Most pathogenic mutations do not alter the membrane topology of the prion protein

The prion protein (PrP), a glycolipid-anchored membrane glycoprotein, contains a conserved hydrophobic sequence that can span the lipid bilayer in either direction, resulting in two transmembrane forms designated (PrP)-Pr-Ntm and (PrP)-Pr-Ctm. Previous studies have shown that the proportion of (PrP)-Pr-Ctm is increased by mutations in the membrane-spanning segment, and it has been hypothesized that (PrP)-Pr-Ctm represents a key intermediate in the pathway of prion-induced neurodegeneration, To further test this idea, we have surveyed a number of mutations associated with familial prion diseases to determine whether they alter the proportions of (PrP)-Pr-Ntm and (PrP)-Pr-Ctm produced in vitro, in transfected cells, and in transgenic mice. For the in vitro experiments, PrP mRNA was translated in the presence of murine thymoma microsomes which, in contrast to the canine pancreatic microsomes used in previous studies, are capable of efficient glycolipidation, We confirmed that mutations within or near the transmembrane domain enhance the formation of (PrP)-Pr-Ctm, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, thus exhibiting an unusual, dual mode of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts of (PrP)-Pr-Ctm and (PrP)-Pr-Ntm, arguing against the proposition that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases.