Amino Acids of an Extracellular Domain of the Na , K - ATPase a - Subunit Are Sufficient for Assembly with the Na , K - ATPase p - Subunit

Chimeric cDNAs encoding a sarcoplasmiclendoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase a-subunit were constructed to seek the minimal region of the a-subunit sufficient for assembly with the Na,K-ATPase @-subunit. cDNAs encoding a chimera and the chicken @-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken @-subunit. A chimera containing 26 amino acyl residues of the Na,K-ATPase al-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asnss4 to Ala9lS) that replaced the corresponding avian SERCAl Ca-ATPase amino acyl residues (Thrs7I to Thrss8) was able to assemble with the chicken @-subunit. This a-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full-length @-subunit also assembled with a @-subunit chimera that retained only the ectodomain of the chicken @l-subunit. These results suggest that the Na,K-ATPase a-subunit has the same topology in the membrane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membrane-spanning domains, and that the aminoacyl residues between membrane domains H7 and H8 are involved in assembly with the /3-subunit in the extracellularflumenal space.