Analysis of Arf interaction with GGAs in vitro and in vivo

Small GTPases of the ADP-ribosylation factor (Arf) family regulate membrane traffic and dynamics in eukaryotic cells. GGAs (Golgi-localizing, gamma-adaptin ear homology domain, Arf-binding proteins) are a family of monomeric clathrin adaptor proteins that were originally identified as proteins interacting with Arfs and found to associate mainly with membranes of the trans-Golgi network (TGN). Like other adaptor and coat proteins, membrane association of GGAs is regulated by Arfs in a GTP-dependent manner. Together with or independent of the adaptor protein complex AP-1, GGAs mediate sorting of transmembrane proteins, including mannose 6-phosphate receptors, between the TGN and endosomes by clathrin-coated vesicles. This chapter describes methods to examine the interaction between Arfs and GGAs and to analyze the cellular function of GGAs regulated by Arfs.