Integrity of H1 helix in prion protein revealed by molecular dynamic simulations to be especially vulnerable to changes in the relative orientation of H1 and its S1 flank

In the template-assistance model, normal prion protein (PrPC), the pathogenic cause of prion diseases such as Creutzfeldt-Jakob in human, bovine spongiform encephalopathy in cow, and scrapie in sheep, converts to infectious prion (PrPSc) through an autocatalytic process triggered by a transient interaction between PrPC and PrPSc. Conventional studies suggest the S1-H1-S2 region in PrPC to be the template of S1-S2 β-sheet in PrPSc, and the conformational conversion of PrPC into PrPSc may involve an unfolding of H1 in PrPC and its refolding into the β-sheet in PrPSc. Here we conduct a series of simulation experiments to test the idea of transient interaction of the template-assistance model. We find that the integrity of H1 in PrPC is vulnerable to a transient interaction that alters the native dihedral angles at residue Asn¹⁴³, which connects the S1 flank to H1, but not to interactions that alter the internal structure of the S1 flank, nor to those that alter the relative orientation between H1 and the S2 flank.     

Functional assessment of surface loops: deletion of eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae

Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.     

Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone.

A conformational transition between the normal cellular prion protein (PrPC) and the beta-sheet-rich pathological isoform (PrPSc) is a central event in the pathogenesis of spongiform encephalopathies. The prion infectious agent seems to contain mainly, if not exclusively, PrPSc, which has the ability to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce the PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse PrPC with partially purified mouse PrPSc. After 48 h of incubation with a 10-fold molar excess of PrPSc, the cellular protein acquired PK-resistance resembling a PrPSc-like state. Time course experiments suggest that the conversion follows a stepwise mechanism involving kinetic intermediates. The conversion was induced by PrPSc extracted from mice infected with two different prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are present in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed under the same conditions using purified proteins without cell-lysate. However, when PrPC-depleted cell-lysate was added to the purified proteins the conversion was recovered. These findings provide direct evidence for the participation of a chaperone-like activity involved in catalyzing the conversion of PrPC into PrPSc.     

The deletion of amino acids 114-121 in the TM1 domain of mouse prion protein stabilizes its conformation but does not affect the overall structure

A mutant of mouse prion protein (PrPC) carrying a deletion of residues 114-121 (PrPDelta114-121) has previously been described to lack convertibility into the scrapie-associated isoform of PrP (PrPSc) and to exhibit a dominant-negative effect on the conversion of wild-type PrPC into PrPSc in living cells. Here we report the characterization of recombinantly expressed PrPDelta114-121 by Fourier-transformation infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. The analysis of spectra revealed an increased antiparallel beta-sheet content in the deletion mutant compared to wild-type PrPC. This additional short beta-sheet stabilized the fold of the mutant protein by DeltaDeltaG(0)'=3.4+/-0.3 kJ mol(-1) as shown by chemical unfolding experiments using guanidine hydrochloride. Secondary structure predictions suggest that the additional beta-sheet in PrPDelta114-121 is close to the antiparallel beta-sheet in PrPC. The high-affinity Cu2+-binding site outside the octarepeats, which is located close to the deletion and involves His110 as a ligand, was not affected, as detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting that Cu2+ binding does not contribute to the protection of PrPDelta114-121 from conversion into PrPSc. We propose that the deletion of residues 114-121 stabilizes the mutant protein. This stabilization most likely does not obstruct the interaction of PrPDelta114-121 with PrPSc but represents an energy barrier that blocks the conversion of PrPDelta114-121 into PrPSc.     

Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrPC/PrPSc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.     

正常和反常PRION分子PrP~C和PrP~(Sc)的空间结构模建和电子结构计算

朊蛋白（ＰＲＩＯＮ） 是一种不同于细菌,真菌,病毒的新的病原体。１９９６ 年英国“疯牛病”引起了对ＰＲＩＯＮ 研究的高潮。ＰＲＩＯＮ 分子生物学研究的关键是解释ＰＲＩＯＮ 蛋白构象转换的生物学本质。本文从构象模建,量子化学整体从头算的水平上对两个构象的电子结构进行了计算比较,从中发现了ＰＲＩＯＮ 蛋白分子电子结构上区别于其他分子的特异性, 并对计算所得的活性部位进行了讨论。     

Prions and prion proteins

Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unusual infectious pathogens called prions. There is no evidence for a nucleic acid in the prion, but diverse experimental results indicate that a host-derived protein called PrPSc is a component of the infectious particle. Experiments with scrapie-infected cultured cells show that PrPSc is derived from a normal cellular protein called PrPC through an unknown posttranslational process. We have analyzed the amino acid sequence and posttranslational modifications of PrPSc and its proteolytically truncated core PrP 27-30 to identify potential candidate modifications that could distinguish PrPSc from PrPC. The amino acid sequence of PrP 27-30 corresponds to that predicted from the gene and cDNA. Mass spectrometry of peptides derived from PrPSc has revealed numerous modifications including two N-linked carbohydrate moieties, removal of an amino-terminal signal sequence, and alternative COOH termini. Most molecules contain a glycosylinositol phospholipid (GPI) attached at Ser-231 that results in removal of 23 amino acids from the COOH terminus, whereas 15% of the protein molecules are truncated to end at Gly-228. The structure of the GPI from PrPSc has been analyzed and found to be novel, including the presence of sialic acid. Other experiments suggest that the N-linked oligosaccharides are not necessary for PrPSc formation. Although detailed comparison of PrPSc with PrPC is required, there is no obvious way in which any of the modifications might confer upon PrPSc its unusual physical properties and allow it to act as a component of the prion. If no chemical difference is found between PrPC and PrPSc, then the two isoforms of the prion protein may differ only in their conformations or by the presence of bound cellular components.     

