Myristoylation is required for the intracellular localization and endocytic function of ARF6

ADP-ribosylation factors (ARFs) are modified by a myristate group that is covalently linked to the second glycine residue at the amino terminus. With the recent evidence that ARF6 localizes to the cell periphery and plays a regulatory role in endocytic traffic, we have investigated the role of myristoylation on the membrane association, biological activity, and subcellular distribution of ARF6 in intact cells. A Gly2Ala mutation produced a nonmyristoylated protein that failed to associate with membranes, was cytosolic, and had no effect on endocytic transport. To determine if a different form of lipid modification could restore membrane association and biological activity, a nonmyristoylated ARF6 derivative was constructed that contained a prenyl group at the carboxy terminus. Prenylated ARF6 bound efficiently to membranes, but had no effect on receptor-mediated endocytosis and was mislocalized to distinct intracellular structures. Thus, although prenylation can replace myristoylation for membrane association, the latter appears to be critical for the proper targeting and biological activity of ARF6.