26 AMINO-ACIDS OF AN EXTRACELLULAR DOMAIN OF THE NA,K-ATPASE ALPHA-SUBUNIT ARE SUFFICIENT FOR ASSEMBLY WITH THE NA,K-ATPASE BETA-SUBUNIT

Chimeric cDNAs encoding a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase alpha-subunit were constructe d to seek the minimal region of the alpha-subunit sufficient for assem bly with the Na,K-ATPase beta-subunit. cDNAs encoding a chimera and th e chicken beta-subunit were coexpressed in mammalian cells and assembl y was assayed by immune precipitation of the chimeric subunit with a m onoclonal antibody to the chicken beta-subunit. A chimera containing 2 6 amino acyl residues of the Na,K-ATPase alpha 1-subunit (NDVEDSYGQQWT FEQRKIVEFTCHTA) (Asn(894) to Ala(919)) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr(871) to Thr(898)) was able to assemble with the chicken beta-subunit. This alpha-subunit re gion is predicted to be extracellular, located between membrane-spanni ng domains 7 and 8 (H7-H8). Chimeras that assembled with full-length b eta-subunit also assembled with a beta-subunit chimera that retained o nly the ectodomain of the chicken beta 1-subunit. These results sugges t that the Na,K ATPase alpha-subunit has the same topology in the memb rane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membran e-spanning domains, and that the aminoacyl residues between membrane d omains H7 and H8 are involved in assembly with the beta-subunit in the extracellular/lumenal space.