Identification of the client-binding site on the Golgi membrane protein adaptor Vps74/yGOLPH3

Vps74 and its mammalian counterpart GOLPH3 are COPI associated protein sorting adaptors that function to maintain the cisternal distributions of a diversity of Golgi integral membrane protein 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 loops on the membrane-facing surface, and includes residues mediating binding of GOLPH3s to PI4P, as well as the membrane-binding β hairpin. As an orthogonal approach, we isolated an inhibitory anti-Vps74 nanobody (which also binds to GOLPH3s) with which we corroborate the client-binding site and reveal that Sac1 and Arf1 binding to Vps74 blocks client access. We also identify an additional mode for the recruitment of Vps74 to Golgi membranes whereby the adaptor binds directly to its client N-termini. This study elucidates the molecular mechanism of Vps74 and identifies an inhibitory GOLPH3 nanobody with potential therapeutic applications.