INTRACELLULAR-LOCALIZATION OF NA,K-ATPASE ALPHA-2 SUBUNIT MUTANTS

The Na,K-ATPase is an essential plasma membrane transporter of mammali an 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-ty pe alpha 2 and alpha 2L111R,N122D (alpha 2RD) subunits, The substituti ons, L111R and N122D, lower the affinity of the alpha 2 subunit for th e inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J, Biol. Chem. 266, 16925-16930, 1991) and were introduced in to the alpha 2 subunit to distinguish its enzymatic activity from that of the endogenous alpha 1 subunit of COS-7 cells. The S364P substitut ion is located in the ATP binding site, only five residues from the as partyl residue which is phosphorylated during the catalytic cycle of t he Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha 2RD subuni t, Despite the fact that S364P substitution does not block association of the alpha 2RD subunit with the endogenous beta 1 subunit, it preve nts the alpha 2 and alpha 2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticul um. (C) 1997 Academic Press.