Assays and properties of the Arf GAPs AGAP1, ASAP1, and Arf GAP1.
ADP-ribosylation factors (Arfs) are Ras-like GTP-binding proteins that regulate membrane traffic and actin remodeling. Arf function requires GTP hydrolysis but Arf lacks GTPase activity; consequently, Arf function is dependent on Arf GTPase-activating proteins (GAPs). The Arf GAPs are a structurally diverse group of at least 16 proteins. Several Arf GAPs use a single Arf isoform. However, due to structural differences, the conditions supporting productive interactions between Arf and different Arf GAPs vary. Here, we describe preparation and basic properties of three Arf GAPs. We use these proteins to illustrate assays for Arf GAP activity. Conditions that optimize activity for each GAP are discussed. These methods can be used for the further characterization of Arf-Arf GAP interaction that is necessary for understanding the function of Arf in cellular physiology.
Che
Magnus Mutah
MM
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Nie
Zhongzhen
Z
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D020727:ADP-Ribosylation Factors / Q000032:analysis*; D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis*; D000818:Animals; D005453:Fluorescence; D020549:GTP Phosphohydrolase Activators / Q000032:analysis*; D020690:GTPase-Activating Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D010761:Phosphorus Radioisotopes; D014364:Tryptophan / Q000737:chemistry
C470574:AGAP1 protein, human; C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020549:GTP Phosphohydrolase Activators; D020690:GTPase-Activating Proteins; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D014364:Tryptophan; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors