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Papers In Press, published online ahead of print October 17, 2005
Department of Physiology and Medicine, UCLA, Los Angeles, CA 900073
Corresponding Author: olgav{at}ucla.edu
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 a1 subunit was detected along with the ß2 subunit predominantly on the apical membrane, while the Na,K-ATPase ß1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a YFP-linked Na,K-ATPase ß1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous a1 subunit to the basolateral membrane. The human ß2 subunit has eight N-glycosylation sites, while the ß1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the ß2 subunit were successively introduced in the ß1 subunit by site-directed mutagenesis. The mutated ß1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant ß1 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 ß1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous ß2 subunit and the ß1 mutant with additional glycosylation sites but not for the wild type ß1 subunit. Hence, the N-glycans linked to the ß subunit isoforms of P2-type ATPases contain apical sorting information and the high abundance of the ß2 subunit isoform, which is rich in N-glycans, along with the absence of the ß1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.
J. Biol. Chem, 10.1074/jbc.M508262200
Submitted on July 28, 2005
Revised on October 12, 2005
Accepted on October 14, 2005
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
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