KINETICS STUDY OF CLATHRIN ASSEMBLY IN IN VITRO SYSTEM

Abstract

Clathrin-mediated endocytosis (CME) requires the formation of clathrin-coated pits (CCPs) that can recruit cargo and mediate its uptake into the cell. However, how cargo selection is coupled with CCP assembly is largely unknown. Precise mechanistic understanding would require the functional reconstitution of both CCP assembly and cargo incorporation. To achieve this, I used a cell-free CCP assembly system. Using this system, I confirmed that the de novo assembled clathrin structures do sort cargos. I observed a correlation between the energetic requirement of CCP assembly and cargo capturing efficiency. In absence of the uncoating ATPase hsc70 clathrin assembled into pits but failed to enrich cargo. Consistent with the role of hsc70 in uncoating, single molecule imaging revealed single clathrin dissociation events. Taken together, these results suggest a dynamic coat assembly model where an energy-dependent, rate-limiting uncoating reaction during assembly is critical for cargo selection.