Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump.
Vacuolar H(+)-ATPases (V-ATPases) are essential for acidification of intracellular compartments and for proton secretion from the plasma membrane in kidney epithelial cells and osteoclasts. The cellular proteins that regulate V-ATPases remain largely unknown. A screen for proteins that bind the V-ATPase E subunit using the yeast two-hybrid assay identified the ... cDNA clone coded for aldolase, an enzyme of the glycolytic pathway. The interaction between E subunit and aldolase was confirmed in vitro by precipitation assays using E subunit-glutathione S-transferase chimeric fusion proteins and metabolically labeled aldolase. Aldolase was isolated associated with intact V-ATPase from bovine kidney microsomes and osteoclast-containing mouse marrow cultures in co-immunoprecipitation studies performed using an anti-E subunit monoclonal antibody. The interaction was not affected by incubation with aldolase substrates or products. In immunocytochemical assays, aldolase was found to colocalize with V-ATPase in the renal proximal tubule. In osteoclasts, the aldolase-V-ATPase complex appeared to undergo a subcellular redistribution from perinuclear compartments to the ruffled membranes following activation of resorption. In yeast cells deficient in aldolase, the peripheral V(1) domain of V-ATPase was found to dissociate from the integral membrane V(0) domain, indicating direct coupling of glycolysis to the proton pump. The direct binding interaction between V-ATPase and aldolase may be a new mechanism for the regulation of the V-ATPase and may underlie the proximal tubule acidification defect in hereditary fructose intolerance.
Mesh Terms:
Animals, Binding Sites, Cattle, Cell Membrane, Cell Nucleus, DNA, Complementary, Fructose, Fructose-Bisphosphate Aldolase, Glucose-6-Phosphate Isomerase, Glutathione Transferase, Humans, Immunoblotting, Immunohistochemistry, Kidney, Microsomes, Mutation, Osteoclasts, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proton-Translocating ATPases, Protons, Two-Hybrid System Techniques, Vacuolar Proton-Translocating ATPases
Animals, Binding Sites, Cattle, Cell Membrane, Cell Nucleus, DNA, Complementary, Fructose, Fructose-Bisphosphate Aldolase, Glucose-6-Phosphate Isomerase, Glutathione Transferase, Humans, Immunoblotting, Immunohistochemistry, Kidney, Microsomes, Mutation, Osteoclasts, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proton-Translocating ATPases, Protons, Two-Hybrid System Techniques, Vacuolar Proton-Translocating ATPases
J. Biol. Chem.
Date: Aug. 10, 2001
PubMed ID: 11399750
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