Abstract
Clathrin-mediated endocytosis internalizes proteins and lipids from the cell surface, supporting nutrient uptake, signaling, and membrane trafficking. Recent work has demonstrated that a flexible, liquid-like network of initiator proteins is responsible for catalyzing assembly of clathrin-coated vesicles in diverse organisms including yeast, mammals, and plants. How do cells regulate the assembly of this dynamic network to produce cargo-loaded vesicles? Here we reveal the ability of an endocytic adaptor protein, Epsin1, to conditionally stabilize the initiator protein network, creating a cargo-dependent checkpoint during clathrin-mediated endocytosis. Epsin1 is known to recruit ubiquitylated transmembrane proteins to endocytic sites. Using in vitro assays, we demonstrate that Epsin1 uses competitive binding and steric repulsion to destabilize condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, creating attractive interactions that stabilize condensation. Similarly, in mammalian cells, endocytic dynamics and ligand uptake are disrupted by removal of either ubiquitin or Epsin1. Surprisingly, when Epsin1 and ubiquitin are removed simultaneously, endocytic defects are rescued to near wildtype levels, although endocytic sites lose the ability to distinguish between ubiquitylated and non-ubiquitylated cargos. Taken together, these results suggest that Epsin1 tunes protein condensation to ensure the presence of ubiquitylated cargo during assembly of clathrin-coated vesicles. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions controls the stability of liquid-like protein networks, providing dynamic control over key cellular events.