Kinetic analysis of Arf GAP1 indicates a regulatory role for coatomer.
Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.
Luo
Ruibai
R
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Randazzo
Paul A
PA
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D000595:Amino Acid Sequence; D020756:Coatomer Protein / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D013997:Time Factors; D053835:Unilamellar Liposomes / Q000737:chemistry
C503237:ARFGAP1 protein, human; C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; D053835:Unilamellar Liposomes; D006160:Guanosine Triphosphate