COTRANSLATIONAL FOLDING AND CALNEXIN BINDING DURING GLYCOPROTEIN-SYNTHESIS

To analyze cotranslational folding of influenza hemagglutinin in the e ndoplasmic reticulum of live cells, we used short pulses of radiolabel ing followed by immunoprecipitation and analysis with a two-dimensiona l SDS/polyacrylamide gel system which was nonreducing in the first dim ension and reducing in the second, It separated nascent glycopolypepti des of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epi topes, N-linked glycosylation, and oligosaccharide-dependent binding o f calnexin, a membrane-bound chaperone that binds to incompletely fold ed glycoproteins via partially glucose-trimmed oligosaccharides, When glycosylation or oligosaccharide trimming was inhibited, the folding p athway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin.