beta-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

摘要

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase a- and P-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 Pi-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged Pi-subunits (Piflag) or Myc-tagged Pi-subunits (Pimyc) under the control of a tetracycline-dependent promoter. The induction of Piflag subunit synthesis in MDCK cells, which increases Pi-subunit expression at the plasma membrane by more than twofold, while maintaining stable cti expression levels, revealed that all mature Pi-subunits associate with oti-subunits, and no evidence of "free" Pi-subunits was obtained. Consequently, the ratio of assembled beta_1- to alpha_1-subunits is significantly increased when "extra" p-subunits are expressed. An increased beta_1/alpha_1i 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 pimyc-expressing and Piflag-express-ing MDCK cells show colocalization of beta_1myc and Piflag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the p-subunits. Immunoprecipi-tation using MDCK cells constitutively expressing beta_1myc and tetra-cycline-regulated piflag subunits confirmed p-p-subunit interactions. These results demonstrate that the equimolar ratio of assembled beta_1/alpha_1-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.