CHOLESTEROL DEPLETION AND MODIFICATION OF COOH-TERMINAL TARGETING SEQUENCE OF THE PRION PROTEIN INHIBIT FORMATION OF THE SCRAPIE ISOFORM

After the cellular prion protein (PrPC) transits to the cell surface w here it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Bec ause most GPI-anchored proteins are associated with cholesterol-rich m embranous microdomains, we asked whether such structures participate i n the metabolism of PrPC or the formation of PrPSc. The initial degrad ation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fracti on. Both the formation of PrPSc and the initial degradation of PrPC we re diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polyp eptide did occur within an NH4Cl-sensitive, acidic compartment. Replac ing the GPI addition signal with the transmembrane and cytoplasmic dom ains of mouse CD4 rendered chimeric CD4PrP(C) soluble in cold Triton X -100. Both CD4PrP(C) and truncated PrPC without the GPI addition signa l (Rogers, M., F Yehieley, M. Scott, and S. B. Prusiner, 1993. Proc. N atl. Acad. Sci. USA, 90:3182-3186) were poor substrates for PrPSc form ation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a no n-acidic compartment bound by cholesterol-rich membranes, possibly gly colipid-rich microdomains, where the metabolic fate of PrPC is determi ned. The pathway remains to be identified by which the 17-kD polypepti de and PrPSc are transported to an acidic compartment, presumably endo somes, where the 17-kD polypeptide is hydrolyzed and limited proteolys is of PrPSc produces PrP 27-30.