The Human Rogdi Protein Is the Functional Homologue of Yeast Rav2 and Can Promote V‐ATPase Assembly

Abstract

Mutations in the human Rogdi protein cause Kohlschutter-Tonz syndrome, which is characterized by early-onset seizures, developmental defects, and defective deposition of tooth enamel (amelogenesis imperfecta). The cellular function of Rogdi is not known. The yeast RAVE (regulator of ATPase of vacuoles and endosomes) complex and mammalian Rabconnectin-3 complexes catalyze assembly of certain subpopulations of the V-ATPase proton pump and thus help to regulate pH homeostasis. The yeast RAVE complex is composed of three subunits: Rav1, Rav2, and Skp1. Skp1 is a multifunctional scaffold protein, but both Rav1 and Rav2 act specifically in V-ATPase assembly. Rabconnectin-3 complexes have been reported to contain two larger subunits, Rbcn3α and 3β; both subunits are predicted to have structural similarity to Rav1. No mammalian homologue of yeast Rav2 had been identified. However, despite limited sequence homology, we find that the yeast Rav2 sequence can be modeled with very high confidence on a recent structure of human Rogdi (H. Lee et al. (2017) Sci. Rep. 7:3972). Expression of human Rogdi in yeast complements the V-ATPase-associated growth defects of a yeast rav2∆ mutant. Complementation requires the presence of Rav1, suggesting that Rogdi acts as part of the RAVE complex rather than bypassing RAVE function. Consistent with this, Rogdi copurifies with a FLAG-tagged Rav1 protein. Yeast Rav2 binds to the N-terminal β-propeller region of Rav1. Two-hybrid assays indicate that Rogdi can interact with the same region of Rav1, as well as binding to the N-terminal β-propeller regions of Rbcn3α and 3β isoforms. Based on these data, we hypothesize that Rogdi is a subunit of mammalian Rabconnectin-3 complexes. WDR72 is a possible Rbcn3β isoform in humans. Mutations in the N-terminal β-propeller domain of WDR72 also cause amelogenesis imperfecta, as well as distal renal tubule acidosis, a disease associated with defects in V-ATPase activity. We are testing whether these mutations affect the interaction between Rogdi and WDR72, potentially linking Rogdi with both Rabconnectin-3 and V-ATPase function.