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Am J Physiol Renal Physiol 295: F950-F958, 2008. First published July 16, 2008; doi:10.1152/ajprenal.90258.2008
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Human H⁺ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1

Departments of ¹Medical Genetics, ²Medicine, ⁴Hematology, and ⁶Division of Renal Medicine, Cambridge University, Cambridge; ³Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; and ⁵Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana

Submitted 18 April 2008 ; accepted in final form 14 July 2008

The vacuolar-type ATPase (H⁺ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1–a4. This study investigated two naturally occurring point mutations in a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H⁺ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1's catalytic activity. In yeast expression studies, protein levels, H⁺ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H⁺ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H⁺ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H⁺ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).

kidney; proton pump; yeast; glycolysis; acidosis

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