The Na, K-ATPase Beta1 and Beta2 Subunits Associate with Different Quality Control Pathways in the ER

Abstract

The catalytic Na,K-ATPase α-subunit is not able to exit the ER or catalyze ion transport unless assembled with the β-subunit. However, requirements for the ER exit of the Na,K-ATPase β-subunit that plays an additional, ion-transport-independent, role in intercellular adhesion are not clear. The Na,K-ATPase β1- or β2-subunits and their N-glycosylation-deficient mutants were expressed in renal MDCK cells. Confocal microscopy, immunohistochemistry, and immunoprecipitation were employed to evaluate the role of N-glycans of the β-subunit isoforms in the quality control of the Na,K-ATPase in the ER. Mutagenic removal of as few as two of the eight N-glycosylation sites from the β2-subunit precludes its assembly with the α1 subunit and results in full retention of the unassembled β2-subunit in the ER. However, removal of all three N-glycosylation sites from the β1-subunit only slightly affects its assembly with the α1-subunit and trafficking to the plasma membrane. The β2-subunit binds 4-5-fold more efficiently to the ER lectin chaperone, calnexin, and 2-3-fold less efficiently to the non-lectin ER chaperone, BiP, than the β1-subunit. These results indicate that folding of the β2- and β1-subunits is mediated by lectin and non-lectin chaperones, respectively, consistent with the essential role of N-glycosylation for folding and trafficking of the β2 but not of the β1-subunit. Disruption of the α1-β association by mutations in defined α1-interacting regions of either β1- or β2-subunits results in the ER retention of unassembled mutants, indicating that α-β assembly is essential for the ER export of either β-subunit isoform. In conclusion, the ER quality control system ensures that only properly folded β-subunits assemble with the α-subunits and only assembled α-β complexes are exported to the Golgi and delivered to the plasma membrane.Supported by NIH, DK077149 and DK58333.