Residue Leu973 of the rat α1 subunit of the Na,K-ATPase is located on the cytoplasmic side of the plasma membrane

In a previous study (Yoon, K. L., and Guidotti, G., 1994 J. Biol. Chem. 269, 28249-28258), we indicated that the alpha subunit of the Na,K-ATPase has 4 transmembrane segments in the COOH terminal domain between residues Lys769 and Val939, and that both the NH2-terminus and the COOH-terminus are in the cytosol. However, there was insufficient information to determine whether there are more transmembrane segments between residues Val939 and the COOH-terminal Tyr1018. To investigate this question, we inserted the influenza virus hemagglutinin (HA)-epitope between Leu973 and Arg974 of the alpha 1 chain, expressed the construct in COS-7 and HeLa cells and determined the membrane arrangement by indirect immunofluorescence. The results indicate that Leu973 is not on the extracellular surface of the plasma membrane. Thus, the alpha 1 subunit is likely to possess only four complete transmembrane segments in the COOH terminal domain between residues Lys769 and Tyr1018. (C) 1998 Academic Press.