Multivalency in endocytic accessory proteins orchestrates membrane shape transitions
Fast endophilin-mediated endocytosis (FEME) relies on the coordinated activities of membrane-remodeling proteins and multivalent protein interaction networks. Endophilin is a potent membrane curvature generator through its N-BAR domain and forms multivalent interactions with the proline-rich motifs (PRMs) of lamellipodin via its SH3 domain. Although these interactions are known to promote biomolecular condensation, their functional consequences for membrane remodeling have remained unclear. Here, we investigate how SH3/PRM mediated phase separation regulates endophilin-driven membrane curvature generation. Using purified recombinant proteins, giant unilamellar vesicles, quantitative fluorescence microscopy, and in vitro phase separation assays, we show that multivalent interactions between endophilin and lamellipodin drive the formation of membrane-associated protein-rich phases that strongly suppress endophilin mediated membrane tubulation. Competitive disruption of SH3/PRM interactions with the synaptojanin-derived peptide PP19 (containing only two PRM motifs) rapidly dissolves condensates and restores membrane curvature generation without affecting the intrinsic curvature-generating activity of endophilin. We further demonstrate that this regulatory mechanism is conserved across endophilin A1, A2, and A3 isoforms and depends critically on interaction valency: a multivalent PRM7 construct recapitulates condensate formation and curvature suppression, whereas mono or divalent PRM constructs does not. Together, these findings identify multivalent SH3/PRM interactions as a reversible molecular switch that balances biomolecular condensation and membrane remodeling. Multiple mechanisms, including post-translational modifications, likely regulate the multivalency of these interactions. Our results thus establish a broad functional link between protein phase separation and membrane mechanics, providing a framework for understanding how multivalent interaction networks regulate membrane shaping in multiple endocytic pathways.