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Abstract: TH-OR27

Evolutionary Conserved TLDc Domain Defines a New Class of V-ATPase Interacting Proteins

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Eaton, Amity F., Massachusetts General Hospital, Boston, Massachusetts, United States
  • Brown, Dennis, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Merkulova, Maria, Massachusetts General Hospital, Boston, Massachusetts, United States

Group or Team Name

  • Program in Membrane Biology
Background

Kidney-specific V-ATPase regulates acid-base homeostasis, and its disfunction causes distal renal tubular acidosis (dRTA). We recently found that nuclear receptor coactivator 7 (Ncoa7) interacts with kidney V-ATPase, and its deletion in mice resulted in dRTA. Ncoa7 belongs to a group of proteins playing a role in the oxidative stress response, that contain the evolutionarily conserved TLDc domain. We found that another of these proteins, Oxr1, also interacts with the V-ATPase. Here we asked if other proteins from this family, i.e. Tbc1d24, Tldc1 and Tldc2 interact with V-ATPase in kidney and if their TLDc domains mediate this interaction.

Methods

Interaction between endogenous Tbc1d24, Tldc1 and Tldc2 and V-ATPase was assessed by co-immunoprecipitation (co-IP) and western blotting of mouse kidney lysates. Interaction with the V-ATPase was also studied by GST pull-downs from kidney lysates using purified GST-tagged wild-type TLDc domains of Ncoa7, Oxr1, Tbc1d24, Tldc1 and Tldc2, or mutant TLDc domains of Ncoa7 (G802A, G815A, S817A, G845A, G896A, L926A, E938A) followed by western blotting for B1.

Results

In Co-IP studies of mouse kidney lysates we found that endogenous Tbc1d24 interacted with the B1 subunit isoform of V-ATPase, but not with the more ubiquitous B2 subunit isoform. However, we did not detect any interaction between V-ATPase and endogenous Tldc1 or Tldc2 in Co-IPs, possibly due to low sensitivity of the anti-Tldc1 and anti-Tldc2 antibodies. Additionally, we found that the purified TLDc domains of Ncoa7, Oxr1 and Tldc2, but not Tbc1d24 or Tldc1, interacted with V-ATPase in GST pull-downs. Finally, the G815A, G845A and G896A mutants in evolutionarily conserved regions of the Ncoa7 TLDc domain did not interact with V-ATPase, L926A and E938A mutations resulted in a decreased interaction, while S817A or the non-conserved G802A mutation (used as a positive control), did not decrease interaction at all.

Conclusion

In the kidney, Tbc1d24 and possibly Tldc2, as well as Ncoa7 and Oxr1, interacted with V-ATPase and may play a role in the V-ATPase-dependent regulation of renal acid-base homeostasis. We conclude, that the TLDc motif is a protein-protein interaction domain that defines a new class of V-ATPase interacting regulatory proteins. The evolutionary conserved amino acids within the TLDc domain of Ncoa7 are critical for its interaction with the V-ATPase.

Funding

  • NIDDK Support