Intracellular localization of Na,K-ATPase alpha2 subunit mutants.
The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.
Coppi
M V
MV
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Guidotti
G
G
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D015153:Blotting, Western; D019556:COS Cells; D002458:Cell Fractionation; D004721:Endoplasmic Reticulum / Q000737:chemistry; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009975:Orthomyxoviridae / Q000737:chemistry / Q000235:genetics; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase