British Medical Bulletin

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (34) Request Permissions Disclaimer Google Scholar Articles by Harris, D. A Search for Related Content PubMed PubMed Citation Articles by Harris, D. A Related Collections Immunology Infectious Diseases Social Bookmarking          What's this?

British Medical Bulletin 66:71-85 (2003)
© 2003 Oxford University Press

Trafficking, turnover and membrane topology of PrP

Protein function in prion disease

David A Harris

Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, USA

	Abstract

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Cell biological studies of PrP have contributed enormously to our understanding of prion diseases. Like other membrane proteins, PrP^C is post-translationally processed in the endoplasmic reticulum and Golgi on its way to the cell surface after synthesis. Cell surface PrP^C constitutively cycles between the plasma membrane and early endosomes via a clathrin-dependent mechanism, a pathway consistent with a suggested role for PrP^C in cellular trafficking of copper ions. PrP molecules carrying mutations linked to inherited prion diseases display several abnormalities in their biochemical properties, maturation, and localisation that may explain their pathogenicity. Recent results have clarified the role of the proteasome in degradation of PrP, and the properties of a transmembrane form of PrP which may play a neurotoxic role in prion diseases.

	Introduction

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Prion diseases are rare neurodegenerative disorders that result from the conformational conversion of a normal cell-surface protein (PrP^C) into a protease-resistant, ß-sheet-rich form (PrP^Sc) that is infectious in the absence of nucleic acid. Understanding prion diseases at a mechanistic level requires insights into the cell biological features of PrP^C and PrP^Sc, including how these proteins are synthesised, localised, and metabolised in cells. A great deal is now known about the cell biology of PrP, and several of the most important facets will be reviewed here. This information bears on several outstanding questions in the prion field, including the following: (i) where in the cell does the critical conversion of PrP^C to PrP^Sc occur; (ii) what other cellular molecules play a role in the conversion process; (iii) how does prion propagation damage neurons; and (iv) what is the normal function of PrP^C?

	Cellular trafficking of PrPC

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Biosynthesis of PrP^C

Most PrP^C molecules are normally found on the cell surface where they are attached by virtue of a C-terminal glycosyl-phosphatidylinositol (GPI) anchor¹. The biosynthetic pathway followed by PrP^C is similar to that of other membrane and secreted proteins. PrP^C is synthesised in the rough endoplasmic reticulum (ER), and transits the Golgi on its way to the cell surface. During its biosynthesis in the ER, PrP^C is subject to several post-translational modifications, including cleavage of the N-terminal signal peptide, addition of N-linked oligosaccharide chains at two sites, formation of a single disulphide bond, and attachment of the GPI anchor following cleavage of the C-terminal hydrophobic peptide^1–3. The N-linked oligosaccharide chains added in the ER are of the high-mannose type and are sensitive to digestion by endoglycosidase H. These are subsequently modified in the Golgi to yield complex-type chains that contain sialic acid and are resistant to endoglycosidase H.

Endocytic trafficking of PrP^C

Not all PrP^C molecules remain on the cell surface after their delivery there. Some constitutively cycle between the plasma membrane and an endocytic compartment (Fig. 1). This pathway can be demonstrated by labelling PrP^C molecules on the cell surface with membrane-impermeant iodination or biotinylation reagents⁴. Kinetic analysis of these data demonstrates that PrP^C molecules cycle through the cell with a transit time of 60 min and, during each passage, 1–5% of the molecules undergo proteolytic cleavage near residue 110⁵. Experiments with PrP–GFP fusion proteins demonstrate that a internalised PrP^C co-localises with the endosomal markers transferrin and FM4-64⁶. The endocytic recycling pathway is of interest from the standpoint of prion generation, since there is evidence^7,8 that the initial steps in the conversion of PrP^C into PrP^Sc may take place on the plasma membrane or following internalisation of PrP^C.

View larger version (20K): [in this window] [in a new window]   Fig. 1 Cellular trafficking and cleavage of PrP. After reaching the cell surface, PrPC is internalised into an endocytic compartment from which most of the molecules are recycled intact to the cell surface. A small percentage of the endocytosed molecules are proteolytically cleaved near residue 110, and the N- and C-terminal cleavage products are then externalised. Some of the membrane-anchored protein is released into the extracellular medium by cleavage within the GPI anchor. Reprinted from Shyng et al.4

 
Clathrin-coated pits and vesicles appear to be the morphological structures primarily responsible for endocytic uptake of PrP^C. This conclusion is based on immunogold localisation of PrP^C in these organelles by electron microscopy^9–11, inhibition of PrP^C internalisation by incubation of cells in hypertonic sucrose which disrupts clathrin lattices⁹, and detection of PrP^C in purified preparations of coated vesicles from brain⁹. Particularly compelling evidence for involvement of clathrin has come from experiments in which cells were transfected to express a dominant negative mutant of dynamin I (K44A) that blocks fission of invaginated coated pits from the plasma membrane⁶. In these cells, PrP^C accumulated in structures beneath the plasma membrane that co-labelled with the endocytic marker FM4-64. The N-terminal half of the PrP^C polypeptide chain is essential for efficient clathrin-mediated endocytosis. Deletions within this region diminish internalisation of PrP^C, as assessed by surface labelling^12,13 and by microscopy of PrP–GFP fusion proteins¹⁴.

