Identification of the client-binding site on the Golgi membrane protein adaptor Vps74

Vps74 and its mammalian counterpart GOLPH3 are COPI associated protein sorting adaptors that function to maintain the cisternal distributions of certain Golgi integral membrane protein clients. The GOLPH3 adaptors accommodate a diversity of clients by binding to their short cytoplasmically exposed N-termini. Here, we identify the client-binding site on yeast GOLPH3 (Vps74) which maps to two evolutionarily conserved unstructured regions on the membrane-facing surface of the protein. The client-binding site includes residues previously shown to mediate binding of GOLPH3s to PI4P, as well as the membrane-binding β-hairpin. Clients have varying requirements for the binding interface, and our study thus reconciles how a diversity of client N-termini can be accommodated by Vps74. We establish that the binding site for the PI4P phosphatase Sac1 and the COPI-coatomer associated GTPase Arf1 overlaps with and obscures client access, suggesting a regulatory role for these proteins in opposition to the adaptor. Furthermore, we identify an additional mode for the recruitment of the adaptor from the cytoplasm to Golgi membranes whereby Vps74 binds directly to its client N-termini.