Expression of Functional Na⁺/K⁺-ATPase in Yeast

Abstract

Investigations of the structure and mechanism of the Na⁺/K⁺-ATPase (sodium pump) have been greatly facilitated by the determination of the amino acid sequences of the a and β subunits of the enzyme using recombinant DNA methodologies (13,18–20). The availability of cloned DNAs for different isoforms of each subunit has made it possible for investigators to transfect these DNAs into cells, either in their unmodified forms or in forms containing specific mutations, for subsequent analysis of the structural basis of pump activity. Several heterologous expression systems for Na⁺/K⁺-ATPase have been examined by different investigators, and certain experimental advantages may be attributed to each one of these. Because all animal cells contain endogenous Na⁺/K⁺-ATPase molecules that might contribute to the measured activity in the heterologous expression system, however, the choice of the most appropriate expression cell is not obvious. We had previously observed that the ATPase activity measured in membranes from the yeast Saccharomyces cerevisiae was not inhibited by ouabain and that no highaffinity binding sites for [³H]ouabain could be detected in yeast membranes. These observations indicated that yeast cells do not contain endogenous Na⁺/K⁺-ATPase. Therefore, in order to avoid potential problems that may arise from the heterologous expression of the Na⁺/K⁺-ATPase in the presence of endogenous pump subunits, an expression system for Na⁺/K⁺-ATPase was developed using the yeast Saccharomyces cerevisiae.