[30] Dissociation and reassociation of clathrin

Publisher Summary This chapter describes the dissociation and reassociation of clathrin. The clathrin trimer is capable of assembling into the geodesic dome structure observed on coated vesicles by simply decreasing the pH of clathrin solutions from 7.5 to some value between 6 and 6.8, until chaotropic anions are absent and the ionic strength is not too high. The available procedures for clathrin purification depend on the relative enrichment of this protein by virtue of its tendency to form or remain assembled as macromolecular aggregates under conditions of slightly acidic pH. Cosedimentation of both coated vesicles and clathrin baskets in sucrose gradients allows a further enrichment from the starting homogenate. A second property utilized is clathrin's sensitivity to conditions of basic pH. When assemblies of clathrin are incubated in buffers of low ionic strengths and basic pH, they disassemble into trimers, which can be separated from the bulk of the vesicular membrane and other proteins by sedimentation and column chromatography. There are two principal approaches to clathrin purification described in the literature: one utilizes 2 M urea following low-ionic strength extraction of a crude coated vesicle fraction; the other utilizes a high concentration of Tris buffer and a more highly purified coated vesicle preparation.