SUBCELLULAR COLOCALIZATION OF THE CELLULAR AND SCRAPIE PRION PROTEINSIN CAVEOLAE-LIKE MEMBRANOUS DOMAINS

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumula te primarily in lysosomes, caveolae-like domains (CLDs) have been sugg ested to be the site where PrPC is converted into PrPSc. We report her ein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cel ls contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Trit on X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes re sembling CLDs that were isolated by flotation in sucrose gradients. Si milar results were obtained when CLDs were purified from plasma membra nes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from r edistribution of proteins among subcellular fractions. The caveolar ma rkers ganglioside GM1 and H-ras were found concentrated in the CLD fra ctions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were f ound biotinylated in CLD fractions. Similar results on the colocalizat ion of PrPC and PrPSc were obtained when CLDs were isolated from Syria n hamster brains. Our findings demonstrate that both PrPC and PrPSc ar e present in CLDs and, thus, support the hypothesis that the PrPSc for mation occurs within this subcellular compartment.