Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase β1 subunit results in its clustering in caveolae and apical sorting in HGT-1 cells

In most polarized cells, the Na, K-ATPase is localized on the basolateral plasma membrane. However, an unusual location of the Na, K-ATPase was detected in polarized HGT-1 cells (a human gastric adenocarcinoma cell line). The Na, K-ATPase alpha(1) subunit was detected along with the beta(2) subunit predominantly on the apical membrane, whereas the Na, K-ATPase alpha(1) subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na, K-ATPase alpha(1) subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous alpha(1) subunit to the basolateral membrane. The human beta(2) subunit has eight N-glycosylation sites, whereas the beta(1) isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the beta(2) subunit were successively introduced in the alpha(1) subunit by site-directed mutagenesis. The mutated beta(1) subunits were detected on both apical and basolateral membranes. The fraction of a mutant beta(1) subunit present on the apical membrane increased in proportion to the number of glycosylation sites inserted and reached 80% of the total surface amount for the beta(1) mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous beta(2) subunit and the beta(1) mutant with additional glycosylation sites but not for the wild type beta(1) subunit. Hence, the N-glycans linked to the beta(2) subunit of the Na, K-ATPase contain apical sorting information, and the high abundance of the beta(2) subunit isoform, which is rich in N-glycans, along with the absence of the beta(1) subunit, is responsible for the unusual apical location of the Na, K-ATPase in HGT-1 cells.