Protein-Protein Crowding as a Driving Force for Membrane Bending during Endocytosis

Abstract

Highly curved membranes are essential to many cellular functions, motivating an intense effort to discover the mechanisms that shape them. A conserved feature among many proteins that participate in membrane bending is an amphipathic alpha helix that inserts into one membrane leaflet, attaching the protein to the membrane. These insertions are thought to bend membranes by pushing lipid heads apart like a wedge. First reported for the Epsin 1 N-terminal Homology (ENTH) domain, a protein believed to drive curvature during clathrin-mediated endocytosis, this mechanism is thought to shape a wide range of membrane structures, from trafficking vesicles to viral envelopes. However, recent computational studies have questioned the efficiency of the insertion mechanism, predicting that proteins with amphipathic helices must cover nearly 100% of the membrane surface to generate high curvature, an improbable situation given that cellular membranes are densely populated with a diversity of proteins. How then do proteins with amphipathic helices drive efficient bending of cellular membranes? We show that Epsin1 bends membranes via protein-protein crowding rather than via helix insertion. By correlating membrane tubule formation with FRET (Forster Resonance Energy Transfer) lifetime-based measurements of ENTH density on membrane surfaces, we demonstrate that protein coverage above ∼20% is sufficient to bend membranes. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, lateral steric pressure generated by bound proteins drives bending. Our results suggest that Epsin1's helix insertion functions primarily to achieve high protein-membrane affinity, enabling binding in a crowded environment. These findings call for a reexamination of the insertion hypothesis and demonstrate a new and highly efficient alternative mechanism by which the crowded protein environment on the surface of cellular membranes can directly contribute to membrane shape change.