Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast

Clathrin, forming the triskelion network, orchestrates highly regulated cellular processes facilitating cargo internalization and trafficking in eukaryotes, with its N-terminal domain (NTD) pivotal for adaptor protein (AP) interactions. The NTD contains up to four AP-binding sites, and their roles in preferential occupancy by APs have not been addressed. Here, employing a combination of integrative biophysical and structural approaches together with in vivo functional experiments, we investigated the binding hierarchy and selectivity of adaptors for clathrin, aiming to understand the evolutionary conservation of redundant APs and their specialized roles in endocytosis and cellular trafficking mechanisms. We found that yeast epsin Ent5 displayed the highest affinity for clathrin, indicating its significant role in cellular trafficking processes. Epsins Ent1 and Ent2, which are crucial for endocytosis but described to have redundant functions, revealed distinct binding patterns; Ent1 demonstrated stronger interactions with clathrin than Ent2, explaining its functional divergence towards actin binding. Despite both having actin anchoring domains, since Ent1 is actually more stably recruited by clathrin, it would provide a better actin anchoring function. These results offer molecular insights into AP selectivity, suggesting they competitively bind clathrin while also targeting different clathrin sites.