Human H+ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1.

Abstract

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).

• Authors: Ya Su, Katherine G Blake-Palmer, Sara Sorrell, Babak Javid, Katherine Bowers, Aiwu Zhou, Simon H Chang, Seema Qamar, Fiona E Karet
• Journal: American journal of physiology. Renal physiology (Am J Physiol Renal Physiol) Vol. 295 Issue 4 Pg. F950-8 (Oct 2008) ISSN: 1931-857X [Print] United States
• PMID: 18632794 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Protein Subunits
• Saccharomyces cerevisiae Proteins
• Phosphofructokinase-1
• ATP6V0A4 protein, human
• Vacuolar Proton-Translocating ATPases
• Proton-Translocating ATPases

Topics

• Acidosis, Renal Tubular (metabolism, physiopathology)
• Amino Acid Sequence
• Circular Dichroism
• Glycolysis (physiology)
• Humans
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Phosphofructokinase-1 (genetics, metabolism)
• Protein Subunits (genetics, metabolism)
• Proton-Translocating ATPases (genetics, metabolism)
• Saccharomyces cerevisiae
• Saccharomyces cerevisiae Proteins (genetics, metabolism)
• Surface Plasmon Resonance
• Vacuolar Proton-Translocating ATPases