Molecular requirements for the cell-surface expression of multisubunit ion-transporting ATPases. Identification of protein domains that participate in Na,K-ATPase and H,K-ATPase subunit assembly.
The ion-transporting H,K-ATPase and Na,K-ATPase enzymes are each composed of an alpha and a beta subunit. It is known that assembly of the alpha and beta subunits of the Na,K-ATPase is necessary for the cell-surface delivery of the active enzyme. We have examined the molecular domains involved in the assembly of the H,K-ATPase and Na,K-ATPase alpha and beta subunits by expressing individual subunits and subunit chimeras in transiently transfected COS-1 cells. Our results demonstrate that the H,K-ATPase alpha subunit requires its beta subunit for efficient cell-surface expression, as determined by indirect immunofluorescence. The H,K-ATPase beta protein appears to be able to get to the cell surface unaccompanied by any alpha subunit and appears to localize as well to a population of intracellular vesicles. We find that a transfected chimera encoding the NH2-terminal half of the H,K-ATPase alpha subunit and the COOH-terminal half of the Na,K-ATPase alpha subunit appears to assemble with the endogenous Na,K-ATPase beta subunit and to reach the plasmalemma. Transfection of the complementary alpha chimera requires coexpression with the H,K-ATPase beta subunit in order to attain surface delivery. Thus, it is the COOH-terminal half of the alpha subunit that specifies assembly with a particular beta subunit.
Gottardi
C J
CJ
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Caplan
M J
MJ
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D046911:Macromolecular Substances; D008961:Models, Structural; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D004247:DNA; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase