Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform

After the cellular prion protein (PrP(c)) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrP(c) is either metabolized or converted into the scrapie isoform (PrP(Sc)). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrP(c) or the formation of PrP(Sc). The initial degradation of PrP(c) involves removal of the NH2 terminus of PrP(c) to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrP(Sc) and the initial degradation of PrP(c) were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrP(c) soluble in cold Triton X-100. Both CD4PrP(c) and truncated PrP(c) without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrP(Sc) formation. Thus, it seems likely that both the initial degradation of PrP(c) to the 17-kD polypeptide and the formation of PrP(Sc) occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrP(c) is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrP(Sc) are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrP(Sc) produces PrP 27-30.