The involvement of clathrin-coated pits in endocytosis of PrP^C is unusual since GPI-anchored proteins, like PrP^C, lack a cytoplasmic domain that could interact directly with adapter proteins and clathrin. To explain the association of PrP^C with coated pits, we have postulated¹⁵ the existence of a ‘PrP^C receptor’, a transmembrane protein that has a coated-pit localisation signal in its cytoplasmic domain, and whose extracellular domain binds the N-terminal portion of PrP^C. The identity of the PrP^C receptor, if it exists, remains uncertain, although several candidate proteins have been proposed to be PrP-interacting partners^16,17.

PrP^C is associated with membrane rafts

Although PrP^C molecules undergoing endocytosis are associated with clathrin-coated pits, the majority of the protein in both neuronal and non-neuronal cells is found in detergent-resistant raft domains on the cell surface^18–20. Such domains, which are enriched in sphingolipids, other GPI-anchored proteins, and protein tyrosine kinases, are thought to represent foci for signal transduction events. Indeed, it has been suggested that PrP^C may serve as a signal transducing molecule that is activated by binding of an unidentified extracellular ligand²¹. It has been suggested²² that rafts may also be sites for the conversion of PrP^C to PrP^Sc. The fact that most PrP^C molecules reside in raft domains does not conflict with evidence for association of the protein with coated pits. At any given time, only a small fraction of the PrP^C molecules are undergoing endocytosis, and this subset has presumably left the raft domains to enter coated pits.

PrP^C and cellular trafficking of copper ions

Although the role of PrP^Sc in the disease process has been extensively studied, the normal physiological function of PrP^C has remained enigmatic. The endocytic recycling pathway followed by PrP^C (Fig. 1) suggests a role for the protein in cellular uptake or efflux of an extracellular ligand. One such ligand may be copper ions. PrP^C binds copper with low micromolar affinity, and in a pH-sensitive manner, via the N-terminal histidine-containing octapeptide repeats, and probably via more C-terminal regions of the protein as well^23–26. In addition, several other lines of evidence have emerged in the past few years suggesting a connection between PrP^C and copper metabolism²⁷.

A key observation is that copper dramatically stimulates endocytosis of PrP^C from the cell surface²⁸. This effect is temperature-dependent and is rapidly reversible (within minutes). It is seen with Zn²⁺ as well as Cu²⁺, but not with other transition metals. Copper-induced internalisation of PrP^C depends on the presence of the histidine-containing repeats^14,28,29, implying that the effect is due to binding of the metal to PrP^C rather to some other cellular protein that indirectly modulates endocytosis. PrP^C that is internalised in response to copper is delivered to early endosomes, a subset of Golgi compartments, and possibly to other organelles^6,14,30,31.

Based on the available data, one can envision at least two possible models for the role of PrP^C in the cellular trafficking of copper ions. In an uptake model, PrP^C on the plasma membrane binds Cu²⁺ via the peptide repeats, and then delivers the metal by endocytosis to an acidic, endosomal compartment. Copper ions then dissociate from PrP^C by virtue of the low endosomal pH and, after reduction to Cu⁺, are transported into the cytoplasm by a transmembrane transporter. PrP^C subsequently returns to the cell surface to bind additional copper, and the cycle is repeated. In a second model, PrP^C serves as a receptor that facilitates cellular efflux of copper via the secretory pathway. PrP^C is first delivered via endosomal vesicles to Golgi compartments, where it then serves to bind copper ions that have been pumped into the secretory pathway via the Menkes or Wilson proteins or other transporters.

	Cell biology of mutant PrP

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Familial prion diseases are linked to dominantly inherited mutations in the PrP gene on chromosome 20³². Point mutations in the C-terminal half of the protein are associated with either CJD, GSS, or FFI, while octapeptide insertions in the N-terminal half cause a mixed CJD–GSS phenotype. The presence of the mutation is presumed to favour spontaneous conversion of PrP to the PrP^Sc state without contact with exogenous prions. In order to understand how mutant PrP molecules cause neurological dysfunction in familial prion diseases, we have been analysing the biochemical properties, cellular localisation, and metabolism of these proteins in cultured cells.

Biochemical properties of mutant PrPs

We have constructed stably transfected lines of Chinese hamster ovary (CHO), baby hamster kidney (BHK) and PC12 cells that express murine homologues of mutant PrPs associated with all three familial prion diseases of humans^33–37. We find that each PrP carrying a pathogenic mutation displays three biochemical properties reminiscent of PrP^Sc. These properties include: (i) detergent-insolubility, manifested by sedimentation at 265,000 g from Triton/deoxycholate lysates; (ii) protease-resistance, evident by production of an N-terminally truncated core of 27–30 kDa after treatment with proteinase K; and (iii) PIPLC-resistance, defined by failure of phosphatidylinositol-specific phospholipase C (PIPLC) to release the protein from the cell surface, or to render it hydrophilic in biochemical assays. Several other laboratories have made similar observations^38–44. It thus seems likely that cell culture systems are modelling important features of the PrP^CPrP^Sc conversion process that occur in vivo.

Time course of changes in mutant PrP properties

Using pulse-chase labelling of cultured cells, we have identified three intermediate biochemical steps in the conversion of mutant PrPs to a PrP^Sc-like state⁴⁵. The earliest biochemical change in mutant PrP, which takes place in the ER simultaneously with, or immediately after, synthesis of the polypeptide chain, is the acquisition of PIPLC-resistance. PIPLC resistance reflects conformational alterations at the C-terminus of the protein that render the GPI anchor inaccessible to the phospholipase⁴⁶. The second step in the pathway is acquisition of detergent insolubility, which is not maximal until 1 h of chase³⁵, arguing that it occurs after the acquisition of PIPLC-resistance. Detergent insolubility reflects aggregation of PrP molecules. The third step is acquisition of protease resistance, which is not maximal until several hours after labelling³⁵. Detergent insolubility and protease resistance develop after arrival of the protein at the cell surface, either on the plasma membrane itself or in endocytic compartments. Taken together, the results of these kinetic experiments suggest that mutant PrP molecules begin to misfold early in the secretory pathway, probably while they are being synthesised in the ER. The misfolded proteins then begin to aggregate, with the oligomerisation process occurring over an extended period of time as the molecules reach the cell surface and beyond. Protease resistance develops gradually as the aggregates reach a size or tightness of subunit packing that is sufficient to render the protein inaccessible to protease (except in the region near residue 90).

Subcellular localisation of mutant PrP

We undertook a detailed study of the localisation of mutant PrPs in cultured cells using light and electron microscopic techniques³⁷. To visualise PrP on the cell surface, transfected cells expressing wild-type or mutant PrP were stained with anti-PrP antibody without permeabilisation (Plate IX, surface). Despite equivalent expression levels by Western blotting, virtually all cells synthesising mutant PrP showed much weaker surface staining than those expressing wild-type PrP. To visualise intracellular PrP, fixed cells were permeabilised with Triton X-100 prior to application of primary and secondary antibodies (Plate IX), internal). In cells expressing wild-type PrP, staining was restricted to the perinuclear Golgi, a localisation that is observed for other plasma membrane proteins, and that probably reflects relatively slow transit of secretory proteins through this compartment on their way to the cell surface. In contrast, many cells expressing mutant PrP showed a much more widespread pattern of staining that largely co-localised with the ER marker protein disulphide isomerase. We have observed an ER localisation for four different, disease-associated mutations (PG14, P101L, D177N/M128, and F197S/M128V), in CHO as well as BHK cells, and at both low and high expression levels. Ultrastructural studies, as well as experiments using PrP–EGFP fusion proteins, confirm these immunofluorescence results. Taken together, our results demonstrate that several different pathogenic mutations share the property of impairing delivery of PrP molecules to the plasma membrane, and causing their partial accumulation within the ER. Similar results have been obtained by several other laboratories^{39,42,47–49}.

View larger version (39K): [in this window] [in a new window]   Plate IX Mutant PrP is present at low levels on the cell surface, and is concentrated in the ER. Surface: BHK cells expressing wild-type PrP or PrP carrying a nine-octapeptide insertion linked to CJD (PG14) were stained with anti-PrP antibody prior to fixation. Internal: Cells expressing wild-type PrP (A–F) or PG14 PrP (G–L) were fixed, permeabilised, and stained with an antibody to PrP and an antibody to protein disulphide isomerase (PDI), an ER marker. Cells were viewed with green excitation/emission settings to detect PrP (A, D, G, J) and with red excitation/emission settings to detect PDI (B, E, H, K). Merged green and red images are shown in panels C, F, I, L. In the merged images, there is no yellow colour for wild-type PrP, demonstrating the absence of the protein from the ER. In contrast, PG14 PrP extensively co-localises with PDI, producing a yellow colour throughout the cytoplasm, except in a region near the nucleus which appears green due to the presence of the protein in the Golgi. Adapted from Ivanova et al.37

 
Delayed biosynthetic maturation of mutant PrP

To complement these cellular localisation studies, we have carried out a detailed analysis of the maturation and turnover of mutant PrP molecules using pulse-chase metabolic labelling⁵⁰. The key observation is that mutant PrPs become endoglycosidase H-resistant more slowly than wild-type PrP, both in transfected CHO cells and in cultured cerebellar granule neurons. These results indicate that the mutant proteins are delayed in their transit through the ER or early Golgi compartments. Mutant PrPs do not seem to be irreversibly retained in the ER, since a substantial percentage of the initially labelled protein (50–70%) eventually becomes endoglycosidase H-resistant, an efficiency that is similar to that for wild-type PrP. The delayed maturation of mutant PrP molecules correlates with their early acquisition of PIPLC-resistance, and likely reflects abnormal folding of the polypeptide chains in the ER. Slow transit through the early secretory pathway is also consistent with the altered distribution of mutant PrPs observed by immunofluorescence microscopy, since at steady state there will be fewer molecules on the cell surface and more in the ER. The combined results of localisation and biosynthetic studies, therefore, suggest that mutant PrP molecules are delayed in their export from the ER. This delay results in a steady-state distribution in which the proteins are concentrated in the ER, and are expressed at lower levels on the cell surface.

A number of inherited human diseases are attributable to defects in export of a mutant protein from the ER^51,52. In some cases, such as hereditary emphysema (PiZ variant) and congenital hypothyroidism, the retained protein accumulates in the ER without being degraded. In these cases, the disease phenotype is due to a toxic effect of the accumulated protein, which stimulates one or more ER stress response pathways. We hypothesise that some inherited prion disorders are members of this second category of ER retention diseases.

	Role of the proteasome in metabolism of PrP

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
PrP is not subject to proteasomal degradation following retrotranslocation

Secretory or membrane proteins that are retained in the ER are sometimes subject to a quality control process by which they are retrotranslocated into the cytoplasm and degraded by the proteasome^53,54. This mechanism is meant to ensure that abnormally folded proteins, or those that are not properly modified or assembled into multi-subunit complexes, do not reach the plasma membrane where they might cause cellular damage. We wondered whether this degradative process might operate on mutant PrP molecules during their protracted residence in the ER. Therefore, we tested the effect of proteasome inhibitors on the turnover of wild-type and mutant PrPs in CHO cells⁵⁰. Surprisingly, neither PSI 1 (Z-Ile-Glu(OtBu)-Ala-Leu-CHO) nor lactacystin had any significant effect on the half-lives or maturation kinetics of wild-type PrP or two different mutant PrPs. We conclude from our studies that, although mutant PrP molecules are delayed in their exit from the ER, they are not substrates for proteasomal degradation via a retrotranslocation pathway.

Untranslocated PrP is degraded by the proteasome

We have discovered that a small fraction of PrP molecules, both wild-type and mutant, is subject to proteasomal degradation in transfected cells, but by a pathway that does not involve retrotranslocation from the ER lumen⁵⁰. Proteasome inhibitors cause the selective accumulation of a form of PrP that is approximately 2 kDa larger than the mature, unglycosylated species (Fig. 2, lanes 2, 6, 10). This larger form, which is the only one that accumulates on Western blots after short-term (< 8 h) treatment of cells with proteasome inhibitors, is turned over rapidly (half-life, 30 min), and its half-life is significantly prolonged by the inhibitors. Our data strongly suggest that the larger, unglycosylated form represents PrP molecules that reside on the cytoplasmic face of the ER membrane, and that have never been translocated into the lumen for further processing. The most decisive observation is that this species reacts with an antibody that is specific for PrP molecules bearing an intact signal peptide (Fig. 2, lanes 4, 8, 12). This feature indicates that the protein has not been exposed to signal peptidase, which resides in the lumen of the ER. Taken together, our results indicate that a small fraction of PrP chains fails to be translocated into the ER lumen during its synthesis, and these molecules remain closely associated with the cytoplasmic face of the ER membrane where they are rapidly degraded by the proteasome. This phenomenon of abortive translocation is not unique to PrP, and is likely to reflect saturation of one or more components of the translocation machinery at the elevated expression levels typical of transfected cells.

View larger version (35K): [in this window] [in a new window]   Fig. 2 An unglycosylated, signal peptide-bearing form of PrP accumulates after treatment of CHO cells with a proteasome inhibitor. Transiently transfected CHO cells expressing wild-type PrP, or PrP carrying either of two disease-associated mutations (PG14, D177N) were treated for 8 h with either ethanol vehicle (– lanes) or with PSI 1 (20 µM) (+ lanes). Cells were then lysed, and PrP analysed by Western blotting using either 3F4 antibody (which recognises an epitope encompassing PrP residues 108–111), or anti-signal peptide antibody (-SP). The white and black arrowheads indicate the positions, respectively, of processed (signal peptide cleaved) and unprocessed (signal peptide-bearing) forms of unglycosylated PrP. These two species are not completely resolved for PG14 PrP because of the higher Mr of this protein. The slightly faster migration of all bands in lane 9 compared to those in lane 10 is an artefact of gel smiling. Reprinted from Drisaldi et al.50

 
Our results challenge several previously published studies on the turnover of PrP^55–58. These studies suggested that a major pathway for PrP turnover involved retrotranslocation from the ER followed by proteasomal degradation, and they proposed that cytosolic PrP may be a key pathogenic intermediate in many prion diseases. The interpretation of some of these experiments is complicated by artifactual effects produced by long-term treatment of cells with proteasome inhibitors⁵⁰.

	Transmembrane PrP

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Three topological forms of PrP

Most PrP^C molecules are attached to the outer leaflet of the plasma membrane through a C-terminal glycosyl-phosphatidylinositol anchor (this topology is designated ^SecPrP; see Fig. 3). However, when PrP is synthesised in vitro, in transfected cells or in mouse brain, some of the molecules assume a transmembrane orientation^59–66. These species, designated ^NtmPrP and ^CtmPrP, span the lipid bilayer once via a highly conserved hydrophobic region in the centre of the molecule (amino acids 111–134), with either the N-terminus or C-terminus, respectively, on the extracytoplasmic side of the membrane (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3 Three topological forms of PrP.

 
^NtmPrP and ^CtmPrP are generated in small amounts (< 10% of the total) as part of the normal biosynthesis of wild-type PrP in the endoplasmic reticulum. However, mutations within or near the transmembrane domain, including A117V and P105L mutations linked to GSS as well as several ‘artificial’ mutations not seen in human patients, increase the relative proportion of ^CtmPrP to as much as 20–30% of the total^{59,61,63–65}. Current evidence^61,64 indicates that the membrane topology of PrP is determined by competition at the translocon between two conflicting topological determinants in the polypeptide chain: (i) the signal sequence (residues 1–22) that directs translocation of the N-terminus of the polypeptide chain across the membrane to produce ^SecPrP or ^NtmPrP; and (ii) the central hydrophobic domain (residues 111–134) that acts as a type II signal-anchor sequence, directing translocation of the C-terminus across the membrane to produce ^CtmPrP.

