Cells have evolved a variety of assembly chaperones to aid in the difficult process of forming macromolecular complexes in a crowded cytoplasm. Assembly of adaptor protein complex 2 (AP-2), the primary cargo adaptor in clathrin-mediated endocytosis, is regulated by the chaperones AAGAB and CCDC32, whose deletion causes loss of all AP-2 subunits in vivo. AAGAB and CCDC32 are thought to act sequentially to assemble the AP-2 tetramer from its constituent heterodimers. However, the molecular requirements and structural consequences of CCDC32 interaction with AP-2 are not yet understood. Here, using in vitro reconstitution and integrative structural analysis, we describe the molecular mechanism of CCDC32-mediated AP-2 assembly. First, CCDC32 interacts with the appendage domain of the AP-2 α subunit, using the same binding site as canonical endocytic regulators in addition to a novel, yet highly conserved pocket on α. CCDC32 contains cargo sorting motifs normally found in trans-membrane cargo and binds to AP-2 heterodimers using canonical cargo-binding sites. Additionally, two amphipathic helices in CCDC32 bind to the α/σ2 heterodimer. Surprisingly, in solution, we find that CCDC32 prevents complex assembly and actively disassembles AP-2 tetramers. Inhibition requires the amphipathic helices of CCDC32, which also mediate binding to 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. Using cryo-EM, we visualize an assembly intermediate that mimics the conformation of AP-2 found in vesicles, with CCDC32 bound at both cargo binding sites and both membrane-binding sites, suggesting that assembly leads to deposition of active complexes on the plasma membrane.
Sloan et al. (2026) studied this question.