Separation of Timescales for Endophilin Dimerization and Membrane Binding

Abstract

The membrane association of endophilin is a pivotal step in clathrin-mediated endocytosis. In this process, the basis for the functional role of endophilin is believed to involve the promotion of membrane curvature, which in turn depends on membrane shaping by the dimeric structure of endophilin. Thus, the dynamic nature of endophilin-membrane association and dimerization are functionally important. However, little is known about the timescales and factors determining the kinetics of the interactions involved. To illuminate these aspects, we study the kinetics and equilibria of endophilin N-BAR dimerization and membrane binding. We determine the dimerization equilibrium constant by using subunit exchange FRET. We characterize N-BAR membrane association, under conditions where the dimeric species predominates, by stopped-flow, observing prominent electrostatic sensitivity. Our results suggest that membrane insertion of amphipathic helices rapidly follows association, in a non-rate-limiting manner. Relative to membrane binding, we find that dimerization is governed by far slower kinetics. Thus, monomer-dimer exchange does not contribute to the kinetic mechanism of membrane binding. These results underscore a separation of timescales for endophilin dimerization and membrane binding, which may facilitate temporal regulation of functional membrane processes.