A proposed pathogenic role of ^CtmPrP

It has been hypothesised that ^CtmPrP is a key pathogenic intermediate in both familial and infectiously acquired prion diseases. One piece of evidence for a role in familial forms comes from transgenic mice that synthesise PrP molecules carrying the A117V mutation, or one of the other ^CtmPrP-favouring mutations^59,63. Animals expressing these mutant proteins above a threshold level synthesise ^CtmPrP in their brains, and spontaneously develop a scrapie-like neurological illness but without PrP^Sc detectable by Western blotting or infectivity assays. Evidence for a role of ^CtmPrP in infectiously acquired prion diseases comes from mice in which a wild-type hamster PrP transgene serves as a reporter of ^CtmPrP formation⁶³. When these animals are inoculated with mouse prions, the amounts of ^CtmPrP as well as PrP^Sc in the brain are found to increase during the course of the infection. This result has been interpreted to indicate that PrP^Sc induces formation of ^CtmPrP, which is then the proximate cause of neurodegeneration. Thus, the amount of ^CtmPrP can be increased either directly by mutations in the PrP molecule, or indirectly via formation of PrP^Sc.

The cell biology of ^CtmPrP

To investigate further the hypothesis that ^CtmPrP plays an important role in the pathogenesis of prion diseases, it is necessary to characterise the cell biological properties of this form, since very little is known about its localisation, metabolism, or mode of synthesis and processing in cells. Part of the difficulty in addressing these issues has been that it was not possible to produce ^CtmPrP in the absence of the other two topological variants (^NtmPrP and ^SecPrP). We have overcome this limitation by identifying mutations in PrP that cause the protein to be synthesised exclusively with the ^CtmPrP topology.

The starting point for these studies was our discovery of a novel structural feature of ^CtmPrP that had not been previously appreciated – ^CtmPrP has an uncleaved, N-terminal signal peptide⁶⁶. This feature can be rationalised by the fact that the N-terminus of the polypeptide chain does not enter the ER lumen where signal peptidase is located. We reasoned that mutations in the signal peptide itself might influence the amount of ^CtmPrP. Consistent with this idea, we found that the substitution of a charged residue for a hydrophobic residue within the signal sequence (L9R) markedly increased the proportion of ^CtmPrP to 50% after in vitro translation. Combining this mutation with 3AV, a mutation within the transmembrane domain, to create L9R/3AV resulted in a protein that was synthesised exclusively as ^CtmPrP, in both in vitro translation reactions and transfected cells⁶⁶.

The availability of L9R/3AV PrP provided us for the first time the ability to analyse the properties of ^CtmPrP in a cellular context in the absence of the other two topological variants⁶⁶. By labelling cells expressing L9R/3AV PrP with [³H]-palmitate, we demonstrated that ^CtmPrP contains a GPI anchor. This result implies that ^CtmPrP has an unusual, dual mode of membrane attachment, including both a membrane-spanning domain and a C-terminal GPI anchor. We also found that L9R/3AV PrP (and hence ^CtmPrP) is absent from the cell surface, and is completely retained in the ER when expressed in transfected cells. This observation suggests the hypothesis that ^CtmPrP is toxic because it stimulates the activation of pro-apoptotic, ER stress-response pathways.

Most pathogenic mutations do not alter the membrane topology of PrP

Mutations associated with familial prion diseases are found throughout the length of the PrP sequence³². Although mutations in or around the central, hydrophobic region were known to increase the amount of ^CtmPrP, the effect of mutations outside of this area had not been examined. Therefore, we carried out in vitro translations of PrP mRNA encoding disease-associated mutations that lie both N- and C-terminal to the central, hydrophobic segment⁶⁵. We found that the proportion of ^CtmPrP was not increased over wild-type levels by any of the mutations outside of the central, hydrophobic domain. These results argue against the idea that ^CtmPrP is an obligate toxic intermediate in all forms of familial prion diseases.

	Conclusions

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
The cell biological results discussed here shed light on several outstanding problems in prion biology. First, elucidation of a clathrin-mediated endocytic pathway for PrP^C has suggested a possible function for the protein in the trafficking of copper ions. Second, studies of the metabolism of PrP molecules carrying pathogenic mutations has revealed that these molecules misfold soon after their synthesis and exit the ER more slowly than wild-type molecules. Nevertheless, neither mutant nor wild-type PrP is subject to retrotranslocation from the ER followed by proteasomal degradation. These observations suggest that accumulation of misfolded PrP in the ER lumen, rather than in the cytosol, is the likely pathogenic event in a subset of familial prion diseases. Finally, recent studies of ^CtmPrP have revealed several previously anticipated features of this isoform, including the presence of an uncleaved signal peptide and retention of the protein along the secretory pathway. Although ^CtmPrP is not elevated in all forms of prion disease, it may nevertheless be a key toxic intermediate in some. It seems certain that further studies of the cell biology of PrP will continue to enrich our understanding of these unusual neurodegenerative disorders.

	Acknowledgements

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Work in the Harris laboratory is supported by grants from the National Institutes of Health.

