An intrinsic membrane glycoprotein with cytosolically oriented n-linked sugars.
We demonstrate that the Na(+)-pump alpha-subunit polypeptide is glycosylated by using bovine milk galactosyltransferase, a specific enzyme which attaches galactose to terminal N-acetylglucosamine residues. The galactose acceptor sites are available for glycosylation only after permeabilization of right-side-out vesicles prepared from kidney outer medulla; therefore, the oligosaccharide moieties are facing the cytoplasm of the cell. We further show that the oligosaccharides are bound to asparagine residues of the alpha-subunit polypeptide, since the protein-carbohydrate linkage is hydrolyzed by peptide-N glycosidase F (an enzyme specific for N-linked sugars). Thus, the Na(+)-pump alpha subunit is a glycoprotein with its N-linked oligosaccharide moieties located at the cytosolic face of the cell membrane. Intrinsic membrane glycoproteins with such an oligosaccharide-protein linkage and cell membrane orientation have not been previously reported, to our knowledge.
Pedemonte
C H
CH
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
Sachs
G
G
Kaplan
J H
JH
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D006020:Glycopeptides / Q000302:isolation & purification; D006031:Glycosylation; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008892:Milk / Q000201:enzymology; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*
D006020:Glycopeptides; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D005700:Galactosyltransferases; D000254:Sodium-Potassium-Exchanging ATPase