Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.
cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.
Renaud
K J
KJ
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Inman
E M
EM
Fambrough
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D002678:Chimera; D002872:Chromosome Deletion; D003593:Cytoplasm / Q000201:enzymology*; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011233:Precipitin Tests; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004247:DNA; D006026:Glycoside Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase