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.