The highways and byways of prion protein trafficking

Review

TRENDS in Cell Biology

Vol.15 No.2 February 2005

The highways and byways of prion protein trafficking Vincenza Campana1,2, Daniela Sarnataro1 and Chiara Zurzolo1,2 1

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita` degli Studi di Napoli Federico II, 80131 Napoli, Italy 2 Unite´ de Trafic Membranaire et Pathoge´ne`se, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

Prions are defined as infectious agents that comprise only proteins and are responsible for transmissible spongiform encephalopathies (TSEs) – fatal neurodegenerative diseases that affect humans and other mammals and include Creutzfeldt–Jacob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. Prions have been proposed to arise from the conformational conversion of the cellular prion protein PrPC to a misfolded form termed PrPSc that precipitates into aggregates and fibrils. The conversion process might be triggered by interaction of the infectious form with the cellular form or it might result from a mutation in the gene encoding PrPC. Exactly how and where in the cell the interaction and the conversion of PrPC to PrPSc occur, however, remain controversial. Recent studies have shed light on the intracellular trafficking of PrPC, the role of protein missorting and the cellular factors that are thought to be required for the conformational conversion of prion proteins.

Introduction The prion or PrPSc is the causative agent of transmissible spongiform encephalopathies (TSEs) – neurodegenerative disorders that include scrapie in sheeps and goats, bovine spongiform encephalopathies, chronic wasting disease in deer and elk, and Creutzfeldt–Jakob disease in humans. The etiology of these pathologies can be infectious, sporadic or genetic. For each of them, the ‘protein-only’ hypothesis proposes that the prion is identical to a conformational isoform of the cellular prion protein PrPC [1]. PrPC is a cell-surface glycosylphosphatidylinositol (GPI)-anchored protein that is normally expressed in neurons, various non-neuronal tissues and leukocytes [2]. In comparison to PrPC, the PrPSc form has a higher content of b-sheet structure and is partially resistant to digestion with proteinase K [1]. In the most accredited model of prion formation and replication, a direct interaction between the pathogenic PrPSc template and the endogenous PrPC substrate is proposed to drive the formation of nascent infectious prions in the case of infectious diseases [1]. In genetic forms of TSEs, by contrast, alterations in PrPC conformation might be Corresponding author: Zurzolo, C. ([email protected]). Available online 6 January 2005

directly and spontaneously caused by a genetic mutation present in Prpn, the gene encoding PrPC [1,3]. It is likely that unknown cellular factors are necessary to generate the infectious form [1]; indeed, only recently has it been possible to generate the infectious agent in vitro [4]. The normal function of PrPC remains unknown, although its conservation in different species infers that it has some relevance in basic physiological processes. PrPC has been proposed to be involved in several functions: (i) copper and/or zinc ion transport or metabolism [5–7]; (ii) protection from oxidative stress [7]; (iii) cellular signaling [8,9]; (iv) membrane excitability and synaptic transmission [10,11]; (v) neuritogenesis [12]; and (vi) apoptosis [8,13]. These functions could be achieved through the interaction of PrPC with different partners, ligands and/or effectors, such as laminin, the chaperone BiP, glial fibrillary acidic protein (GFAP) and Bcl-2 (reviewed in Ref. [14]), at different locations in the cell (Table 1). Whether the impairment of one or more of these functions, or the gain of a new function by PrPSc, is related to the development of disease is not known [15,16]. Like other membrane proteins, PrPC is synthesized in the rough endoplasmic reticulum (ER) and travels through the Golgi apparatus to the plasma membrane (Figure 1). During its biosynthesis, PrPC undergoes several posttranslational modifications, including cleavage of the amino (N)-terminal signal peptide, addition of N-linked oligosaccharide chains, formation of a disulfide bond and attachment of a GPI anchor [3], which facilitates its association with specific lipid membrane domains called ‘rafts’ (Box 1). The precise localization of PrPC in neurons remains enigmatic, however, owing to conflicting data obtained by different techniques (Table 1). Whereas some data indicate that PrPC has predominantly a plasma membrane location – in particular in the axon, where it seems to have a preference for the synaptic membrane and shows no expression on synaptic vesicles or in the cytoplasm (discussed in Ref. [17]) – other data suggest that PrPC is present on all biosynthetic and endocytic transport membranous structures and in the cytosol of a subpopulation of neurons in the hippocampus, thalamus and neocortex [17] (Figure 1). Because of the lack of antibodies specific to PrPSc, the subcellular distribution of PrPSc has been extremely difficult to assess and a range of discordant studies indicate that it has a wide distribution, in particular at

