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J Cell Biol. Apr 1, 1995; 129(1): 121–132.
PMCID: PMC2120366

Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform [published erratum appears in J Cell Biol 1995 Jul;130(2):501]

Abstract

After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderson RG, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. [PubMed]
  • Anderson RG. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10909–10913. [PMC free article] [PubMed]
  • Barry RA, Prusiner SB. Monoclonal antibodies to the cellular and scrapie prion proteins. J Infect Dis. 1986 Sep;154(3):518–521. [PubMed]
  • Barry RA, Vincent MT, Kent SB, Hood LE, Prusiner SB. Characterization of prion proteins with monospecific antisera to synthetic peptides. J Immunol. 1988 Feb 15;140(4):1188–1193. [PubMed]
  • Borchelt DR, Scott M, Taraboulos A, Stahl N, Prusiner SB. Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol. 1990 Mar;110(3):743–752. [PMC free article] [PubMed]
  • Borchelt DR, Taraboulos A, Prusiner SB. Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J Biol Chem. 1992 Aug 15;267(23):16188–16199. [PubMed]
  • Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. [PubMed]
  • Büeler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell. 1993 Jul 2;73(7):1339–1347. [PubMed]
  • Butler DA, Scott MR, Bockman JM, Borchelt DR, Taraboulos A, Hsiao KK, Kingsbury DT, Prusiner SB. Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol. 1988 May;62(5):1558–1564. [PMC free article] [PubMed]
  • Carlson GA, Ebeling C, Yang SL, Telling G, Torchia M, Groth D, Westaway D, DeArmond SJ, Prusiner SB. Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5690–5694. [PMC free article] [PubMed]
  • Caughey B, Raymond GJ. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem. 1991 Sep 25;266(27):18217–18223. [PubMed]
  • Caughey B, Race RE, Ernst D, Buchmeier MJ, Chesebro B. Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells. J Virol. 1989 Jan;63(1):175–181. [PMC free article] [PubMed]
  • Caughey B, Raymond GJ, Ernst D, Race RE. N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol. 1991 Dec;65(12):6597–6603. [PMC free article] [PubMed]
  • Cerneus DP, Ueffing E, Posthuma G, Strous GJ, van der Ende A. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol. J Biol Chem. 1993 Feb 15;268(5):3150–3155. [PubMed]
  • Chang WJ, Rothberg KG, Kamen BA, Anderson RG. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J Cell Biol. 1992 Jul;118(1):63–69. [PMC free article] [PubMed]
  • Cinek T, Horejsí V. The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol. 1992 Oct 1;149(7):2262–2270. [PubMed]
  • DeArmond SJ, Yang SL, Lee A, Bowler R, Taraboulos A, Groth D, Prusiner SB. Three scrapie prion isolates exhibit different accumulation patterns of the prion protein scrapie isoform. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6449–6453. [PMC free article] [PubMed]
  • Deddish PA, Skidgel RA, Kriho VB, Li XY, Becker RP, Erdös EG. Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. J Biol Chem. 1990 Sep 5;265(25):15083–15089. [PubMed]
  • Gabriel JM, Oesch B, Kretzschmar H, Scott M, Prusiner SB. Molecular cloning of a candidate chicken prion protein. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9097–9101. [PMC free article] [PubMed]
  • Gasset M, Baldwin MA, Lloyd DH, Gabriel JM, Holtzman DM, Cohen F, Fletterick R, Prusiner SB. Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10940–10944. [PMC free article] [PubMed]
  • Giloh H, Sedat JW. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. [PubMed]
  • Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. [PubMed]
  • Haraguchi T, Fisher S, Olofsson S, Endo T, Groth D, Tarentino A, Borchelt DR, Teplow D, Hood L, Burlingame A, et al. Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch Biochem Biophys. 1989 Oct;274(1):1–13. [PubMed]
  • Harris DA, Huber MT, van Dijken P, Shyng SL, Chait BT, Wang R. Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry. 1993 Feb 2;32(4):1009–1016. [PubMed]
  • Harris DA, Falls DL, Johnson FA, Fischbach GD. A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7664–7668. [PMC free article] [PubMed]
  • Heider JG, Boyett RL. The picomole determination of free and total cholesterol in cells in culture. J Lipid Res. 1978 May;19(4):514–518. [PubMed]
  • Hooper NM, Bashir A. Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. Biochem J. 1991 Dec 15;280(Pt 3):745–751. [PMC free article] [PubMed]
  • Hooper NM, Hryszko J, Turner AJ. Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme. Biochem J. 1990 Apr 15;267(2):509–515. [PMC free article] [PubMed]
  • Kascsak RJ, Rubenstein R, Merz PA, Tonna-DeMasi M, Fersko R, Carp RI, Wisniewski HM, Diringer H. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol. 1987 Dec;61(12):3688–3693. [PMC free article] [PubMed]
  • Keller GA, Siegel MW, Caras IW. Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways. EMBO J. 1992 Mar;11(3):863–874. [PMC free article] [PubMed]
  • Kitamoto T, Ohta M, Doh-ura K, Hitoshi S, Terao Y, Tateishi J. Novel missense variants of prion protein in Creutzfeldt-Jakob disease or Gerstmann-Sträussler syndrome. Biochem Biophys Res Commun. 1993 Mar 15;191(2):709–714. [PubMed]
