Subunit requirements for expression of functional sodium pumps in yeast cells

Na+/K(+)-ATPase from animal cell membranes is known to consist of an alpha-subunit and a beta-subunit. Amino acids within the alpha-subunit have been shown to participate in the catalytic functions of the enzyme and in the binding of cardioactive steroids. Although the function of the beta-subunit is not known, expression of both alpha- and beta-subunits is required for the functional enzyme. A putative third subunit, the gamma-subunit, has been suggested to be a part of the functional Na+/K(+)-ATPase complex, based on experiments showing that both the catalytic alpha-subunit and a small peptide of M(r) = 11,000 can be labeled by a photoreactive ouabain analog. Although the primary structure for the putative gamma-subunit from rat and sheep was recently deduced from cDNA clones, participation of this small protein in the catalytic activity of the Na+/K(+)-ATPase has not been demonstrated. In experiments described here, the heterologous expression of Na+/K(+)-ATPase in yeast cells was used to investigate whether the gamma-subunit is an essential component of the Na+/K(+)-ATPase. Yeast cells do not contain an endogenous Na+/K(+)-ATPase. The alpha- and beta-subunits or the alpha-, beta- and the putative gamma-subunits of Na+/K(+)-ATPase were expressed in the yeast Saccharomyces cerevisiae and ouabain-sensitive ATPase, p-nitrophenylphosphatase, and 86Rb uptake activities were measured either in membranes prepared from transformed yeast cells, or in intact yeast cells. Nontransformed yeast cells or yeast cells transformed with the gamma-subunit alone served as controls. Northern analysis and Western blots demonstrated that yeast cells do not contain an endogenous peptide with significant sequence homology to the putative gamma-subunit. Yeast samples containing only Na+/K(+)-ATPase alpha and beta subunits were capable of ouabain-inhibitable enzymatic activity and 86Rb transport. No gamma-subunit-dependent differences in the measured enzymatic activities or transport properties were detected in the different samples. These observations establish that the alpha beta-subunit complex is the minimum structural unit required for all the ouabain-sensitive reactions of Na+/K(+)-ATPase.