Separation of Membrane Trafficking and Actin Remodeling Functions of ARF6 with an Effector Domain Mutant

Abstract

The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions. To identify sequences in ARF6 that are necessary for these distinct functions, we examined the behavior of a chimeric protein of ARF1 and ARF6. The 1-6 chimera (with the amino half of ARF1 and the carboxyl half of ARF6) localized like ARF6 in HeLa cells and moved between the endosome and PM, but it did not form protrusions, an ARF6 effector function. Two residues in the amino-terminal half of ARF6, Q37 and S38, when substituted into the 1-6 chimera allowed protrusion formation, whereas removal of these residues from ARF6 resulted in an inability to form protrusions. Interestingly, expression of 1-6 in cells selectively inhibited protrusions induced by wild-type ARF6 but had no effect on ARF6-regulated membrane movement or Rac-induced ruffling. Thus, we have uncoupled two functions of ARF6, one involved in membrane trafficking, which is necessary for Rac ruffling, and another involved in protrusion formation.

The small GTPase ADP‐ribosylation factor 6 negatively regulates dendritic spine formation

“…Subcellular localization and physiological function of class II of ARFs are largely unknown. Of three classes of ARF, the class III of ARF, ARF6, localizes at the plasma membrane and is implicated in the regulation of cortical actin dynamics and plasma membrane recycling [19]. In the hippocampus, ARF6 mRNA is expressed in CA1-3 pyramidal cell layers and the dentate gyrus [20].…”

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons

“…6C,D). Arfs 34 constitute a family of Ras-related, low molecular mass ( 20 kDa) GTP-binding proteins that act as molecular switches for membrane and vesicle traffic as well as actin dynamics in cells 35 . In a first step, the interaction of C9ORF72 with these small GTPases was confirmed by co-precipitation analyses, using antibodies against Arf6 (Fig.…”

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

“…In both arf1-16 and arf1-17 alleles, glutamic acid at position 41 is mutated to valine. Recently, Al-Awar et al (2000) showed that Q37 of mammalian ARF6, corresponding to E41 of yeast Arf1p, is critical for its effector function, formation of protrusion, suggesting that this position represents a site of interaction with effector molecules. arf1-16 has one additional mutation, D129E, which is in the consensus GTP-binding domain, NKXD.…”