Synthesis and trafficking of prion proteins in cultured cells

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrP^Sc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN₂a) and hamster brain (ScHaB) cells synthesize PrP^Sc from the normal PrP isoform (PrP^C) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrP^Sc. Removal of BFA after the chase permitted synthesis of PrP^Sc to resume. BFA also blocked the export of nascent PrP^C to the cell surface but did not alter the distribution of intracellular deposits of PrP^Sc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrP^Sc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrP^Sc and that the synthesis of PrP occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrP^Sc.