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Table 1. Controversial issues in prion research: cellular distribution, transconformation site and interacting moleculesa Cellular compartment ER

Suggested role in transconformation? Yes [3,25,47]

PrP isoforms

Techniques

PrPC

Biochemistry [47,53]; IF after induction of retrotransport by BFA[47] and constitutively active Rab6 [25] Biochemistry [47,74]; IF [75]; EM [75]

PrP mutant or PrPSc-like conformer Golgi

Unknown

PrPC PrPSc PrP mutant or PrPSc-like conformer

Molecular partners in compartmentb BiP

BiP, CNX, CLT [47]

Biochemistry [47,53]; IF [33,47,75]; immunohistochemistry in brain [76] IF [77] Biochemistry, IF and EM [75]

Unknown Unknown Unknown

Endolysosomes

Yes [20,21,24,25]

PrPC PrPSc

EMc; immunofluorescence [33] EM [17,20,21]

Unknown Unknown

Exosomes

Yes [38]

PrPC PrPSc

EM [38] EM [38]

Unknown Unknown

Plasma membrane

Yes [18,19]

PrPC

Biochemistry [53]; IF [53]; biochemistry, immunohistochemistry on synaptosomes and ultrastructural study in cerebellumc Biochemistry [18]; EM [19] IF [75]

ST1, GAG, Laminin, Laminin R, Aplp1 Unknown Unknown

EM and biochemistry [34]d

Unknown

EM [35]; biochemistry [36,37]

Caveolin-1, CK1 CK2, N-CAM Unknown

PrPSc PrP mutant or PrPSc-like conformer Clathrin-coated pits Caveolae and CLD

Yes [34] Yes [35–37]

PrPC PrP

C

PrPSc C

Axonal or synaptic membrane

Unknown

PrP

Nucleus

Unknown

PrPSc

Cytosol

e

Yes

C

PrP PrP mutant or PrPSc-like conformer

Biochemistry [36] Free-floating immunohistochemistry, EM and biochemistryc

Unknown

IF and biochemistry [78]

Unknown

Quantitative EM [17] Biochemistry and IF [22,79]; immunohistochemistry [80]

Unknown Unknown

a

Abbreviations: BFA, brefeldin A; CK, casein kinase; CLT, calreticulin; CNX, calnexin; EM, electron microscopy; GAG, glycosaminoglycan; IF, immunofluorescence; N-CAM, neural cell adhesion molecule 1; ST1, stress-inducible protein 1. b Other proposed partners of prion proteins with undefined localization (reviewed in Ref. [14]) are Pint1, Bcl-2, nucleic acid, Nrf2 and Hsp60 (with PrPC); GFAP (with PrPC, PrPSclike conformers and PrPSc); plasminogen (with PrPSc extracellularly and in rafts); p75 (with PrPC fragment); synapsin 1b and Grb2 (with PrPC in intracellular vesicles); and reggie-1, lck and fyn (with PrPC in rafts) [81]. c Discussed in Ref. [17]. d Discussed in Ref. [3]. e Discussed in Refs [27,28].

Box 1. Membrane microdomains: rafts and detergent-resistant membranes The concept of rafts Building on the fluid-mosaic model proposed by Singer and Nicolson, later studies indicate that the plasma membrane is a mosaic of compartments maintained by the active cytoskeleton network. Rafts represent a membrane microdomain, wherein lipids of specific chemistry can dynamically associate with each other to form platforms that segregate specific membrane proteins [42,67]. Currently, there are several hypotheses concerning the nature of rafts (reviewed in Refs [41,67]). Simons and Ikonen [42] have proposed that rafts are relatively small structures (w50 nm) derived from the segregation of specific lipids. Alternatively, rafts have been viewed as lipid shells – small dynamic assemblies in which ‘raft’ proteins are preferentially associated with specific types of lipid [68]. We have recently proposed that raft formation can be regulated by the presence of core proteins and by the oligomeric status of low-affinity raft proteins [46,69].

