Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

CIMR University of Cambridge Biomedical Campus Hills Road Cambridge CB2 0XY UK

Czech Academy of Sciences Institute of Information Theory and Automation Pod Vodarenskou vezi 4 182 08 Prague 8 Czech Republic

Department of Cell Biology University of Pittsburgh School of Medicine 3500 Terrace Street Pittsburgh PA USA

Institute for Biochemistry 1 Medical Faculty University of Cologne Joseph Stelzmann Straße 52 50931 Cologne Germany

Intracellular Trafficking Laboratory Transdisciplinary Biology Research Program Rajiv Gandhi Centre for Biotechnology Thiruvananthapuram Kerala India

Kennedy Institute of Rheumatology University of Oxford Roosevelt Drive Oxford OX3 7FY UK

Max Planck Institute of Biochemistry 82152 Martinsried Germany

MRC LMB Cambridge Biomedical Campus Cambridge CB2 0QH UK

Rosalind Franklin Institute Harwell Campus Didcot UK

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Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes