Prion diseases propagate by converting a normal glycoprotein of the host, PrP C , into a pathogenic prion' conformation. Several misfolding mutants of PrP C are degraded through the ER-associated degradation (ERAD)±proteasome pathway. In their infectious form, prion diseases such as bovine spongiform encephalopathy involve PrP C of wild-type sequence. In contrast to mutant PrP, wild-type PrP C was hitherto thought to be stable in the ER and thus immune to ERAD. Using proteasome inhibitors, we now show that~10% of nascent PrP C molecules are diverted into the ERAD pathway. Cells incubated with N-acetyl-leucinal-leucinal-norleucinal (ALLN), lactacystin or MG132 accumulated both detergent-soluble and insoluble PrP species. The insoluble fraction included an unglycosylated 26 kDa PrP species with a protease-resistant core, and a M r`l adder' that contained ubiquitylated PrP. Our results show for the ®rst time that wild-type PrP C molecules are subjected to ERAD, in the course of which they are dislocated into the cytosol and ubiquitylated. The presence of wild-type PrP molecules in the cytosol may have potential pathogenic implications.

Prions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrP C , into a ␤-sheet-rich conformer, PrP Sc . Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrP Sc ) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion "rods" are arguably the purest prion preparation available. The only proteinaceous component of rods is PrP Sc . Mouse neuroblastoma N2a, hypothalamus GT1-1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4°C) and internalized them (at 37°C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1-1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrP Sc synthesis and as a receptor for productive prion uptake.The transmissible spongiform encephalopathies that comprise infectious, familial, and sporadic neurodegenerations such as Creutzfeldt-Jakob disease of humans (1), scrapie of sheep, and bovine spongiform encephalopathy (2) are caused by prions (3). These proteinaceous agents are thought to propagate by refolding a normal cell surface glycoprotein of the host, the cellular prion protein (PrP C ) 1 , into an abnormal ␤-sheetrich (4, 5) conformation (reviewed in Ref. 6). The resulting pathological conformer, PrP Sc , is in turn the only known component of the infectious prion. The formation of PrP Sc is thought to involve a direct contact between "seed" PrP Sc and "substrate" PrP C (7, 8) and probably involves cellular cofactors (9) including the laminin receptors (10 -12) and cellular heparan sulfate proteoglycans (13-15).Although several cell lines are susceptible to prion infection (16) (reviewed in Ref. 17), the molecular mechanisms involved remain largely obscure. Infection is usually started by exposing cells to prion-infected material, such as brain homogenate. Many cell types (including cell lines (18) and primary dendritic cells (19)) can internalize prion-infected material, but the cellular receptors for prions have not been identified. One factor that is likely to complicate the study of how ...

During prion diseases, the host protein PrPC is refolded into an abnormal conformer "prion" PrP Sc . Histological and pharmacological data have suggested that glycosaminoglycans may be involved in the development of prion diseases. Here we present the first direct evidence that cellular glycosaminoglycans play a role in the biogenesis of PrP

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.

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