β-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 α- and β-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 β₁-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged β₁-subunits (β₁flag) or Myc-tagged β₁-subunits (β₁myc) under the control of a tetracycline-dependent promoter. The induction of β₁flag subunit synthesis in MDCK cells, which increases β₁-subunit expression at the plasma membrane by more than twofold, while maintaining stable α₁expression levels, revealed that all mature β₁-subunits associate with α₁-subunits, and no evidence of “free” β₁-subunits was obtained. Consequently, the ratio of assembled β₁- to α₁-subunits is significantly increased when “extra” β-subunits are expressed. An increased β₁/α₁stoicheometry 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 β₁myc-expressing and β₁flag-expressing MDCK cells show colocalization of β₁myc and β₁flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the β-subunits. Immunoprecipitation using MDCK cells constitutively expressing β₁myc and tetracycline-regulated β₁flag subunits confirmed β-β-subunit interactions. These results demonstrate that the equimolar ratio of assembled β₁/α₁-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.