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 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.
Peters
P J
PJ
Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
Hsu
V W
VW
Ooi
C E
CE
Finazzi
D
D
Teal
S B
SB
Oorschot
V
V
Donaldson
J G
JG
Klausner
R D
RD
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment
D017931:DNA Primers; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors