Kohlschütter-Tönz protein ROGDI is the homolog of yeast Rav2 and a novel Rabconnectin-3 subunit

Abstract

V-ATPases are rotary proton pumps that are extraordinarily well-conserved among eukaryotes. V-ATPases function primarily to acidify intracellular compartments, critical to maintaining cellular homeostasis. The V-ATPase-generated proton gradient provides the optimal environment for lysosomal catabolism and drives intracellular protein trafficking. V-ATPases serve important functions throughout the human body. For example, V-ATPase activity energizes the active transport of neurotransmitters into synaptic vesicles, regulates the acid/base balance in the kidney, and helps the immune system recognize invading pathogens. However, when V-ATPase activity is inappropriately increased or decreased, these processes are affected, and disease can result. V-ATPases are composed of peripheral V₁ and integral membrane V₀ subcomplexes; V₁ hydrolyzes ATP and transmits rotation to V₀, which moves protons across a membrane. V-ATPase activity is regulated in part through the reversible association of the V₁ subcomplex and V₁C subunit from V₀. Upon disassembly, both V₁ and V₀ are catalytically inactivated. In yeast, the RAVE complex catalyzes the efficient reassembly of V-ATPases. Rabconnectin-3 is the human homolog of the RAVE complex and functions similarly. Mutations in the Rabconnectin-3 complex can reduce V-ATPase activity through decreased assembly, which leads to disease. Both Rabconnectin-3 subunits share substantial homology with the RAVE subunit Rav1. We have identified the poorly characterized protein ROGDI as the mammalian homolog of the yeast RAVE subunit, Rav2. ROGDI shares strong functional and structural homology with yeast Rav2. Expression of ROGDI in a rav2Δ yeast strain partially rescues the growth phenotype characteristic of RAVE mutants. ROGDI binds to the structurally conserved N-terminal β-sheet rich domain. AlphaFold3 modeling predicts that ROGDI binds between the Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 and V-ATPase subunits. Additionally, ROGDI is present alongside V-ATPase and Rabconnectin-3 subunits on lysosomal membranes. This indicates that, like RAVE and Rav2, Rabconnectin-3 and ROGDI localize intracellular regions rich in V-ATPases. Identifying ROGDI as a novel Rabconnectin-3 subunit is a substantial step forward in our understanding of Rabconnectin-3 and how it influences V-ATPase activity.