INOSITOL HEXAKISPHOSPHATE BINDS TO CLATHRIN ASSEMBLY PROTEIN 3(AP-3/AP180) AND INHIBITS CLATHRIN CAGE ASSEMBLY IN-VITRO

We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP(6)). The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180. This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles. The isolated protein-bound InsP(6) with a dissociation constant of 1.2 mu M and a stoichiometry of 0.9 mel of InsP(6) bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP(6) with a similar affinity. InsP(6) inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages. We speculate that InsP(6) and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.