Transmembrane topology of alpha- and beta-subunits of Na+,K+-ATPase derived from beta-galactosidase fusion proteins expressed in yeast

Various models of the transmembrane topology of the Na+,KC-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 Arg(60) (LTTAR(60)), Glu(116) (AATEE(116)), Ala(247) (VEGTA(247)), Leu(311) (YTWEL(311)), Ala(444) (VAGDA(444)), Ala(789) (IFIIA(789)) Met(809) (LGTDM(809)), Asp(884) (RVTWD(884)), IIe(946) (MKNKI(946)), and Arg(972) (GVALR(972)) of the sheep alpha(1)-subunit and at Pro(236) (LGGYP(236)) 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 alpha(1)/beta-galacosidase 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 Pro(236) 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 alpha(1)-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.