):3723-36. doi: 10.1091/mbc.11.11.3723.
The heterotetrameric adaptor protein complex AP-3 has been shown to function in the sorting of proteins to the endosomal/lysosomal system. However, the mechanism of AP-3 recruitment onto membranes is poorly understood, and it is still uncertain whether AP-3 nucleates clathrin-coated vesicles. Using purified components, we show that AP-3 and clathrin are recruited onto protein-free liposomes and Golgi-enriched membranes by a process that requires ADP-ribosylation factor (ARF) and GTP but no other proteins or nucleotides. The efficiency of recruitment onto the two sources of membranes is comparable and independent of the composition of the liposomes. Clathrin binding occurred in a cooperative manner as a function of the membrane concentration of AP-3. Thin-section electron microscopy of liposomes and Golgi-enriched membranes that had been incubated with AP-3, clathrin, and ARF.GTP showed the presence of clathrin-coated buds and vesicles. These results establish that AP-3-containing clathrin-coated vesicles form in vitro and are consistent with AP-3-dependent protein transport being mediated by clathrin-coated vesicles.
异源四聚体衔接蛋白复合物AP-3已被证明在蛋白质分选至内体/溶酶体系统中发挥作用。然而，AP-3募集到膜上的机制仍知之甚少，并且AP-3是否形成网格蛋白包被小泡也仍不确定。我们使用纯化的组分表明，AP-3和网格蛋白通过一种需要ADP-核糖基化因子（ARF）和GTP但不需要其他蛋白质或核苷酸的过程被募集到无蛋白脂质体和富含高尔基体的膜上。募集到这两种膜来源上的效率相当，且与脂质体的组成无关。网格蛋白的结合以协同方式发生，是AP-3膜浓度的函数。对已与AP-3、网格蛋白和ARF·GTP一起孵育的脂质体和富含高尔基体的膜进行超薄切片电子显微镜观察，显示存在网格蛋白包被的芽和小泡。这些结果表明含AP-3的网格蛋白包被小泡在体外形成，并且与网格蛋白包被小泡介导的AP-3依赖性蛋白质转运一致。
