A clathrin-binding site in the hinge of the β2 chain of mammalian AP-2 complexes

Abstract

The assembly of cytosolic clathrin into the cytoplasmic face of coated pits and coated vesicles appears to be driven by the clathrin-associated protein (AP) complexes. We have previously shown that one of the large chains of the AP complexes, the β chain, is sufficient to drive coat assembly in vitro. This chain consists of two domains, the amino-terminal trunk and the carboxyl-terminal ear, linked by a 'hinge.' We report here that presence of the hinge in recombinant β trunk or in recombinant β ear fragments is essential for driving in vitro assembly of clathrin into coats. We have also used a binding assay to map the clathrin-binding site by nested deletion of hinge sequences to a 50-residue region in the center of the hinge. This sequence is conserved in all known β sequences from multicellular organisms. The interaction of a single β hinge with a clathrin triskelion is weak, and we propose that recruitment of cytosolic clathrin to a forming coated pit involves simultaneous contacts between the legs of single clathrin trimers and the β hinges of two or three membrane-bound AP complexes. Uncoating is likely to require interruption of these contacts.