  • Kocisko DA, Come JH, Priola SA, Chesebro B, Raymond GJ, Lansbury PT, Caughey B. Cell-free formation of protease-resistant prion protein. Nature. 1994 Aug 11;370(6489):471–474. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Langan TJ, Volpe JJ. Obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in glial primary cultures. J Neurochem. 1986 Apr;46(4):1283–1291. [PubMed]
  • Mayor S, Rothberg KG, Maxfield FR. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. [PubMed]
  • McKinley MP, Taraboulos A, Kenaga L, Serban D, Stieber A, DeArmond SJ, Prusiner SB, Gonatas N. Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells. Lab Invest. 1991 Dec;65(6):622–630. [PubMed]
  • Meyer RK, McKinley MP, Bowman KA, Braunfeld MB, Barry RA, Prusiner SB. Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2310–2314. [PMC free article] [PubMed]
  • Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, et al. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10962–10966. [PMC free article] [PubMed]
  • Pan KM, Stahl N, Prusiner SB. Purification and properties of the cellular prion protein from Syrian hamster brain. Protein Sci. 1992 Oct;1(10):1343–1352. [PMC free article] [PubMed]
  • Prusiner SB. Molecular biology of prion diseases. Science. 1991 Jun 14;252(5012):1515–1522. [PubMed]
  • Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10608–10612. [PMC free article] [PubMed]
  • Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983 Dec;35(2 Pt 1):349–358. [PubMed]
  • Prusiner SB, Scott M, Foster D, Pan KM, Groth D, Mirenda C, Torchia M, Yang SL, Serban D, Carlson GA, et al. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell. 1990 Nov 16;63(4):673–686. [PubMed]
  • Raeber AJ, Borchelt DR, Scott M, Prusiner SB. Attempts to convert the cellular prion protein into the scrapie isoform in cell-free systems. J Virol. 1992 Oct;66(10):6155–6163. [PMC free article] [PubMed]
  • Rogers M, Serban D, Gyuris T, Scott M, Torchia T, Prusiner SB. Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. J Immunol. 1991 Nov 15;147(10):3568–3574. [PubMed]
  • Rogers M, Yehiely F, Scott M, Prusiner SB. Conversion of truncated and elongated prion proteins into the scrapie isoform in cultured cells. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3182–3186. [PMC free article] [PubMed]
  • Rothberg KG, Ying YS, Kolhouse JF, Kamen BA, Anderson RG. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. [PMC free article] [PubMed]
  • Rothberg KG, Ying YS, Kamen BA, Anderson RG. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. [PMC free article] [PubMed]
  • Safar J, Roller PP, Gajdusek DC, Gibbs CJ., Jr Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J Biol Chem. 1993 Sep 25;268(27):20276–20284. [PubMed]
  • Scott MR, Köhler R, Foster D, Prusiner SB. Chimeric prion protein expression in cultured cells and transgenic mice. Protein Sci. 1992 Aug;1(8):986–997. [PMC free article] [PubMed]
  • Scott M, Groth D, Foster D, Torchia M, Yang SL, DeArmond SJ, Prusiner SB. Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell. 1993 Jun 4;73(5):979–988. [PubMed]
  • Serban D, Taraboulos A, DeArmond SJ, Prusiner SB. Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology. 1990 Jan;40(1):110–117. [PubMed]
  • Shyng SL, Heuser JE, Harris DA. A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J Cell Biol. 1994 Jun;125(6):1239–1250. [PMC free article] [PubMed]
  • Simons K, van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988 Aug 23;27(17):6197–6202. [PubMed]
  • Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry. 1993 Mar 2;32(8):1991–2002. [PubMed]
  • Stahl N, Borchelt DR, Hsiao K, Prusiner SB. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell. 1987 Oct 23;51(2):229–240. [PubMed]
  • Tagliavini F, Prelli F, Ghiso J, Bugiani O, Serban D, Prusiner SB, Farlow MR, Ghetti B, Frangione B. Amyloid protein of Gerstmann-Sträussler-Scheinker disease (Indiana kindred) is an 11 kd fragment of prion protein with an N-terminal glycine at codon 58. EMBO J. 1991 Mar;10(3):513–519. [PMC free article] [PubMed]
  • Taraboulos A, Raeber AJ, Borchelt DR, Serban D, Prusiner SB. Synthesis and trafficking of prion proteins in cultured cells. Mol Biol Cell. 1992 Aug;3(8):851–863. [PMC free article] [PubMed]
  • Taraboulos A, Rogers M, Borchelt DR, McKinley MP, Scott M, Serban D, Prusiner SB. Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8262–8266. [PMC free article] [PubMed]
  • Telling GC, Scott M, Hsiao KK, Foster D, Yang SL, Torchia M, Sidle KC, Collinge J, DeArmond SJ, Prusiner SB. Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9936–9940. [PMC free article] [PubMed]
  • Wandinger-Ness A, Bennett MK, Antony C, Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. [PMC free article] [PubMed]
  • Westaway D, Goodman PA, Mirenda CA, McKinley MP, Carlson GA, Prusiner SB. Distinct prion proteins in short and long scrapie incubation period mice. Cell. 1987 Nov 20;51(4):651–662. [PubMed]
  • Westaway D, DeArmond SJ, Cayetano-Canlas J, Groth D, Foster D, Yang SL, Torchia M, Carlson GA, Prusiner SB. Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion proteins. Cell. 1994 Jan 14;76(1):117–129. [PubMed]
  • Yanagishita M. Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. J Biol Chem. 1992 May 15;267(14):9505–9511. [PubMed]
  • Ying YS, Anderson RG, Rothberg KG. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins. Cold Spring Harb Symp Quant Biol. 1992;57:593–604. [PubMed]

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