GTP HYDROLYSIS BY ADP-RIBOSYLATION FACTOR IS DEPENDENT ON BOTH AN ADP-RIBOSYLATION FACTOR GTPASE-ACTIVATING PROTEIN AND ACID PHOSPHOLIPIDS

ADP-ribosylation factor (ARF) is a 21-kDa GTP binding protein that reg ulates eukaryotic membrane traffic. Both the binding and hydrolysis of GTP by ARF have been shown to be necessary for this function. However , purified mammalian ARF lacks intrinsic GTPase activity (<0.0015 min( -1)). We document the presence, in bovine brain extracts, of a protein with the predicted properties for an ARF GTPase activating protein (A RF GAP). This activity was highly dependent on phospholipids. An acid phospholipid fraction from bovine brain (containing primarily phosphat idylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate , phosphatidylinositol, and phosphatidylserine) had no effect on intri nsic GTPase activity of purified ARF but increased the ARF GAP activit y of bovine brain homogenates about 8-fold. This dependence on acid ph ospholipids was retained after >100-fold purification of ARF GAP, maki ng it, likely, an inherent property of this reaction. PIP2 alone stimu lated ARF GAP activity up to 30-fold with a half-maxmal effect at 100- 300 mu M but had no effect on the GTPase rate of ARF alone. Phosphatid ylinositol 4-phosphate was also active but had only 50% of the maximal effect and twice the EC(50) of PIP2. Phosphatidylserine, phosphatidyl ethanolamine, phosphatidylcholine, phosphatidylinositol, and diacylgly cerol either alone or in the presence of ARF GAP do not stimulate ARF GTPase activity. ARF proteins have been identified recently as regulat ors of phospholipase D. The product of the phospholipase D reaction, p hosphatidic acid, stimulated ARF GAP approximately 5-fold and reduced the PIP2 concentration needed for GAP stimulation about 6-fold. The su bstrate of phospholipase D, phosphatidylcholine, inhibited ARF GAP act ivity, but this inhibition seen with phosphatidylcholine was partially reversed by phosphatidic acid. A feedback loop for the coordinate reg ulation of phospholipase D and ARF activities is proposed.