A Phosphotyrosine Switch for Cargo Sequestration at Clathrin-coated Buds

Abstract

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 β2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs β-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the β2 appendage platform. Tyr-888 is a critical constituent of this spatially confined β2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed β2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the β2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and β-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the β2 platform-binding site and, hence, cargo sorting.