Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form ((PrP)-Pr-Sec), two transmembrane forms ((PrP)-Pr-Ntm and (PrP)-Pr-Ctm), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, (PrP)-Pr-Ctm and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of (PrP)-Pr-Ctm. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of (PrP)-Pr-Ctm and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the (PrP)-Pr-Ctm topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases (PrP)-Pr-Ctm. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes (PrP)-Pr-Ctm and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of (PrP)-Pr-Ctm and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that (PrP)-Pr-Ctm and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.