title: Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump.
abstract: 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.
authors.affiliation: Departments of Medicine and Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA. luming@medicine.ufl.edu
authors.forename: M
authors.initials: M
authors.lastname: Lu
authors.forename: L S
authors.initials: LS
authors.lastname: Holliday
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: W A
authors.initials: WA
authors.lastname: Dunn
authors.forename: S L
authors.initials: SL
authors.lastname: Gluck
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus; D018076:DNA, Complementary / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D010010:Osteoclasts / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons; D020798:Two-Hybrid System Techniques; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D018076:DNA, Complementary; D011522:Protons; D005632:Fructose; D005982:Glutathione Transferase; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005634:Fructose-Bisphosphate Aldolase; D005956:Glucose-6-Phosphate Isomerase