26 - Expression and Properties of ADP-Ribosylation Factor (ARF6) in Endocytic Pathways

This chapter describes approaches to studying adenosine diphosphate (ADP)-ribosylation factor 6 (ARF6) roles in membrane trafficking, which is integral to the additional effects of ARF6 observed on the actin cytoskeleton. A convenient method, for studying ARF6 and novel guanosine-5'-triphosphate (GTP)ases in general, is to use transient expression in cells of wild-type and mutant GTPases. In this way, the consequences of expression of GTP-binding defective (i.e., inactive, guanidine diphosphate (GDP)-bound form) and GTPase-defective (i.e., constitutively active, GTP-bound form) mutants can be observed in cells and compared with that observed by expression of the wild-type ARF6. It is suggested that HeLa cells can serve as a model for studying alternative endocytic pathways. Studies in HeLa cells have been facilitated by the identification of reversible, pharmacologic reagents that mimic the effects of the GTPbinding defective ARF6-T27N and GTPase-defective ARF6-Q67L mutants. This has allowed studying the trafficking from the PM through the ARF6 endosome in cells expressing the wild-type protein.