In vitro assays of Arf1 interaction with GGA proteins.
ADP-ribosylation factor 1 (Arf1) is a GTP-binding protein that regulates membrane traffic. This function of Arf1 is, at least in part, mediated by Arf1 x GTP binding to coat proteins such as coatomer, clathrin adaptor protein (AP) complexes 1 and 3, and gamma-adaptin homology-Golgi associated Arf-binding (GGA) proteins. Binding to Arf1 x GTP recruits these coat proteins to membranes, leading to the formation of transport vesicles. Whereas coatomer and the AP complexes are hetero-oligomers, GGAs are single polypeptide chains. Therefore, working with recombinant GGAs is straightforward compared to the other Arf1 effectors. Consequently, the GGAs have been used as a model for studying Arf1 interactions with effectors and as reagents to determine Arf1 x GTP levels in cells. In this chapter, we describe in vitro assays for analysis of GGA interaction with Arf1 x GTP and for determining intracellular Arf1 x GTP levels.
Yoon
Hye-Young
HY
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Bonifacino
Juan S
JS
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000032:analysis / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D010761:Phosphorus Radioisotopes; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013462:Sulfur Radioisotopes
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors