Abstract
Various models of the transmembrane topology of the Na+,K+-ATPase predict either 8 or 10 membrane spans for the alpha-subunit and one to three membrane spans for the beta-subunit. Structure/function analysis, however, requires precise knowledge about the folding of enzymes. Therefore, the intention of this work was to establish a transmembrane topology model for the subunits of Na+,K+-ATPase. The bacterial enzyme beta-galactosidase was fused to the C termini of truncated alpha- and beta-subunits of Na+,K+-ATPase. Fusions were generated at Arg60 (LTTAR60), Glu116 (AATEE116), Ala247 (VEGTA247), Leu311 (YTWEL311), Ala444 (VAGDA444), Ala789 (IFIIA789), Met809 (LGTDM809), Asp884 (RVTWD884), Ile946 (MKNKI946), and Arg972 (GVALR972) of the sheep alpha1-subunit and at Pro236 (LGGYP236) of the dog beta-subunit. The fusion constructs were expressed in yeast cells for studies on the localization of the fused reporter enzyme. Activity measurements of the reporter enzyme revealed that only intracellular fusion sites lead to active beta-galactosidase. Indirect immunofluorescence microscopy with cells expressing alpha1/beta-galactosidase and beta/beta-galactosidase hybrid proteins demonstrated that inactive beta-galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side. The data obtained suggest that Pro236 of the beta-subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the alpha-subunit of the Na+,K+-ATPase consists of 10 membrane-associated spans. They also suggest that a stretch of the alpha1-subunit between membrane spans M7 and M8 might be hidden within the membrane, surrounded by the other hydrophobic spans, in analogy to the P-loop of Na+ or K+ channels and to the "hourglass" structure of water channels.
Highlights
Naϩ,Kϩ-ATPase is an enzyme embedded in plasma membranes of animal cells
This approach is most promising for the analysis of transmembrane topology, the results obtained can be contro
A reporter enzyme was fused at certain locations along the ␣1-subunit of sheep kidney Naϩ,Kϩ-ATPase, and its activity was localized after expression in the yeast S. cerevisiae
Summary
Naϩ,Kϩ-ATPase (sodium pump; EC 3.6.1.37) is an enzyme embedded in plasma membranes of animal cells. The amino-terminal third of the ␣-subunit transverses the membrane four times [9] This is followed by a large hydrophilic third of the protein that has been identified as the ATP-binding domain of the sodium pump. The detection of single amino acids on either the extracellular or intracellular surface of the plasma membrane was used together with the hydropathy data as a basis for the analysis of the transmembrane topology. Asn831 is the amino-terminal amino acid of a 19-kDa hydrophobic fragment derived from the ␣-subunit that is involved in Rbϩ occlusion [18, 19] This fragment can be generated when Naϩ,Kϩ-ATPase in inside-out vesicles is extensively digested by trypsin. This approach is most promising for the analysis of transmembrane topology, the results obtained can be contro-
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