Evolving nature of the AP2 a-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoat-ing. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein inter-action hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to dis-tinct subdomains on a-appendages, called ‘top ’ and ‘side’ sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversi-ble interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered a(appendage)-hubs have al-tered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving direc-tionality to vesicle assembly