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. Such a scaffold is spontaneously generated by clathrin. Conjectures that the attachment of clathrin was the cause of the change in curvature were challenged on theoretical grounds, and also by the discovery of a number of clathrin-associated proteins with the capacity to induce membrane curvature. 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.