Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]

Abstract

Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino-or the carboxy-tertninal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

“…PrP contains a 22-residue N-terminal signal sequence, a potential transmembrane domain from residues 113 to 135, N-linked glycosylation sites at residues 181 and 197, and a C-terminal signal for glycolipid anchor addition (32). Analysis of early translocation intermediates of these chimeric constructs (at 91 maa, before synthesis of the transmembrane domain or glycosylation sites) revealed that, as with the other substrates analyzed, the nascent chain was well protected from PK when the pPrl signal sequence was used but poorly protected when the p␤L and pIgG signal sequences were used (Fig.…”

Synthesis and trafficking of prion proteins in cultured cells.

“…Because no scrapiespecific covalent modifications have been detected to date on PrP' (Prusiner, 1991;Stahl and Prusiner, 1991), it is unlikely that a missing cofactor functions in covalently modifying PrPSC. More likely, the conversion of PrP to PrPSc involves misfolding, aberrant membrane insertion (Hay et al, 1987), or aggregation with other molecules. It is possible that the ER-Golgi of BFA-treated cells are unable to synthesize de novo PrPSc, because this compartment lacks pre-existing PrP' that is needed to direct this process (Figure 2A).…”

Specific Features of the Prion Protein Transmembrane Domain Regulate Nascent Chain Orientation

“…The prion protein (PrP) can be synthesized in mul-tiple topological forms (15)(16)(17)(18). During co-translational translocation at the ER, some PrP nascent chains pass completely through the translocon to generate secretory PrP ( Sec PrP), others integrate into the lipid bilayer with their N terminus in the ER lumen ( Ntm PrP), and still others integrate in the opposite orientation ( Ctm PrP; see Fig.…”