	Footnotes

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 
Correspondence to: Dr David A Harris, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 So. Euclid Ave, St Louis, MO 63110, USA

	References

Top Footnotes Abstract Introduction Cellular trafficking of PrPC Cell biology of mutant... Role of the proteasome... Transmembrane PrP Conclusions Acknowledgements References

 

1. Stahl N, Borchelt DR, Hsiao K, Prusiner SB. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 1987; 51: 229–49[CrossRef][Web of Science][Medline]
2. Turk E, Teplow DB, Hood LE, Prusiner SB. Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 1988; 176: 21–30[Web of Science][Medline]
3. Haraguchi T, Fisher S, Olofsson S et al. Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch Biochem Biophys 1989; 274: 1–13[CrossRef][Web of Science][Medline]
4. Shyng SL, Huber MT, Harris DA. A prion protein cycles between the cell surface and an endocytic compartment in cultured neuroblastoma cells. J Biol Chem 1993; 268: 15922–8[Abstract/Free Full Text]
5. Harris DA, Huber MT, van Dijken P, Shyng S-L, Chait BT, Wang R. Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry 1993; 32: 1009–16[CrossRef][Medline]
6. Magalhães AC, Silva JA, Lee KS et al. Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein. J Biol Chem 2002; 277: 33311–8[Abstract/Free Full Text]
7. Borchelt DR, Taraboulos A, Prusiner SB. Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J Biol Chem 1992; 267: 16188–99[Abstract/Free Full Text]
8. 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; 266: 18217–23[Abstract/Free Full Text]
9. Shyng SL, Heuser JE, Harris DA. A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J Cell Biol 1994; 125: 1239–50[Abstract/Free Full Text]
10. Madore N, Smith KL, Graham CH et al. Functionally different GPI proteins are organized in different domains on the neuronal surface. EMBO J 1999; 18: 6917–26[CrossRef][Web of Science][Medline]
11. Lainé J, Marc ME, Sy MS, Axelrad H. Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum. Eur J Neurosci 2001; 14: 47–56[CrossRef][Web of Science][Medline]
12. Shyng SL, Moulder KL, Lesko A, Harris DA. The N-terminal domain of a glycolipid-anchored prion protein is essential for its endocytosis via clathrin-coated pits. J Biol Chem 1995; 270: 14793–800[Abstract/Free Full Text]
13. Nunziante M, Gilch S, Schatzl HM. Essential role of the prion protein N-terminus in subcellular trafficking and half-life of PrP^C. J Biol Chem 2002; 10.1074/jbc.M206313200
14. Lee KS, Magalhaes AC, Zanata SM, Brentani RR, Martins VR, Prado MA. Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells. J Neurochem 2001; 79: 79–87[CrossRef][Web of Science][Medline]
15. Harris DA, Gorodinsky A, Lehmann S, Moulder K, Shyng S-L. Cell biology of the prion protein. Curr Top Microbiol Immunol 1996; 207: 77–93[Web of Science][Medline]
16. Gauczynski S, Peyrin JM, Haik S et al. The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein. EMBO J 2001; 20: 5863–75[CrossRef][Web of Science][Medline]
17. Zanata SM, Lopes MH, Mercadante AF et al. Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. EMBO J 2002; 21: 3307–16[CrossRef][Web of Science][Medline]
18. Gorodinsky A, Harris DA. Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J Cell Biol 1995; 129: 619–27[Abstract/Free Full Text]
19. Vey M, Pilkuhn S, Wille H et al. Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci USA 1996; 93: 14945–9[Abstract/Free Full Text]
20. Naslavsky N, Stein R, Yanai A, Friedlander G, Taraboulos A. Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem 1996; 272: 6324–31
21. Mouillet-Richard S, Ermonval M, Chebassier C et al. Signal transduction through prion protein. Science 2000; 289: 1925–8[Abstract/Free Full Text]
22. Taraboulos A, Scott M, Semenov A et al. Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J Cell Biol 1995; 129: 121–32[Abstract/Free Full Text]
23. Stöckel J, Safar J, Wallace AC, Cohen FE, Prusiner SB. Prion protein selectively binds copper(II) ions. Biochemistry 1998; 37: 7185–93[CrossRef][Medline]
24. Kramer ML, Kratzin HD, Schmidt B et al. Prion protein binds copper within the physiological concentration range. J Biol Chem 2001; 276: 16711–9[Abstract/Free Full Text]
25. Burns CS, Aronoff-Spencer E, Dunham CM et al. Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry 2002; 41: 3991–4001[CrossRef][Medline]
26. Jackson GS, Murray I, Hosszu LL et al. Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci USA 2001; 98: 8531–5[Abstract/Free Full Text]
27. Brown LR, Harris DA. The prion protein and copper: what is the connection? In: Massaro EJ. (ed) Handbook of Copper Pharmacology and Toxicology. Totowa: Humana, 2002; 103–13
28. Pauly PC, Harris DA. Copper stimulates endocytosis of the prion protein. J Biol Chem 1998; 273: 33107–10[Abstract/Free Full Text]
29. Perera WS, Hooper NM. Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region. Curr Biol 2001; 11: 519–23[CrossRef][Web of Science][Medline]
30. Brown LR, Harris DA. Copper and zinc cause delivery of the prion protein from the plasma membrane to a subset of early endosomes and the Golgi. J Neurochem 2003; In Press
31. Marella M, Lehmann S, Grassi J, Chabry J. Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release. J Biol Chem 2002; 277: 25457–64[Abstract/Free Full Text]
32. Young K, Piccardo P, Dlouhy S, Bugiani O, Tagliavini F, Ghetti B. The human genetic prion diseases. In: Harris DA. (ed) Prions: Molecular and Cellular Biology. Wymondham, UK: Horizon Scientific, 1999; 139–75
33. Lehmann S, Harris DA. A mutant prion protein displays an aberrant membrane association when expressed in cultured cells. J Biol Chem 1995; 270: 24589–97[Abstract/Free Full Text]
34. Lehmann S, Harris DA. Mutant and infectious prion proteins display common biochemical properties in cultured cells. J Biol Chem 1996; 271: 1633–7[Abstract/Free Full Text]
