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

The prion protein (PrP), a glycolipid-anchored membrane glycoprotein, conta ins a conserved hydrophobic sequence that can span the lipid bilayer in eit her 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 th e pathway of prion-induced neurodegeneration, To further test this idea, we have surveyed a number of mutations associated with familial prion disease s 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. Fo r the in vitro experiments, PrP mRNA was translated in the presence of muri ne thymoma microsomes which, in contrast to the canine pancreatic microsome s used in previous studies, are capable of efficient glycolipidation, We co nfirmed 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 mutati ons 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 transmembran e PrP plays an obligate role in the pathogenesis of prion diseases.