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

Abstract

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 PrP 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 PrP 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.