title: beta-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells.
abstract: In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase alpha- and beta-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase beta1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged beta1-subunits (beta1flag) or Myc-tagged beta1-subunits (beta1myc) under the control of a tetracycline-dependent promoter. The induction of beta1flag subunit synthesis in MDCK cells, which increases beta1-subunit expression at the plasma membrane by more than twofold, while maintaining stable alpha1 expression levels, revealed that all mature beta1-subunits associate with alpha1-subunits, and no evidence of "free" beta1-subunits was obtained. Consequently, the ratio of assembled beta1- to alpha1-subunits is significantly increased when "extra" beta-subunits are expressed. An increased beta1/alpha1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured beta1myc-expressing and beta1flag-expressing MDCK cells show colocalization of beta1myc and beta1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the beta-subunits. Immunoprecipitation using MDCK cells constitutively expressing beta1myc and tetracycline-regulated beta1flag subunits confirmed beta-beta-subunit interactions. These results demonstrate that the equimolar ratio of assembled beta1/alpha1-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607-7170, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Clifford
authors.forename: Jack H
authors.initials: JH
authors.lastname: Kaplan
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: C503199:Na K ATPase beta2 subunit, rat; D021122:Protein Subunits; D014415:Tunicamycin; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase