title: 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.affiliation: Department of Medical Genetics, Cambridge University, Cambridge Institute for Medical Research, Addenbrooke's Hospital Box 139, Cambridge, CB2 0XY, UK.
authors.forename: Ya
authors.initials: Y
authors.lastname: Su
authors.forename: Katherine G
authors.initials: KG
authors.lastname: Blake-Palmer
authors.forename: Sara
authors.initials: S
authors.lastname: Sorrell
authors.forename: Babak
authors.initials: B
authors.lastname: Javid
authors.forename: Katherine
authors.initials: K
authors.lastname: Bowers
authors.forename: Aiwu
authors.initials: A
authors.lastname: Zhou
authors.forename: Simon H
authors.initials: SH
authors.lastname: Chang
authors.forename: Seema
authors.initials: S
authors.lastname: Qamar
authors.forename: Fiona E
authors.initials: FE
authors.lastname: Karet
mesh_terms: D000141:Acidosis, Renal Tubular / Q000378:metabolism / Q000503:physiopathology*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006019:Glycolysis / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D010732:Phosphofructokinase-1; C490181:ATP6V0A4 protein, human; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases