Characterisation of human H⁺-ATPase a4 subunit

Su, Ya (2004). Characterisation of human H⁺-ATPase a4 subunit. PhD thesis The Open University.



The vacuolar H+-ATPase (or V-ATPases) are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialised sites such as kidney epithelial cells and osteoclasts, as well as acidifying intracellular compartments. The 100 kDa polytopic a subunit of this group of ATPases is suggested to play important roles in proton translocation, assembly, and targeting as well as coupling of ATP hydrolysis and proton transport of the V-ATPase. In man, different a subunit paralogues are encoded by four genes. ATP6V0A4 encodes a4, which is dominantly expressed apically in a-intercalated cells in both human and mouse kidney. I sought binding partners for a4 in order to address its potential role in the V-ATPase complex. Random peptide phage display analysis using a4's C terminus as a target protein revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by phage ELISA. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from human kidney membrane proteins. An in vitro PFK-1 pull-down assay showed that this interaction is also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalised with a4 in α-IC in the collecting ducts of human kidney. These findings indicate a direct link between V-ATPase and glycolysis, via the C-terminus of the pump's a subunit, and suggest a novel regulatory mechanism between V-ATPase function and energy supply. This interaction between the a subunit and PFK-1 also provides new evidence that the C-terminus of this subunit lies cytoplasmically in vivo. Finally, SPR analysis suggests a possible alteration of the a4/PFK-1 interaction by the mutation (G820R) within the a4(C) region identified from a patient with rdRTA, providing a potential mechanism for disease.

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