Detergent-resistant membranes One of the main methods used to prove the existence of raft domains in cell membranes has been extraction by non-ionic detergents such as Triton X-100 [41,67]. It is not possible, however, to compare the www.sciencedirect.com

domains isolated by detergents with the raft structures that occur in native membranes, which are much more complex in terms of composition, thermodynamics and organization [41,67]. Thus, an association with detergent-resistant membranes can define only a biochemical characteristic of the molecule itself and cannot provide information regarding the organization of components in domains existing in living cells.

Rafts in living cells Other techniques have been recently developed to study lipiddependent organization in correlation with the cellular functions of putative raft components in living cell membranes [67]. Quantitative measurements of membrane dynamics are now possible by singleparticle tracking techniques, optical trapping by laser tweezers, fluorescence recovery after photobleaching and fluorescence resonance energy transfer (reviewed in Ref. [67]). These different approaches suggest that rafts range in size from a few molecules to 200 nm. Overall, the emerging concept is that rafts are likely to be small and dynamic entities in the resting state, which can coalesce in response to different stimuli into larger functional units [41,46,69].

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PrPC PrPSc Mutant PrP Misfolded PrP

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(ii)

Recycling endosome Raft (i)

Rab4 Rab5

(i) Aggregates (iv) Rab6

(iii) Early endosome

Golgi

Late endosome Lysosome ER

Proteasome

Exosome

TRENDS in Cell Biology C

Sc

Sc

C

Figure 1. Intracellular trafficking of PrP and PrP , and possible pathways of PrP formation. PrP is synthesized in the ER, in which it acquires posttranslational modifications and in which misfolded PrP and hereditary pathological mutants (mutant PrP) are partially degraded by the proteasome after retrotranslocation through the cytosol. PrPC has also been found in the neuronal cytosol and, under some experimental conditions, misfolded PrP aggregates are also present in the cytosol. After ER quality control, PrPC is transported through the Golgi apparatus to the cell surface, at which it associates with rafts and is internalized by clathrin- and/or caveolin-dependent mechanisms. Blocking transport of PrPC to the plasma membrane and rerouting it to lysosomes for degradation (i), and releasing nascent PrPC from the cell surface (ii) prevent the formation of PrPSc. A reduction in PrPC internalization (iii) also decreases PrPSc formation. Both PrP isoforms are found in Rab5-positive early endosomes, pass through late endosomes and are totally (PrPC) or partially (PrPSc) degraded in acidic lysosomes. Moreover, part of the PrPC pool is recycled back to the plasma membrane in a Rab4-dependent pathway, and both PrPC and PrPSc are found associated with exosomal membranes in the extracellular medium of infected cells. Finally, the ER has been postulated to have a role in prion conversion by amplifying PrPSc formation after the Rab6a-dependent retrograde transport of PrPC (iv).

the plasma membrane [18,19] and in the endolysosomal compartment [20,21] (Figure 1). Furthermore, in vitro studies show that under some conditions PrPSc-like conformers – that is, prion protein conformers that are not necessarily infective – accumulate in the cytosol of infected cells [22]. Characterizing the exact intracellular localization of PrPC and PrPSc is important for identifying the intracellular compartment and the mechanisms that underlie prion formation. The possibility that misfolding and/or misfunction of PrPC correlate with defects in its trafficking is supported by several studies in which the intracellular localization of some inherited pathological PrP mutants (prion protein molecules with a single or insertional mutation in the gene encoding PrPC) have been shown to be altered [3]. It is not yet clear, however, whether mislocalization is the cause or the effect of misfolding and/or misfunction. Consequently, it is important to understand the relationships between the intracellular trafficking, proper protein folding and function of PrPC (Figure 1). In this review, we analyze the controversial information gathered so far on the intracellular localization and transport of prion protein and discuss the role of these processes in the conformational conversion of PrPC to PrPSc. The ER sorting station One of the main issues in prion research is where PrPC– PrPSc transconformation – that is, the conformational www.sciencedirect.com

