ABSTRACT
Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous µ1, µ2, µ3, and µ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on µ2 and µ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the µ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the µ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The µ3A C-terminal domain consists of an immunoglobulin-like ß-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on µ3A subdomain A, at a location similar to the YXXØ-binding site on µ2 but not µ4. The binding sites on µ3A and µ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the µ3A site and show that this protein binds YXXØ signals with 14-19 µm affinity. The surface electrostatic potential of µ3A is less basic than that of µ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.