The vacuolar-type ATPase (H⁺ATPase) is a ubiquitously expressed multi-subunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in man has four forms, a1-a4. This study investigated two naturally occurring point mutations in a4's C-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 C-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).