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Volume 271, Number 46, Issue of November 15, 1996 pp. 29312-29320
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Transmembrane Topology of - and -Subunits of Na⁺,K⁺-ATPase Derived from -Galactosidase Fusion Proteins Expressed in Yeast

(Received for publication, February 2, 1996, and in revised form, August 8, 1996)

From the Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Federal Republic of Germany

Various models of the transmembrane topology of the Na⁺,K⁺-ATPase predict either 8 or 10 membrane spans for the -subunit and one to three membrane spans for the -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 -galactosidase was fused to the C termini of truncated - and -subunits of Na⁺,K⁺-ATPase. Fusions were generated at Arg⁶⁰ (LTTA⁶⁰), Glu¹¹⁶ (AATE¹¹⁶), Ala²⁴⁷ (VEGT²⁴⁷), Leu³¹¹ (YTWE³¹¹), Ala⁴⁴⁴ (VAGD⁴⁴⁴), Ala⁷⁸⁹ (IFII⁷⁸⁹), Met⁸⁰⁹ (LGTD⁸⁰⁹), Asp⁸⁸⁴ (RVTW⁸⁸⁴), Ile⁹⁴⁶ (MKNK⁹⁴⁶), and Arg⁹⁷² (GVAL⁹⁷²) of the sheep ₁-subunit and at Pro²³⁶ (LGGY²³⁶) of the dog -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 -galactosidase. Indirect immunofluorescence microscopy with cells expressing ₁/-galactosidase and /-galactosidase hybrid proteins demonstrated that inactive -galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side. The data obtained suggest that Pro²³⁶ of the -subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the -subunit of the Na⁺,K⁺-ATPase consists of 10 membrane-associated spans. They also suggest that a stretch of the 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.

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