REGULATION OF AP-3 FUNCTION BY INOSITIDES

As part of the growing effort to understand the role inositol phosphat es and inositol lipids play in the regulation of vesicle traffic withi n nerve terminals, we determined whether or not the synapse-specific c lathrin assembly protein AP-3 can interact with inositol lipids. We fo und that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate ( DiC(8)-PtdIns(3,4,5)P-3) was only 7.5-fold weaker a ligand than D-myo- inositol hexakisphosphate in assays that measured the displacement of D-myo-[H-3]inositol hexakisphosphate. In functional as says we found t hat both of these ligands inhibited clathrin assembly, but DiC(8)-PtdI ns(3,4,5)P-3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC(8)-PtdIns(3,4,5)P-3 that establish specificity. Dioctanoyl-phosphatidylinositol 3,4-bisphospha te, which does not have a 5-phosphate, and 4,5-O-bisphosphoryl D-myo-i nosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate, which does not h ave a 3-phosphate, were, respectively, a-fold and 4 fold less potent t han DiC(8)-PtdIns(3,4,5)P-3 as inhibitors of clathrin assembly. Deacyl ation of DiC(8)-PtdIns(3,4,5)P-3 reduced its affinity for AP 3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosp hatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosp hate were both not significant inhibitors of clathrin assembly. It the refore appears that the interactions of inositides with AP-3 should no t be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be media ted by hydrophobic interactions with the fatty-acyl chains of the inos itol lipids.