Reconstitution of clathrin-coated bud and vesicle formation with minimal components

During the process of clathrin-mediated endocytosis an essentially planar area of membrane has to undergo a gross deformation to form a spherical bud. Three ways have been recognized by which membranes can be induced to transform themselves locally from a planar state to one of high curvature: a change in lipid distribution between the leaflets, insertion of a protein into one leaflet and formation of a protein scaffold over the surface(1). Such a scaffold is spontaneously generated by clathrin(2-5). Conjectures that the attachment of clathrin was the cause of the change in curvature were challenged on theoretical grounds(6), and also by the discovery of a number of clathrin-associated proteins with the capacity to induce membrane curvature(7-9). We have now developed a cell-free system that has enabled us to demonstrate that clathrin polymerization alone is sufficient to generate spherical buds in a membrane. This process is reversible, as shown by the reassimilation of the buds into the planar membrane when the intra-clathrin contacts are dissociated by the chaperone Hsc70. We further show that the final step in the formation of coated vesicles ensues when clathrin-coated buds are released through the action of dynamin.