title: 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-terminal 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.
mesh_terms: D002462:Cell Membrane / Q000648:ultrastructure; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis; D006031:Glycosylation; D008565:Membrane Proteins; D011328:Prions; D011499:Protein Processing, Post-Translational
chemical_list: D006023:Glycoproteins; D008565:Membrane Proteins; D011328:Prions; D004247:DNA; D010450:Endopeptidases; D019286:Endopeptidase K