Arf GAP2 is positively regulated by coatomer and cargo.
Arf GAP2 is one of four Arf GAPs that function in the Golgi apparatus. We characterized the kinetics of Arf GAP2 and its regulation. Purified Arf GAP2 had little activity compared to purified Arf GAP1. Of the potential regulators we examined, coatomer had the greatest effect, stimulating activity one to two orders of magnitude. The effect was biphasic, with half-maximal activation observed at 50 nM coatomer and activation peaking at approximately 150 nM coatomer. Activation by coatomer was greater for Arf GAP2 than has been reported for Arf GAP1. The effects of phosphoinositides and changes in vesicle curvature on GAP activity were small compared to coatomer; however, both increased coatomer-dependent activity. Peptides from p24 cargo proteins increased Arf GAP2 activity by an additional 2- to 4-fold. The effect of cargo peptide was dependent on coatomer. Overexpressing the cargo protein p25 decreased cellular Arf1*GTP levels. The differential sensitivity of Arf GAP1 and Arf GAP2 to coatomer could coordinate their activities. Based on the common regulatory features of Arf GAP1 and 2, we propose a mechanism for cargo selection in which GTP hydrolysis triggered by cargo binding to the coat protein is coupled to coat polymerization.
Luo
Ruibai
R
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA.
Ha
Vi Luan
VL
Hayashi
Ryo
R
Randazzo
Paul A
PA
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D020756:Coatomer Protein / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology; D051381:Rats; D013997:Time Factors
C503237:ARFGAP1 protein, human; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D006160:Guanosine Triphosphate; D020727:ADP-Ribosylation Factors