Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase beta1 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 alpha1 subunit was detected along with the beta2 subunit predominantly on the apical membrane, whereas the Na,K-ATPase beta1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na,K-ATPase beta1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous alpha1 subunit to the basolateral membrane. The human beta2 subunit has eight N-glycosylation sites, whereas the beta1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the beta2 subunit were successively introduced in the beta1 subunit by site-directed mutagenesis. The mutated beta1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant beta1 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 beta1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous beta2 subunit and the beta1 mutant with additional glycosylation sites but not for the wild type beta1 subunit. Hence, the N-glycans linked to the beta2 subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the beta2 subunit isoform, which is rich in N-glycans, along with the absence of the beta1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.
Vagin
Olga
O
Department of Physiology, School of Medicine at UCLA, Los Angeles, California 90073, USA. olgav@ucla.edu
Turdikulova
Shahlo
S
Sachs
George
G
D000251:Adenosine Triphosphatases / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
C503551:ATP1B1 protein, human; D001426:Bacterial Proteins; D051242:Caveolin 1; D018076:DNA, Complementary; D003902:Detergents; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D017830:Octoxynol; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase