OVEREXPRESSION OF WILD-TYPE AND MUTANT ARF1 AND ARF6 - DISTINCT PERTURBATIONS OF NONOVERLAPPING MEMBRANE COMPARTMENTS

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 cytoso lic coat proteins coatomer (COPI) and gamma-adaptin with the Golgi com plex, the functions of the other ARF proteins have not been defined. H ere, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to aff ect predictably the assembly of COPI and gamma-adaptin with Golgi memb ranes in cells, ARF6 is localized to the endosomal/plasma membrane sys tem and has no effect on these Golgi-associated coat proteins. By immu ne-electron microscopy, the wild-type ARF6 protein is observed along t he plasma membrane and associated with endosomes, and overexpression o f ARF6 does not appear to alter the morphology of the peripheral membr ane system. In contrast, overexpression of ARF6 mutants predicted eith er to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-de fective mutant is localized to the plasma membrane and its overexpress ion results in a profound induction of extensive plasma membrane vagin ations and a depletion of endosomes. Conversely, the GTP binding-defec tive ARF6 mutant is present exclusively in endosomal structures, and i ts overexpression results in a massive accumulation of coated endocyti c structures.