Abstract 1510 The Kohlschutter-Tonz syndrome protein Rogdi may act as a Rabconnectin-3 subunit to help support V-ATPase assembly

V-ATPases are highly conserved organellar proton pumps important for a range of processes including protein trafficking, ion and metabolite transport, neurotransmitter uptake, and pH regulation. Many of these functions are regulated by reversible disassembly of V-ATPases but this process is not fully understood. V-ATPases are composed of two subcomplexes: V1 resides on the cytosolic face of the membrane and contains the sites of ATP hydrolysis and V0 is an integral membrane complex containing the proton pore. In response to nutritional signals, V1 dissociates from V0 and the activity of each complex is silenced. Reassembly requires the RAVE complex in yeast and Rabconnectin-3 complexes in higher eukaryotes. In yeast, RAVE binds to both the V1 and V0 subunits, bringing the subcomplexes together in response to cellular signals for reassembly. The RAVE complex consists of three subunits: Rav1, which serves as a scaffold, Rav2, and Skp1. In contrast, only two Rabconnectin-3 subunits, Rbcn3α and β, have been identified in higher eukaryotes. Both Rabconnectin-3 subunits share broad structural homology with Rav1 while Rbcn3α shares additional sequence homology. Despite the conserved structure and function of both Rabconnectin-3 and the V-ATPase, no mammalian Rav2 homolog had been identified. We identified the ROGDI protein as a potential Rav2 homolog based on homology modeling. Mutations in ROGDI result in Kohlschutter-Tonz syndrome, which is characterized by early epilepsy and other clinical features also associated mutations in V-ATPase and Rabconnectin-3 subunits. Human ROGDI and yeast Rav2 share extensive structural homology but little sequence homology. However, yeast Rav2 binds to the N-terminal beta propeller region of Rav1, and ROGDI interacts with a structurally similar region of human Rbcn3α and β isoforms, as well as Rav1, in yeast two-hybrid assays. Expression of ROGDI in a rav2Δ mutant yeast strain partially rescues growth defects of the mutant, supporting functional similarity between Rav2 and ROGDI. Molecular modeling using AlphaFold/Collabfold suggests that ROGDI might bridge the N-terminal β-propeller domains of the Rbcn3α and β subunits in Rabconnectin-3 complexes. In a mouse breast cancer cell line, DMXL1, one of the two Rbcn3α isoforms in mammals, localizes predominantly to endolysosomal compartments, which are enriched in V-ATPases. In addition, DMXL1 co-immunoprecipitates ROGDI along with the Rbcn3β isoform WDR7, and the catalytic V1 A subunit from these cells. Taken together, these data support an emerging structural picture of the Rabconnectin-3 complex, which includes the ROGDI protein as a subunit and the functional homologue of yeast Rav2. This work was supported by the NIH grant R35GM145256 to PMK.