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 (InsP6). 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 InsP6 with a dissociation constant of 1.2 microM and a stoichiometry of 0.9 mol of InsP6 bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP6 with a similar affinity. InsP6 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 InsP6 and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.
Norris
F A
FA
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Ungewickell
E
E
Majerus
P W
PW
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; D010833:Phytic Acid; D014357:Trypsin