Intracellular Location Of Newly Synthesized Interferon In Human Fs-4 Cells

The distribution of interferon was examined in fractions of human FS-4 diploid fibroblasts induced with polyinosinate-polycytidylate [poly(I) · poly(C)]. In order to synchronize interferon production in the cultures, cells were first induced with poly(I) · poly(C) in the presence of the protein synthesis inhibitor, cycloheximide. After 4 hr, protein synthesis was allowed to resume by removing cycloheximide. Cells were harvested for the analysis of intracellular interferon at 20 min after the removal of cycloheximide, i.e., before substantial quantities of newly synthesized interferon could reach the extracellular environment. Much of the intracellular interferon present in the postnuclear supernatant (PNS) was associated with a membrane fraction that formed a single peak in sucrose density gradients. Treatment with pancreatic ribonuclease which abolished free polysomes did not result in a marked alteration of the distribution of interferon activity in the sucrose gradient, whereas treatment of the PNS with deoxycholate (0.5%) resulted in the release of all interferon activity from the membrane. Unlike free interferon, membrane-associated interferon was resistant to treatment with trypsin-chymotrypsin. Treatment with puromycin in high salt, which caused release on membrane-bound ribosomes, failed to remove interferon from the membrane fraction. These observations suggest that the membrane-associated interferon was contained inside membraneous vesicles. In addition to the interferon found in membrane-bound form, some interferon activity in the PNS of induced cells was present in free form. However, available evidence strongly suggests that this “free” interferon is an artifact due to the release of interferon from membraneous vesicles during the cell homogenization procedure. It is concluded that probably all interferon in this cell system is synthesized on membrane-bound polysomes, discharged into the lumina of the rough endoplasmic reticulum, and further processed during passage through various intracellular membrane compartments.