The assembly factor Dmxl1 is required for H+(V)-ATPase assembly and function in mammals in vivo

Abstract

The V-ATPase (proton pump) generates electrochemical gradients across cell membranes, driving essential biological processes. Critically, the two V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in serious pathologies such as cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting intercalated cells (IC) in the kidney. However, very little is known about the molecular regulation of V-ATPase in mammals. We have identified a novel interactor of the IC-enriched B1 subunit of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 protein (Dmxl1), aka Rabconnectin-3A (Rbcn3A). The yeast homologue of Dmxl1, Rav1p, is part of a protein complex that catalyzes the reversible assembly of the V1 and VO domains, thus regulating proton pumping activity. We, therefore, hypothesized that Dmxl1 is an assembly factor for the V-ATPase in mammalian cells. We generated kidney IC-specific Dmxl1 knockout (KO) mice and they had a high urine pH, similar to B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting of kidney lysates showed decreased B1 V-ATPase expression, but other V-ATPase subunits were normal. B1 (V1 domain) and a4 (VO domain) subunits were immunolocalized in ICs and apical localization quantified using FIJI. Both B1 and a4 were more intracellular in Dmxl1 KO ICs relative to control mice. A Pearson’s correlation analysis of B1 and a4 staining revealed a significant decrease in colocalization in Dmxl1 KO ICs, suggesting reduced B1/a4 assembly. In parallel, subcellular fractionation experiments revealed less V1 domain-associated B1 subunit in the membrane fraction relative to the cytosol. Furthermore, a Proximity Ligation Assay (PLA) was performed using probes against B1 and a4 to confirm decreased assembly of the two domains in KO ICs. When the two probes are within 40 μM of each other, like when the domains are assembled, this results in a fluorescent PLA punctum. As expected, we observed a significant decrease in the area of PLA puncta per cell in KO animals relative to control animals. These data demonstrate that the V-ATPase is less assembled in Dmxl1 KO ICs, relative to controls. We propose that loss of Dmxl1 reduces assembly of the V-ATPase holoenzyme, thereby inhibiting its proton pumping function. Dmxl1 may mediate recruitment of the V1 domain to the membrane and facilitate its assembly with the transmembrane VO domain. In the absence of Dmxl1, V1 is instead targeted for degradation, explaining the inability of these mice to properly acidify their urine. We conclude that Dmxl1 is a bona-fide mammalian V-ATPase assembly factor that functions similarly to its yeast homolog, Rav1p in affecting V-ATPase assembly. NIH/NIDDK R01DK121848 to DB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.