Source data for Winkley and Kane, The ROGDI protein mutated in Kohlschutter-Tonz syndrome is a novel subunit of the Rabconnectin-3 complex implicated in V-ATPase assembly

V-ATPases are highly conserved ATP driven rotary proton pumps found widely among eukaryotes. V-ATPases are composed of two subcomplexes: V1 and V0. V-ATPase activity is regulated in part through a process called reversible disassembly, during which V1 physically separates from V0 and both subcomplexes become inactive. Reassociation of V1 to V0 reactivates the complex for ATP-driven proton pumping and organelle acidification. V-ATPase reassembly in S. cerevisiae requires the RAVE complex (Rav1, Rav2, and Skp1) and higher eukaryotes, including humans, utilize the Rabconnectin-3 complex. Mammalian Rabconnectin-3 has two subunits: Rabconnectin-3α and Rabconnectin-3β. Rabconnectin-α isoforms are homologous to Rav1, but there is no known Rav2 homolog and the molecular basis of the interaction between the Rabconnectin-3α and β subunits is unknown. We identified ROGDI as a Rav2 homolog and novel Rabconnectin-3 subunit. ROGDI mutations cause Kohlschutter-Tonz syndrome, an epileptic encephalopathy with amelogenesis imperfecta with parallels to V-ATPase-related disease. ROGDI shares extensive structural homology with yeast Rav2 and binds to the N-terminal domains of both Rabconnectin-3 α and β in a manner similar to Rav2 binding to Rav1. Molecular modeling suggests that ROGDI may bridge the two Rabconnectin-3 and subunits. ROGDI co-immunoprecipitates with Rabconnectin-3 subunits from detergent solubilized lysates and is present with them in immunopurified lysosomes. In immunofluorescence microscopy, ROGDI partially localizes with Rabconnectin-3a in highly acidic, perinuclear lysosomes. The discovery of ROGDI as a novel Rabconnectin-3 interactor sheds new light on both Kohschutter-Tonz syndrome and the mechanisms behind mammalian V-ATPase regulation.