The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins

A F Eaton,M Merkulova,D Brown

doi:10.1038/s41598-021-01809-y

A F Eaton, M Merkulova + Show 1 more

Open Access

https://doi.org/10.1038/s41598-021-01809-y

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Abstract

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved “TLDc” domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

Highlights

The vacuolar ATPase (V-ATPase, known as the H +-ATPase) is a multisubunit, transmembrane protein complex whose function is to catalyze ATP hydrolysis and harness the released energy to actively pump protons across biological membranes
Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase, using a combination of pull-down and co-IP assays
In detailed studies using purified domains of these proteins, and site-specific mutagenesis of the TLDc domain of nuclear receptor coactivator 7 (Ncoa7), we found that this interaction depends on distinct amino acid residues within the TLDc domain itself

Summary

Introduction

The vacuolar ATPase (V-ATPase, known as the H +-ATPase) is a multisubunit, transmembrane protein complex whose function is to catalyze ATP hydrolysis and harness the released energy to actively pump protons across biological membranes. Ncoa and Oxr are members of a protein family that contain a conserved “TLDc” domain on their C-terminus, and all have been shown to play a role in protecting cells from oxidative stress. Domain, Tbc1d24, was detected in our proteomics study, but did not reach the threshold to be reliably considered as a V-ATPase-interacting protein, possibly due to the high stringency of the protein–protein interaction scoring that we applied in order to avoid possible false positives9 In mammals, this family includes Tldc ( known as Meak7) and T ldc210. More recently, loss of Ncoa in humans was linked to autism spectrum disorders, and the mechanism remains unknown, it could be due to decreased neuroprotection, similar to Oxr119 It is not known if Tldc and Tldc play essential roles in protection from oxidative stress in vivo

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