Assembly of Na,K-ATPase alpha-subunit isoforms with Na,K-ATPase beta-subunit isoforms and H,K-ATPase beta-subunit.
cDNA encoding an epitope tag was joined to cDNAs encoding the chicken Na,K-ATPase beta 1 and beta 2 and H,K-ATPase beta-subunits to allow recognition of these beta-subunits with the same monoclonal antibody during assembly assays. cDNAs encoding chicken Na,K-ATPase alpha 1, alpha 2, or alpha 3 and Na,K-ATPase beta 1 or beta 2 or H,K-ATPase beta-subunits were transiently coexpressed in mammalian cells. Subunit assembly was assayed by immune precipitation of alpha-isoforms with a monoclonal antibody to the epitope-tagged beta-subunits. Each of the chicken alpha-isoforms assembled with each of the Na,K-ATPase beta-subunits and the H,K-ATPase beta-subunit. Each of the epitope-tagged beta-subunits also assembled with a Na,K-ATPase/Ca-ATPase chimera that retained only 26 amino acids of the Na,K-ATPase alpha-subunit, demonstrating that all three beta-subunits recognize this same alpha-subunit assembly site.
Lemas
M V
MV
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Yu
H Y
HY
Takeyasu
K
K
Kone
B
B
Fambrough
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002645:Chickens; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis; D016259:Genes, myc; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase