Sla2 is a core interaction hub for clathrin light chain and the Pan1/End3/Sla1 complex

The interaction network of Sla2, a vital endocytic mid-coat adaptor protein, undergoes constant rearrangement. Sla2 serves as a scaffold linking the membrane to the actin cytoskeleton, with its role modulated by the clathrin light chain (CLC), which inhibits Sla2's function under certain conditions. We show that Sla2 has two independent binding sites for CLC: one previously described in homologs of fungi (Sla2) and metazoa (Hip1R), and a second found only in Fungi. We present the structural model of the Sla2 actin-binding domains in the context of regulatory structural domains by cryoelectron microscopy. We provide an interaction map of Sla2 and the regulatory proteins Sla1 and Pan1, predicted by AI modeling and confirmed by molecular biophysics techniques. Pan1 may compete with CLC for the conserved Sla2-binding site. These results enhance the mapping of crucial interactions at endocytic checkpoints and highlight the divergence between Metazoa and Fungi in this vital process.