Seminar

Unravelling the clathrin cage - structural and dynamic measurements of

CORINNE SMITH, PhD

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, Hsc70 and the J-domain-containing protein, auxilin, in an ATP-dependent process. Clathrin forms complex polyhedral cage structures in vitro and the manner in which auxilin and Hsc70, which are relatively small in comparison to the cages, can bring about the disassembly of these structures is the focus of this work. 3D cryo-electron microscopy techniques have allowed the structure of clathrin cages bound to auxilin and Hsc70 to be elucidated but questions remain as to precisely how Hsc70 and auxilin achieve release of clathrin triskelions from the cage. Using systematic global fitting of perpendicular light scattering data, we have carried out a kinetic analysis of the mechanism by which Hsc70 and auxilin act to disassemble clathrin cages. We find that the simplest model which fits all the data requires Hsc70 to bind to clathrin-auxilin and hydrolyse ATP in a three-stage sequential mechanism.