GLYCOSYLATION IS ESSENTIAL FOR BIOSYNTHESIS OF FUNCTIONAL GASTRIC H-ATPASE IN INSECT CELLS(,K+)

The role of N-linked glycosylation in the functional properties of gas tric H+,K+-ATPase has been examined with tunicamycin and 1-deoxymannoj irimycin, inhibitors of glycoprotein biosynthesis and glycoprotein pro cessing respectively. Tunicamycin completely abolished both K+-stimula ted and )-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080) -sensitive ATPase activity and SCH 28080-sensitive phosphorylation cap acity. The expression level of both H+,K+-ATPase subunits remained una ffected. 1-Deoxymannojirimycin clearly affected the structure of the N -linked oligosaccharide moieties without affecting specific phosphoryl ation capacity. Purification of the functional recombinant enzyme from nonfunctional H+,K+-ATPase subunits coincided with purification of gl ycosylated beta-subunits and not of non-glycosylated beta-subunits. Tr ansport of the H+,K+-ATPase beta-subunit to the plasma membrane but no t its ability to assemble with the alpha-subunit depended on N-glycosy lation events. We conclude that the acquisition, but not the exact str ucture, of N-linked oligosaccharide moieties, is essential for biosynt hesis of functional gastric H+,K+-ATPase in insect cells.