正常和反常PRION分子PrP~C和PrP~(Sc)的空间结构模建和电子结构计算

朊蛋白（ＰＲＩＯＮ）是一种不同于细菌,真菌,病毒的新的病原体。１９９６年英国“疯牛病”引起了对ＰＲＩＯＮ研究的高潮。ＰＲＩＯＮ分子生物学的关键是解释ＰＲＩＯＮ蛋白构象转换的生物不本质。本文从构象模建,量子化学整体从头算的水平上对两个构象的电子结构进行了计算比较,从中发现了ＰＲＩＯＮ蛋白分子电子结构上区别于其他分子的特异性,并对计算所得的活性部位进行了讨论。     

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]

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.     

Cationic lipopolyamines induce degradation of PrPSc in scrapie-infected mouse neuroblastoma cells

In prion diseases the endogenous prion protein (PrPC) is converted into an abnormally folded isoform, denoted PrPSc, which represents the major component of infectious scrapie prions. The mechanism of the conversion is largely unknown, but the conversion is thought to occur after PrPC has reached the plasma membrane. Here we show that exogenous administration of the cationic lipopolyamine DOSPA interfered with the accumulation of PrPSc in scrapie-infected neuroblastoma cells. Structural analysis of the compounds tested revealed that inhibition of PrPSc was specific for lipids with a headgroup composed of the polyamine spermine and a quarternary ammonium ion between the headgroup and the lipophilic tail. The cationic lipopolyamine DOSPA induced the cellular degradation of preexisting PrPSc aggregates within 12 hours and interfered with the de novo synthesis of PrPSc. Biosynthesis of PrPC, or the assembly of sphingolipid-cholesterol microdomains (rafts) on the plasma membrane, were not affected by this inhibitor. After removal of DOSPA and replating into normal medium propagation of PrPSc commenced, although initially at a reduced rate. Incubation of ScN2a cells in free spermidine had no inhibitory effect on the accumulation of PrPSc. Our results indicate that membrane targeting of a small polyamine molecule creates a potent inhibitor of PrPSc propagation and offers the possibility to degrade preexisting PrPSc aggregates in living cells.     

Comparative computational analysis of prion proteins reveals two fragments with unusual structural properties and a pattern of increase in hydrophobicity associated with disease-promoting mutations

Prion diseases are a group of neurodegenerative disorders associated with conversion of a normal prion protein, PrPC, into a pathogenic conformation, PrPSc. The PrPSc is thought to promote the conversion of PrPC. The structure and stability of PrPC are well characterized, whereas little is known about the structure of PrPSc, what parts of PrPC undergo conformational transition, or how mutations facilitate this transition. We use a computational knowledge-based approach to analyze the intrinsic structural propensities of the C-terminal domain of PrP and gain insights into possible mechanisms of structural conversion. We compare the properties of PrP sequences to those of a PrP paralog, Doppel, and to the distributions of structural propensities observed in known protein structures from the Protein Data Bank. We show that the prion protein contains at least two sequence fragments with highly unusual intrinsic propensities, PrP(114-125) and helix B. No segments with unusual properties were found in Doppel protein, which is topologically identical to PrP but does not undergo structural rearrangements. Known disease-promoting PrP mutations form a statistically significant cluster in the region comprising helices B and C. Due to their unusual properties, PrP(114-125) and the C terminus of helix B may be considered as primary candidates for sites involved in conformational transition from PrPC to PrPSc. The results of our study also show that most PrP mutations associated with neurodegenerative disorders increase local hydrophobicity. We suggest that the observed increase in hydrophobicity may facilitate PrP-to-PrP or/and PrP-to-cofactor interactions, and thus promote structural conversion.     

Retrovirus infection strongly enhances scrapie infectivity release in cell culture

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.     

Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.     

Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.     

Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.     

The AGAAAAGA palindrome in PrP is required to generate a productive PrPSc-PrPC complex that leads to prion propagation

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.     

Purification and properties of the cellular and scrapie hamster prion proteins

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.     

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.     

The role of electrostatic interaction in triggering the unraveling of stable helix 1 in normal prion protein. A molecular dynamics simulation investigation

The conversion of normal prion protein (PrPC) into scrapie isoform (PrPSc) is a key event in the pathogenesis of prion diseases. However, the conversion mechanism has given rise to much controversy. For instance, there is much debate on the behavior of helix 1 (H1) in the conversion. A series of experiments demonstrated that H1 in isolated state was very stable under a variety of conditions. But, other experiments indicated that helices 2 and 3 rather than H1 were retained in PrPSc. In this paper, molecular dynamics (MD) simulation is employed to investigate the dynamic behavior of H1. It is revealed that although the helix 1 of Human PrPC (HuPrPC) is very stable in the isolated state, it becomes unstable when incorporated into native HuPrPC, which likely results from the long-range electrostatic interaction between Asp147 and Arg208 located in the helices 1 and 3, respectively. This explanation is supported by experimental evaluation and MD simulation on D147N mutant of HuPrPC that the mutant becomes a little more stable than the wild type HuPrPC. This finding not only help to reconcile the existing debate on the role of helix 1 in the PrPC-->PrPSc transition, but also reveals a possible mechanism for triggering the PrPC-->PrPSc conversion.     

Evidence for synthesis of scrapie prion proteins in the endocytic pathway.

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.