The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (α) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the α subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat α1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitiope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the α subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.
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