Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2.

Abstract

Clathrin-coated vesicles mediate membrane cargo transportation from the plasma membrane, the trans-Golgi network, the endosome, and the lysosome. Heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) are bridges that link cargo-loaded membranes to clathrin coats. Assembly of AP2 was previously considered to be spontaneous; however, a recent study found AP2 assembly is a highly orchestrated process controlled by alpha and gamma adaptin binding protein (AAGAB). Evidence shows that AAGAB controls AP1 assembly in a similar way. Insights into the orchestrated assembly process and three-dimensional structures of assembly intermediates are only emerging. Here, we describe a protocol for reconstitution and purification of the complexes containing AAGAB and AP1 or AP2 subunits, known as AP1 and AP2 hemicomplexes. Our purification routinely yields milligrams of pure complexes suitable for structural analysis by X-ray crystallography and electron microscopy.