Studies on the membrane topology of the (Na,K)-ATPase.
The topology of the alpha 1 and beta 1 subunits of the rat (Na,K)-ATPase has been studied by insertion of epitope(s): at the NH2 terminus and COOH terminus and between Glu-117 and Glu-118, Lys-828 and Arg-829, Gln-900 and Trp-901, and Val-939 and Phe-940 of the alpha subunit; and at the NH2 terminus and COOH terminus and between Glu-228 and Tyr-229 of the beta subunit. The epitope-tagged alpha 1 constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na,K)-ATPase. The epitope-tagged beta constructs were transiently expressed in Cos-7 cells. The membrane arrangement of the epitopes was revealed by indirect immunofluorescence with cells expressing the (Na,K)-ATPase chains. The results indicate that the alpha subunit has 4 transmembrane segments in the COOH-terminal membrane-bound domain between residues 760 and 938, and that both the NH2 terminus and the COOH terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH terminus. The beta subunit has only one transmembrane-spanning region with the NH2 terminus in the cytosol and the COOH terminus on the extracytoplasmic surface of the plasma membrane.
Yoon
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KL
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Guidotti
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000737:chemistry*; D014162:Transfection
D017931:DNA Primers; D000939:Epitopes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase