Differential roles of N‐glycans linked to the Na,K‐ATPase β1 and β2 subunits in sorting of the pump in polarized HGT‐1 cells

In most polarized cells, the Na,K‐ATPase is localized on the basolateral membrane. We showed that in polarized HGT‐1 cells (parietal cell‐derived carcinoma line) the Na,K‐ATPase is sorted to the apical membrane due to the high abundance of the β2 subunit and absence of the β1 subunit. Expressed YFP‐linked Na,K‐ATPase β1 subunit localized exclusively to the lateral membranes accompanied by partial redistribution of the endogenous α1 subunit to the lateral surface as detected by confocal microscopy and surface biotinylation. Hence, sorting information present in the β isoform overrides sorting information in the α subunit in HGT‐1 cells, which makes these cells a good model for studies of sorting of the Na,K‐ATPase β isoforms as compared to MDCK and LLCPK cells in which basolateral sorting of the α1 subunit prevails. The β1 isoform has 3 N‐glycosylation sites, while the β2 subunit has 8 sites. Sequential mutagenic removal of N‐glycosylation sites from the β1 subunit resulted in gradual loss of subunit from the lateral membranes and intracellular endosomal localization of the non‐glycosylated mutant. Sequential mutagenic addition of the N‐glycosylation sites homologous to the ones present in the β2 subunit promoted apical sorting of the mutant β1 subunit and gradually increased the fraction of mutant protein on the apical membrane that reached 80% of the total surface amount for the β1mutant with 5 additional sites. Therefore, N‐glycans on the β1 subunit are essential for stable expression of the pump in the lateral membranes of HGT‐1 cells while the N‐glycans unique to the β2 subunit isoform contain apical sorting information as do N‐glycans in the H,K‐ATPase β subunit.