Sla2 is a core interaction hub for Clathrin Light Chain and the Pan1/End3/Sla1 Complex

The interaction network of Sla2, a vital adaptor protein in the endocytic mid-coat, undergoes constant rearrangement incorporating or replacing interacting proteins over time. Sla2 serves as a scaffold linking the membrane to the actin cytoskeleton, with this role modulated by 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 electron cryo-microscopy. We provide an interaction map of Sla2 and the regulatory proteins Sla1 and Pan1, predicted by AI modelling and confirmed by molecular biophysics techniques. Pan1 competes with CLC for the conserved binding site on Sla2. These results enhance the mapping of crucial interactions at endocytic checkpoints and highlight the divergence between Metazoa and Fungi in this vital process. Teaser Sla2 forms complexes with three regulatory proteins in the endocytic pit, two of which compete for the same site