35. Lehmann S, Harris DA. Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform. Proc Natl Acad Sci USA 1996; 93: 5610–4[Abstract/Free Full Text]
36. Chiesa R, Harris DA. Nerve growth factor-induced differentiation does not alter the biochemical properties of a mutant prion protein expressed in PC12 cells. J Neurochem 2000; 75: 72–80[CrossRef][Web of Science][Medline]
37. Ivanova L, Barmada S, Kummer T, Harris DA. Mutant prion proteins are partially retained in the endoplasmic reticulum. J Biol Chem 2001; 276: 42409–21[Abstract/Free Full Text]
38. Priola SA, Chesebro B. Abnormal properties of prion protein with insertional mutations in different cell types. J Biol Chem 1998; 273: 11980–5[Abstract/Free Full Text]
39. Singh N, Zanusso G, Chen SG et al. Prion protein aggregation reverted by low temperature in transfected cells carrying a prion protein gene mutation. J Biol Chem 1997; 272: 28461–70[Abstract/Free Full Text]
40. Zanusso G, Petersen RB, Jin T et al. Proteasomal degradation and N-terminal protease resistance of the codon 145 mutant prion protein. J Biol Chem 1999; 274: 23396–404[Abstract/Free Full Text]
41. Lorenz H, Windl O, Kretzschmar HA. Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases. J Biol Chem 2002; 277: 8508–16[Abstract/Free Full Text]
42. Capellari S, Parchi P, Russo CM et al. Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain. Am J Pathol 2000; 157: 613–22[Abstract/Free Full Text]
43. Capellari S, Zaidi SI, Long AC, Kwon EE, Petersen RB. The Thr183Ala mutation, not the loss of the first glycosylation site, alters the physical properties of the prion protein. J Alzheimers Dis 2000; 2: 27–35[Medline]
44. Gauczynski S, Krasemann S, Bodemer W, Weiss S. Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance. J Cell Sci 2002; 115: 4025–36[Abstract/Free Full Text]
45. Daude N, Lehmann S, Harris DA. Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells. J Biol Chem 1997; 272: 11604–12[Abstract/Free Full Text]
46. Narwa R, Harris DA. Prion proteins carrying pathogenic mutations are resistant to phospholipase cleavage of their glycolipid anchors. Biochemistry 1999; 38: 8770–7[CrossRef][Medline]
47. Jin T, Gu Y, Zanusso G et al. The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome. J Biol Chem 2000; 275: 38699–704[Abstract/Free Full Text]
48. Petersen RB, Parchi P, Richardson SL, Urig CB, Gambetti P. Effect of the D178N mutation and the codon 129 polymorphism on the metabolism of the prion protein. J Biol Chem 1996; 271: 12661–8[Abstract/Free Full Text]
49. Negro A, Ballarin C, Bertoli A, Massimino ML, Sorgato MC. The metabolism and imaging in live cells of the bovine prion protein in its native form or carrying single amino acid substitutions. Mol Cell Neurosci 2001; 17: 521–38[CrossRef][Web of Science][Medline]
50. Drisaldi B, Stewart RS, Adles C et al. Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither mutant nor wild-type PrP undergoes retrotranslocation prior to proteasomal degradation. J Biol Chem 2003; 278: 21732–21743[Abstract/Free Full Text]
51. Aridor M, Balch WE. Integration of endoplasmic reticulum signalling in health and disease. Nat Med 1999; 5: 745–51[CrossRef][Web of Science][Medline]
52. Kim PS, Arvan P. Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr Rev 1998; 19: 173–202[Abstract/Free Full Text]
53. Tsai B, Ye Y, Rapoport TA. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 2002; 3: 246–55[CrossRef][Web of Science][Medline]
54. Bonifacino JS, Weissman AM. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev Biol 1998; 14: 19–57[CrossRef][Web of Science][Medline]
55. Ma J, Lindquist S. Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci USA 2001; 98: 14955–60[Abstract/Free Full Text]
56. Ma J, Wollmann R, Lindquist S. Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 2002; 298: 1781–5[Abstract/Free Full Text]
57. Ma J, Lindquist S. Conversion of PrP to a self-perpetuating PrP^Sc-like conformation in the cytosol. Science 2002; 298: 1785–8[Abstract/Free Full Text]
58. Yedidia Y, Horonchik L, Tzaban S, Yanai A, Taraboulos A. Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. EMBO J 2001; 20: 5383–91[CrossRef][Web of Science][Medline]
59. Hegde RS, Mastrianni JA, Scott MR et al. A transmembrane form of the prion protein in neurodegenerative disease. Science 1998; 279: 827–34[Abstract/Free Full Text]
60. Hölscher C, Bach UC, Dobberstein B. Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus. J Biol Chem 2001; 276: 13388–94[Abstract/Free Full Text]
61. Kim SJ, Rahbar R, Hegde RS. Combinatorial control of prion protein biogenesis by the signal sequence and transmembrane domain. J Biol Chem 2001; 276: 26132–40[Abstract/Free Full Text]
62. Rutkowski DT, Lingappa VR, Hegde RS. Substrate-specific regulation of the ribosome-translocon junction by N-terminal signal sequences. Proc Natl Acad Sci USA 2001; 98: 7823–8[Abstract/Free Full Text]
63. Hegde RS, Tremblay P, Groth D, DeArmond SJ, Prusiner SB, Lingappa VR. Transmissible and genetic prion diseases share a common pathway of neurodegeneration. Nature 1999; 402: 822–6[CrossRef][Medline]
64. Kim SJ, Hegde RS. Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel. Mol Biol Cell 2002; 13: 3775–86[Abstract/Free Full Text]
65. Stewart RS, Harris DA. Most pathogenic mutations do not alter the membrane topology of the prion protein. J Biol Chem 2001; 276: 2212–20[Abstract/Free Full Text]
66. Stewart RS, Drisaldi B, Harris DA. A transmembrane form of the prion protein contains an uncleaved signal peptide and is retained in the endoplasmic reticulum. Mol Biol Cell 2001; 12: 881–9[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				M. Jeffrey, C. Goodsir, G. McGovern, S. J. Barmada, A. Z. Medrano, and D. A. Harris Prion Protein with an Insertional Mutation Accumulates on Axonal and Dendritic Plasmalemma and Is Associated with Distinctive Ultrastructural Changes Am. J. Pathol., September 1, 2009; 175(3): 1208 - 1217. [Abstract] [Full Text] [PDF]

