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 constructed to seek the minimal region of the alpha-subunit sufficient for assembly with the Na,K-ATPase beta-subunit. cDNAs encoding a chimera and the chicken beta-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken beta-subunit. A chimera containing 26 amino acyl residues of the Na,K-ATPase alpha 1-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asn894 to Ala919) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr871 to Thr898) was able to assemble with the chicken beta-subunit. This alpha-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full-length beta-subunit also assembled with a beta-subunit chimera that retained only the ectodomain of the chicken beta 1-subunit. These results suggest that the Na,K-ATPase alpha-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 beta-subunit in the extracellular/lumenal space.
Lemas
M V
MV
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Hamrick
M
M
Takeyasu
K
K
Fambrough
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000096:biosynthesis / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase