(56d) Structured and Intrinsically Disordered Domains within Amphiphysin1 Work Together to Sense and Drive Membrane Curvature

Many cellular processes, including clathrin-mediated endocytosis (CME), require remodeling of the plasma membrane. During CME, specialized proteins sense and amplify fluctuations in membrane curvature and recruit clathrin to eventually form clathrin-coated vesicles. Amphiphysin1, a multi-domain endocytic protein, contains an N-terminal BAR (Bin/amphiphysin/Rvs) domain, which facilitates membrane binding, and a largely disordered C-terminal domain, which contains binding motifs for clathrin and other endocytic proteins. Many studies attribute membrane curvature sensing and tubule generation to the BAR domain, but neglect contributions from the disordered domain. Interestingly, we have recently demonstrated that the disordered domains amplify membrane curvature sensing and generation. However, the mechanisms responsible for this phenomenon remain unclear. Here we elucidated the biophysical role of the disordered domain by successively truncating it. Using quantitative fluorescence and electron microscopy, our results demonstrate that the disordered domains begin to significantly impact membrane remodeling once their projected areas exceed that of the BAR domain. Steric interactions with the membrane and neighboring proteins give rise to increased curvature sensing and membrane vesiculation, respectively. Interestingly, full-length Amphiphysin1 is also able to impart substantial curvature sensitivity upon clathrin. Specifically, in the presence of Amphiphysin1, clathrin becomes a potent sensor of curvature, with even greater curvature sensitivity than that of Amphiphysin1 itself, suggesting a synergistic interaction between the two proteins. Similarly, when an artificial membrane-binding tag was used to assemble clathrin directly on membrane surfaces, clathrin’s curvature sensitivity decreased substantially, suggesting that the interaction with Amphiphysin1 strongly promotes curvature sensing by clathrin. These results indicate a cooperative binding effect, where the avidity between Amphiphysin1 and clathrin increases as the density of Amphiphysin1 on the membrane surface increases. These findings elucidate the synergy between structured and disordered protein domains in membrane remodeling and curvature sensing during CME.