Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

Ruibai Luo,Paul A Randazzo

doi:10.1074/jbc.m802268200

Ruibai Luo, Paul A Randazzo

Open Access

https://doi.org/10.1074/jbc.m802268200

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Abstract

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.

Highlights

Arf family GTP-binding proteins are critical regulators of membrane traffic and the actin cytoskeleton (1, 2)
The first hypothesis is that Arf GAP1 is regulated by curvature of the lipid bilayer
The second is that coatomer provides the catalytic residue to the Arf11⁄7GTP1⁄7Arf GAP1 complex to initiate GTP hydrolysis

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Plasmids for the bacterial expression of [⌬17]Arf, Arf, and N-myristoyltransferase have been described previously (27–29). Fractions containing Arf GAP1 were applied to a 1-ml of HisTrap HP column (GE Healthcare) and eluted using an imidazole gradient from 20 to 600 mM in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 10% glycerol. To determine the C50 values, Arf GAPs were titrated into a reaction mixture containing 0.5 ␮M myrArf11⁄7[␣-32P]GTP and LUVs prepared as indicated. Time courses of GTP hydrolysis under conditions in which the GAP was limiting were performed by removing samples from a reaction after variable times, stopping the reaction as for the C50 determination, and measuring relative levels of GDP and GTP bound to Arf. 0.5 ␮M of [1–257]Arf GAP1 or [1– 415]Arf GAP1-His were incubated with 0.2 ␮M of coatomer and sucrose-loaded LUVs in 25 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM MgCl2 and 1 mM EDTA for 5 min at 30 °C. All data presented are either the summary of the indicated number of experiments or representative data of at least two experiments with similar results

RESULTS

DISCUSSION

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