SUBUNIT REQUIREMENTS FOR EXPRESSION OF FUNCTIONAL SODIUM PUMPS IN YEAST-CELLS

Na+/K+-ATPase from animal cell membranes is known to consist of an alp ha-subunit and a beta-subunit. Amino acids within the alpha-subunit ha ve 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-subu nits is required for the functional enzyme. A putative third subunit, the gamma-subunit, has been suggested to be a part of the functional N a+/K+-ATPase complex, based on experiments showing that both the catal ytic alpha-subunit and a small peptide of M(r) = 11000 can be labeled by a photoreactive ouabain analog. Although the primary structure for the putative gamma-subunit from rat and sheep was recently deduced fro m cDNA clones, participation of this small protein in the catalytic ac tivity 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 essentia l component of the Na+/K+-ATPase. Yeast cells do not contain an endoge nous 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-nitrop henylphosphatase, and Rb-86 uptake activities were measured either in membranes prepared from transformed yeast cells, or in intact yeast ce lls. Nontransformed yeast cells or yeast cells transformed with the ga mma-subunit alone served as controls. Northern analysis and Western bl ots demonstrated that yeast cells do not contain an endogenous peptide with significant sequence homology to the putative gamma-subunit. Yea st samples containing only Na+/K+-ATPase alpha and beta subunits were capable of ouabain-inhibitable enzymatic activity and Rb-86 transport. No gamma-subunit-dependent differences in the measured enzymatic acti vities or transport properties were detected in the different samples. These observations establish that the alpha beta-subunit complex is t he minimum structural unit required for all the ouabain-sensitive reac tions of Na+/K+-ATPase.