An intrinsic membrane glycoprotein with cytosolically oriented N-linked sugars

Abstract

We demonstrate that the Na+-pump α-sub-unit 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 aparagine residues of the α-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 α 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.