				S. Hornemann, C. von Schroetter, F. F. Damberger, and K. Wuthrich Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution J. Biol. Chem., August 21, 2009; 284(34): 22713 - 22721. [Abstract] [Full Text] [PDF]

				M. W. van der Kamp and V. Daggett The consequences of pathogenic mutations to the human prion protein Protein Eng. Des. Sel., August 1, 2009; 22(8): 461 - 468. [Abstract] [Full Text] [PDF]

				S. Paquet, N. Daude, M.-P. Courageot, J. Chapuis, H. Laude, and D. Vilette PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells J. Virol., October 1, 2007; 81(19): 10786 - 10791. [Abstract] [Full Text] [PDF]

				V. Campana, A. Caputo, D. Sarnataro, S. Paladino, S. Tivodar, and C. Zurzolo Characterization of the Properties and Trafficking of an Anchorless Form of the Prion Protein J. Biol. Chem., August 3, 2007; 282(31): 22747 - 22756. [Abstract] [Full Text] [PDF]

				E. Maas, M. Geissen, M. H. Groschup, R. Rost, T. Onodera, H. Schatzl, and I. M. Vorberg Scrapie Infection of Prion Protein-deficient Cell Line upon Ectopic Expression of Mutant Prion Proteins J. Biol. Chem., June 29, 2007; 282(26): 18702 - 18710. [Abstract] [Full Text] [PDF]

				C. Hetz, J. Castilla, and C. Soto Perturbation of Endoplasmic Reticulum Homeostasis Facilitates Prion Replication J. Biol. Chem., April 27, 2007; 282(17): 12725 - 12733. [Abstract] [Full Text] [PDF]

				C. R Trevitt and J. Collinge A systematic review of prion therapeutics in experimental models Brain, September 1, 2006; 129(9): 2241 - 2265. [Abstract] [Full Text] [PDF]

				V. Campana, D. Sarnataro, C. Fasano, P. Casanova, S. Paladino, and C. Zurzolo Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum J. Cell Sci., February 1, 2006; 119(3): 433 - 442. [Abstract] [Full Text] [PDF]

				E. Cancellotti, F. Wiseman, N. L. Tuzi, H. Baybutt, P. Monaghan, L. Aitchison, J. Simpson, and J. C. Manson Altered Glycosylated PrP Proteins Can Have Different Neuronal Trafficking in Brain but Do Not Acquire Scrapie-like Properties J. Biol. Chem., December 30, 2005; 280(52): 42909 - 42918. [Abstract] [Full Text] [PDF]

				E. Morel, T. Andrieu, F. Casagrande, S. Gauczynski, S. Weiss, J. Grassi, M. Rousset, D. Dormont, and J. Chambaz Bovine Prion Is Endocytosed by Human Enterocytes via the 37 kDa/67 kDa Laminin Receptor Am. J. Pathol., October 1, 2005; 167(4): 1033 - 1042. [Abstract] [Full Text] [PDF]

				A. Cardinale, I. Filesi, V. Vetrugno, M. Pocchiari, M.-S. Sy, and S. Biocca Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation J. Biol. Chem., January 7, 2005; 280(1): 685 - 694. [Abstract] [Full Text] [PDF]

				M. Moudjou, E. Treguer, H. Rezaei, E. Sabuncu, E. Neuendorf, M. H. Groschup, J. Grosclaude, and H. Laude Glycan-Controlled Epitopes of Prion Protein Include a Major Determinant of Susceptibility to Sheep Scrapie J. Virol., September 1, 2004; 78(17): 9270 - 9276. [Abstract] [Full Text] [PDF]

				S. Paquet, E. Sabuncu, J.-L. Delaunay, H. Laude, and D. Vilette Prion Infection of Epithelial Rov Cells Is a Polarized Event J. Virol., July 1, 2004; 78(13): 7148 - 7152. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (34) Request Permissions Disclaimer Google Scholar Articles by Harris, D. A Search for Related Content PubMed PubMed Citation Articles by Harris, D. A Related Collections Immunology Infectious Diseases Social Bookmarking          What's this?

Online ISSN 1471-8391 - Print ISSN 0007-1420

Copyright © 2010 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics