Post-translational folding of influenza hemagglutinin in isolated endoplasmic reticulum-derived microsomes.
The folding of influenza hemagglutinin was analyzed after in vitro translation and translocation into dog pancreas microsomes. Ectodomain folding of this membrane glycoprotein involves the formation of six intrachain disulfide bonds. After translation under reducing conditions, the folding process was initiated by the addition of oxidized glutathione or diamide. For correct folding a reduction-oxidation potential of -310 to -210 mV had to be reached in the bulk solution. At lower values, or after addition of other oxidants such as NAD or NADP, no HA disulfides formed. At more oxidizing values interchain disulfide-cross-linked aggregates were generated. Judging by their electrophoretic gel mobility and immunoreactivity, the folding intermediates observed in microsomes were indistinguishable from those previously seen in the endoplasmic reticulum of live cells. The kinetics of folding was also similar, but the efficiency being 43% was somewhat lower. The folding process was dependent on lumenal factors within the rough endoplasmic reticulum vesicles and also on some macromolecular component(s) present in the reticulocyte lysate. The results showed that dog pancreas microsomes provide a useful system for protein folding studies.
Marquardt
T
T
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Hebert
D N
DN
Helenius
A
A
D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000378:metabolism; D010084:Oxidation-Reduction; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral