Open Accessβ-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells
Author(s) -
Rebecca J. Clifford,
Jack H. Kaplan
Publication year - 2008
Publication title -
american journal of physiology. renal physiology./american journal of physiology. renal physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.335
H-Index - 169
eISSN - 1931-857X
pISSN - 1522-1466
DOI - 10.1152/ajprenal.90406.2008
Subject(s) - protein subunit , immunoprecipitation , tunicamycin , microbiology and biotechnology , colocalization , v atpase , biology , flag (linear algebra) , cell culture , chemistry , biochemistry , gene , mathematics , unfolded protein response , endoplasmic reticulum , pure mathematics , genetics , algebra over a field
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.