Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and Goat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) synthesis. PtdIns(4,5)P-2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphalidylinositol 4-kinase and a soluble type I phosphatidylinositol I-phosphate B-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate B-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4,5)P-2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P-2 functions, at least in part, to activate the PtdIns(4,5)P-2-dependent phospholipase D1, These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.