The Evolutionarily Conserved TLDc Domain Defines a New Class of V‐ATPase Interacting Proteins

Abstract

The kidney-specific V-ATPase regulates physiological acid-base homeostasis, and its dysfunction causes distal renal tubular acidosis (dRTA). We recently found that nuclear receptor coactivator 7 (NCOA7) interacts with the V-ATPase in the kidney, and its deletion in mice resulted in dRTA. NCOA7 belongs to a group of five proteins that contain the evolutionarily conserved TLDc domain, which are known to play a role in the oxidative stress response, and like the V-ATPase, mutations in TLDc proteins lead to serious human neurological diseases. In addition to NCOA7, we found that the TLDc protein, OXR1, interacts with the kidney V-ATPase. Here we asked if the other proteins that make up this family, TBC1D24, TLDC1 and TLDC2, also, interact with the kidney V-ATPase and whether this interaction is mediated by their TLDc domains. Interactions with the V-ATPase were assessed by GST pull-down using mouse kidney lysates and purified GST-tagged TLDc domains of NCOA7, OXR1, TBC1D24, TLDC1 and TLDC2 or the NCOA7 TLDc domain harboring pathogenic mutations (G802A, G815A, S817A, G845A, G896A, L926A, E938A), followed by western blotting. These experiments revealed that the purified TLDc domains of NCOA7, OXR1 and TLDC2, but not TBC1D24 or TLDC1, interact with the kidney-enriched B1 isoform of the V-ATPase. Moreover, the G815A, G845A and G896A mutants found in evolutionarily conserved regions of the NCOA7 TLDc domain did not interact with V-ATPase, L926A and E938A mutations decreased interaction, while S817A or the non-conserved G802A mutation, did not attenuate interaction at all. Since we could not identify an interaction between purified TBC1D24 or TLDC1 and the kidney V-ATPase we investigated the interactions between endogenous TBC1D24, TLDC1 and V-ATPase by co-immunoprecipitation (co-IP) and western blotting. In co-IP studies using mouse kidney lysate, we found that endogenous TBC1D24 interacts with the B1 isoform of the V-ATPase, but not with the more ubiquitous B2 isoform. On the other hand, we did not detect any interaction between endogenous TLDC1 and either the B1 or B2 isoform of the V-ATPase in kidney lysates. Because we were unable to detect TLDC1 in kidney lysates, we performed a co-IP using mouse lung lysate, which expresses high levels of TLDC1. In lung we found that TLDC1 interacts with the B2, but not the B1 subunit of the V-ATPase. However, B1 was expressed at low levels in the lung, which could explain the lack of detectable interaction with TLDC1. Of note, co-IP experiments using equal parts lung and kidney lysate showed a stronger interaction with B2 than B1, suggesting that TLDC1 may preferentially interact with the B2 subunit of the V-ATPase. In the kidney, NCOA7 and OXR1, as well as TBC1D24 and possibly TLDC2 interacted with the V-ATPase and may play a role in V-ATPase-dependent regulation of renal acid-base homeostasis. Furthermore, the evolutionary conserved amino acids within the TLDc domain of Ncoa7 are critical for its interaction with the kidney V-ATPase. Although TLDC1 was not detectable in the kidney, it interacts with the B2 isoform of the V-ATPase in the lung. We conclude, that the TLDc motif is a protein-protein interaction domain that defines a new class of V-ATPase interacting regulatory proteins.