Cotranslational folding and calnexin binding during glycoprotein synthesis.
To analyze cotranslational folding of influenza hemagglutinin in the endoplasmic reticulum of live cells, we used short pulses of radiolabeling followed by immunoprecipitation and analysis with a two-dimensional SDS/polyacrylamide gel system which was nonreducing in the first dimension and reducing in the second. It separated nascent glycopolypeptides of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epitopes, N-linked glycosylation, and oligosaccharide-dependent binding of calnexin, a membrane-bound chaperone that binds to incompletely folded glycoproteins via partially glucose-trimmed oligosaccharides. When glycosylation or oligosaccharide trimming was inhibited, the folding pathway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin.
Chen
W
W
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510-8002, USA.
Helenius
J
J
Braakman
I
I
Helenius
A
A
D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037281:Calnexin; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004220:Disulfides / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015151:Immunoblotting; D009980:Influenza A virus / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry
D002135:Calcium-Binding Proteins; D004220:Disulfides; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D018832:Molecular Chaperones; D014759:Viral Envelope Proteins; D037281:Calnexin