Peters P J, Hsu V W, Ooi C E, Finazzi D, Teal S B, Oorschot V, Donaldson J G, Klausner R D
Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1995 Mar;128(6):1003-17. doi: 10.1083/jcb.128.6.1003.
The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.
ARF GTP结合蛋白被认为在分泌途径中作为膜运输的调节因子发挥作用。虽然ARF1蛋白在体外已被证明可介导胞质衣被蛋白（衣被蛋白复合体I，COP1）和γ-衔接蛋白与高尔基体复合体的膜相互作用，但其他ARF蛋白的功能尚未明确。在这里，我们通过用表位标记的ARF进行瞬时转染表明，ARF1定位于高尔基体复合体，并且可以证明其可预测地影响细胞中COP1和γ-衔接蛋白与高尔基体膜的组装，而ARF6定位于内体/质膜系统，对这些与高尔基体相关的衣被蛋白没有影响。通过免疫电子显微镜观察，野生型ARF6蛋白沿质膜分布并与内体相关，ARF6的过表达似乎不会改变外周膜系统的形态。相比之下，预测水解或结合GTP能力较差的ARF6突变体的过表达会改变ARF6的分布并影响内吞途径的结构。GTP水解缺陷型突变体定位于质膜，其过表达会导致广泛的质膜内陷的显著诱导和内体的耗竭。相反，GTP结合缺陷型ARF6突变体仅存在于内体结构中，其过表达会导致被膜内吞结构的大量积累。
