Molecular Mechanism of Clathrin Uncoating

Abstract

Clathrin-coated vesicles mediate vesicular traffic in cells through repeated cycles of coat assembly and disassembly. Following vesicle scission, auxilin recruits the general chaperone Hsc70 to the assembled clathrin lattice, triggering rapid coat disassembly and recycling of coat components. In this dissertation, I combine cryo-electron microscopy with single-molecule fluorescence imaging to define the molecular mechanism of clathrin uncoating. CryoEM structures of the assembled clathrin coat, the coat bound by auxilin, and the coat bound by Hsc70 show how these factors engage the lattice during uncoating. These structures show that the Hsc70 substrate on the clathrin C-terminus extends into a small cavity beneath each coat vertex. Auxilin binds at the vertex and positions its J-domain adjacent to the cavity, where it can recruit Hsc70. Hsc70 then binds the substrate peptide within the cavity, positioned beside lattice contacts formed by multiple triskelions that converge at the vertex. Single-molecule measurements show that full-length auxilin binds stably to the assembled clathrin lattice, even without a vesicle membrane, and that only ~8-10 molecules can bind per coat. Disassembly begins after only ~5 Hsc70 molecules bind the coat and usually initiates by loss of triskelions not bound by Hsc70. We propose that Hsc70 acts locally at a few auxilin- selected vertices, weakening nearby inter-triskelion contacts thereby allowing a small number of molecules to destabilize the entire coat.