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

Su Y, Blake-Palmer KG, Sorrell S, Javid B, Bowers K, Zhou A, Chang SH, Qamar S, Karet FE - Am. J. Physiol. Renal Physiol. (2008)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Cambridge University, Cambridge Institute for Medical Research, Addenbrooke's Hospital Box 139, Cambridge, CB2 0XY, UK.

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- 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).

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Confirmation of yeast H+ATPase/Pfk interaction and comparison of a-subunit COOH-terminal sequences. A: Co-IP of H+ATPase and Pfk from vacuoles of KEBY9 (a-subunit double deletion) yeast transformed with Vph1p. IP: (+) or (−) indicates inclusion or omission of α-Vma2p; IB: α-Pfk. A single band corresponding to yeast Pfk is seen in the (+) lane only. B: amino acid sequences of the COOH termini from human and yeast orthologs and paralogs are aligned. Residues at the start and end of this domain are numbered. The highly conserved first 17 amino acids are highlighted in gray. Conserved Arg (807 in a4 or 799 in Vph1p) and Gly (820 in a4 or 812 in Vph1p) residues are boxed.
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f1: Confirmation of yeast H+ATPase/Pfk interaction and comparison of a-subunit COOH-terminal sequences. A: Co-IP of H+ATPase and Pfk from vacuoles of KEBY9 (a-subunit double deletion) yeast transformed with Vph1p. IP: (+) or (−) indicates inclusion or omission of α-Vma2p; IB: α-Pfk. A single band corresponding to yeast Pfk is seen in the (+) lane only. B: amino acid sequences of the COOH termini from human and yeast orthologs and paralogs are aligned. Residues at the start and end of this domain are numbered. The highly conserved first 17 amino acids are highlighted in gray. Conserved Arg (807 in a4 or 799 in Vph1p) and Gly (820 in a4 or 812 in Vph1p) residues are boxed.

Mentions: Peptides corresponding to the coding sequences of the last 45 residues of WT human a4 (a4C-WT; Fig. 1) or a4C containing G820R (a4C-GR) with acetylated NH2 termini were synthesized and HPLC-purified by CovalAb UK (Cambridge, UK).


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

Su Y, Blake-Palmer KG, Sorrell S, Javid B, Bowers K, Zhou A, Chang SH, Qamar S, Karet FE - Am. J. Physiol. Renal Physiol. (2008)

Confirmation of yeast H+ATPase/Pfk interaction and comparison of a-subunit COOH-terminal sequences. A: Co-IP of H+ATPase and Pfk from vacuoles of KEBY9 (a-subunit double deletion) yeast transformed with Vph1p. IP: (+) or (−) indicates inclusion or omission of α-Vma2p; IB: α-Pfk. A single band corresponding to yeast Pfk is seen in the (+) lane only. B: amino acid sequences of the COOH termini from human and yeast orthologs and paralogs are aligned. Residues at the start and end of this domain are numbered. The highly conserved first 17 amino acids are highlighted in gray. Conserved Arg (807 in a4 or 799 in Vph1p) and Gly (820 in a4 or 812 in Vph1p) residues are boxed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2576143&req=5

f1: Confirmation of yeast H+ATPase/Pfk interaction and comparison of a-subunit COOH-terminal sequences. A: Co-IP of H+ATPase and Pfk from vacuoles of KEBY9 (a-subunit double deletion) yeast transformed with Vph1p. IP: (+) or (−) indicates inclusion or omission of α-Vma2p; IB: α-Pfk. A single band corresponding to yeast Pfk is seen in the (+) lane only. B: amino acid sequences of the COOH termini from human and yeast orthologs and paralogs are aligned. Residues at the start and end of this domain are numbered. The highly conserved first 17 amino acids are highlighted in gray. Conserved Arg (807 in a4 or 799 in Vph1p) and Gly (820 in a4 or 812 in Vph1p) residues are boxed.
Mentions: Peptides corresponding to the coding sequences of the last 45 residues of WT human a4 (a4C-WT; Fig. 1) or a4C containing G820R (a4C-GR) with acetylated NH2 termini were synthesized and HPLC-purified by CovalAb UK (Cambridge, UK).

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Cambridge University, Cambridge Institute for Medical Research, Addenbrooke's Hospital Box 139, Cambridge, CB2 0XY, UK.

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- 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).

Show MeSH
Related in: MedlinePlus