SummaryClathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

Background: The clathrin adaptor epsin is indispensable for clathrin-mediated endocytosis, but the mechanism by which it regulates clathrin assembly remains unclear.Results: Epsin shows a preference to localize to regions of high membrane curvature.Conclusion: Epsin's membrane curvature sensing directs clathrin assembly to highly curved membranes.Significance: Membrane curvature could determine the hierarchy of molecular interactions during clathrin-mediated membrane budding.

Clathrin-mediated endocytosis sorts the bulk of membrane proteins and is a process that starts with adaptor-induced clathrin assembly. Real-time fluorescence analysis shows that adaptor sorting is determined not by the extent of clathrin recruited or the degree of clathrin clustered but instead by the rate of clathrin assembly.

Clathrin-mediated endocytosis manages the vesicular transport of the bulk of membrane proteins from the plasma membrane and the trans-Golgi network. During this process, discrete sets of adaptor proteins recognize specific classes of membrane proteins, which recruit and assemble clathrin lattices on the membrane. An important determinant to the success of this vesicular transport reaction is the intrinsic ability of adaptors to polymerize clathrin on a membrane surface. Adaptor-induced clathrin assembly has traditionally been analyzed using static electron microscopy-based approaches. Here, we describe a methodology to follow adaptor-induced clathrin assembly in real-time using fluorescence microscopy on a facile model membrane assay system of supported membrane tubes (SMrT). Results from such assays can be conveniently run through routine image analysis procedures to extract kinetic parameters of the clathrin assembly reaction.