POSTTRANSLATIONAL FOLDING OF INFLUENZA HEMAGGLUTININ IN ISOLATED ENDOPLASMIC RETICULUM-DERIVED MICROSOMES

The folding of influenza hemagglutinin was analyzed after in vitro tra nslation and translocation into dog pancreas microsomes. Ectodomain fo lding of this membrane glycoprotein involves the formation of six intr a-chain disulfide bonds. After translation under reducing conditions, the folding process was initiated by the addition of oxidized glutathi one or diamide. For correct folding a reduction-oxidation potential of -310 to -210 mV had to be reached in the bulk solution. At lower valu es, or after addition of other oxidants such as NAD or NADP, no HA dis ulfides formed. At more oxidizing values interchain disulfide-cross-li nked aggregates were generated. Judging by their electrophoretic gel m obility and immunoreactivity, the folding intermediates observed in mi crosomes were indistinguishable from those previously seen in the endo plasmic reticulum of live cells. The kinetics of folding was also simi lar, but the efficiency being 43% was somewhat lower. The folding proc ess was dependent on lumenal factors within the rough endoplasmic reti culum vesicles and also on some macromolecular component(s) present in the reticulocyte lysate. The results showed that dog pancreas microso mes provide a useful system for protein folding studies.