CCDC32 collaborates with the membrane to assemble the AP-2 clathrin adaptor complex

Assembly of adaptor protein complex 2 (AP-2), the primary cargo adaptor in clathrin-mediated endocytosis, is regulated by the chaperones alpha- and gamma-adaptin binding protein (AAGAB) and coiled-coil domain-containing protein 32 (CCDC32), whose deletion causes loss of all AP-2 subunits in vivo. Here, we describe the molecular mechanism of CCDC32-mediated AP-2 assembly. CCDC32 interacts with the appendage domain of the AP-2 α subunit with high affinity, using the same binding site as canonical endocytic regulators in addition to a previously unidentified, yet highly conserved pocket on α. CCDC32 contains cargo sorting motifs normally found in transmembrane cargo and binds to AP-2 heterodimers using canonical cargo-binding sites. In addition, two amphipathic helices in CCDC32 bind to the α/σ2 heterodimer. Unexpectedly, in solution, CCDC32 prevents complex assembly and actively disassembles AP-2 tetramers. Inhibition requires the amphipathic helices of CCDC32, which also mediate binding to phosphatidylinositol 4,5-bisphosphate (PIP₂)-containing membranes. The presence of PIP₂-containing membrane stabilizes the final stages of assembly. We propose that the membrane acts as a molecular switch to release inhibitory interactions, allowing for full complex assembly to proceed.