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 that are composed of two subcomplexes: V and V. V-ATPase activity is regulated in part through reversible disassembly, during which V physically separates from V and both subcomplexes become inactive. Reassociation of V to V reactivates the complex for ATP-driven proton pumping and organelle acidification. V-ATPase reassembly in Saccharomyces 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-3α 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 that has parallels to V-ATPase-related disease. ROGDI shares extensive structural homology with yeast Rav2 and can functionally replace Rav2 in yeast. ROGDI binds to the N-terminal domains of both Rabconnectin-3 α and β, similar to Rav2 binding to Rav1. Molecular modeling suggests that ROGDI may bridge the two Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 subunits from detergent-solubilized lysates and is present with them in immunopurified lysosomes of mammalian cells. In immunofluorescence microscopy, ROGDI partially localizes with Rabconnectin-3α in acidic perinuclear lysosomes. The discovery of ROGDI as a novel Rabconnectin-3 interactor sheds new light on both Kohlschutter-Tonz syndrome and the mechanisms behind mammalian V-ATPase regulation.