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J. Biol. Chem., Vol. 280, Issue 52, 43159-43167, December 30, 2005

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Recombinant Addition of N-Glycosylation Sites to the Basolateral Na,K-ATPase ₁ Subunit Results in Its Clustering in Caveolae and Apical Sorting in HGT-1 Cells^*

From the Department of Physiology, School of Medicine at UCLA and the Department of Veterans Affairs Greater Los Angeles Health Care System, Los Angeles, California 90073

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 ₁ subunit was detected along with the ₂ subunit predominantly on the apical membrane, whereas the Na,K-ATPase 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 ₁ subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous ₁ subunit to the basolateral membrane. The human ₂ subunit has eight N-glycosylation sites, whereas the ₁ isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the ₂ subunit were successively introduced in the ₁ subunit by site-directed mutagenesis. The mutated ₁ subunits were detected on both apical and basolateral membranes. The fraction of a mutant 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 ₁ mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous ₂ subunit and the ₁ mutant with additional glycosylation sites but not for the wild type ₁ subunit. Hence, the N-glycans linked to the ₂ subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the ₂ subunit isoform, which is rich in N-glycans, along with the absence of the ₁ subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.

Received for publication, July 28, 2005 , and in revised form, October 12, 2005.

^* This work was supported in part by National Institutes of Health Grants DK46917, DK58333, and DK53462 and by the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: VAGLAHS/West LA, Bldg. 113, Rm. 324, 11301 Wilshire Blvd., Los Angeles, CA 90073. Tel.: 310-268-4672; Fax: 310-312-9478; E-mail: olgav{at}ucla.edu.

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