Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrP(Sc)) interacts with the substrate cellular PrP (PrP(C)) during conversion into nascent PeP(Sc). While PrP(Sc) appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrP(C) is converted into PeP(Sc). We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrP(C) and PrP(Sc). After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrP(C) (PrP(C)-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrP(C) and PrP(Sc) were found biotinylated in CLD fractions. Similar results on the colocalization of PrP(C) and PrP(Sc) were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrP(C) and PrP(Sc) are present in CLDs and, thus, support the hypothesis that the PrP(Sc) formation occurs within this subcellular compartment.