conversion of PrPC to PrPSc – occurs [3,23] (Figure 1). Formation of PrPSc could take place either at the plasma membrane, where the first contact between endogenous PrPC and exogenous PrPSc is likely to occur, or immediately after its internalization in the endolysosomal compartment [23,24]. Another possibility is that after its internalization PrPSc undergoes retrograde transport to the Golgi apparatus and/or to the ER, thereby perturbing the biosynthesis of newly synthesized prion protein and triggering the formation of the PrPSc isoform from the PrPC precursor. Recent findings have shown that stimulating the retrograde transport and accumulation of PrPC in the ER through overexpression of the constitutively active, small GTPase Rab6a increases the production of PrPSc in infected cells [25], which suggests that the ER might have a significant role in the conversion of PrPC. Such a role of the ER might arise because a high amount of substrate for the conversion reaction travels through this compartment. In addition, it is conceivable that it is easier to transconform the nascent polypeptide than to convert mature, correctly folded forms of the protein. Involvement of the ER in pathological conversion is also supported by earlier studies in which Harris and coworkers demonstrated that the pathological conversion of mutant PrP to PrPSc-like conformers proceeds in a stepwise manner via a series of identifiable biochemical intermediates (reviewed in Ref. [3]). Indeed, it was possible to show by pulse-chase experiments that

Review

TRENDS in Cell Biology

resistance to phosphatidylinositol phospholipase C was the earliest of three PrPSc-related properties to be acquired, being observed a few minutes after cell labeling. These experiments therefore indicated that acquisition of the initial scrapie-related features of mutant PrP occurred in the ER [3]. Furthermore, several studies have shown that the ER-associated degradation pathway is both involved in the degradation of pathogenic PrP mutants that are linked with familial prion diseases and responsible for the normal degradation pathway of misfolded PrP isoforms – that is, uncorrectly folded isoforms of PrPC ([26]; and reviewed in Refs [27,28]). Thus, the available data suggest that the ER has two possible roles in the transconformation of PrPC; first, in genetic prion diseases originating from mutant PrP, it might be directly involved in protein transconformation and consequent prion formation (reviewed in Ref. [3]); second, in infectious prion diseases, it might represent exclusively an amplification compartment for PrPSc produced earlier at other subcellular sites. These two potential roles of the ER reflect the possibility that the mechanism of PrPSc formation differs in the infectious and the genetic forms of the disease. Indeed, in the first model exogenous PrPSc functions as a catalyst for the conversion of endogenous PrPC, whereas in the second model mutant PrP molecules are spontaneously transformed into PrPSc. On the road to the plasma membrane Several studies indicate that the conversion of PrPC to a protease- and phospholipase-resistant state is, in contrast to the previously mentioned models, a posttranslational event that occurs after the protein reaches the cell surface [18,24,29]. Indeed, both the exposure of PrPC to antibodies to PrP and the release PrPC from the cell surface by different methods [18,24] prevent the formation of PrPSc (reviewed in Ref. [30]). Moreover, PrPSc formation is also reduced by preventing PrPC transport to the plasma membrane [31]. Why PrPC must reach the plasma membrane before its conversion is unknown. One possibility is that it acquires the posttranslational modifications that are necessary for its conversion after reaching the plasma membrane. Another is that PrPC functions as a receptor that mediates PrPSc internalization during infection. Alternatively, the lipid and protein environment at the plasma membrane might be favorable for the PrPC–PrPSc interaction and conversion, or a factor needed for the transconformation could be localized specifically at the plasma membrane [1]. Considering these hypotheses, we conclude that both the ER and the plasma membrane are important, but they might be differentially involved in prion formation. Indeed, most of the current data indicate that the first contact between the physiological and pathological forms of PrP in infectious prion diseases occurs at the plasma membrane, whereas the subsequent transconformation could occur either directly on the plasma membrane or after internalization (see below). In both situations, the ER could function in amplifying the PrPSc form after retrograde transport of PrPSc to the ER [25]. In genetic prion diseases, by contrast, the www.sciencedirect.com

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ER could represent the compartment in which mutant PrP molecules are spontaneously transformed to PrPSc or PrPSc-like conformers [3].

Transit through the endocytic compartment PrPC is constitutively internalized in cell cultures and recycles back to the surface [3,23]. The biological role of PrPC internalization is unknown, but internalization has been shown to be inducible by copper and zinc ions and thus could have a physiological function in chelating extracellular copper ions [5,6,32] or in modulating the signaling activity of the protein ([9]; and reviewed in Ref. [23]). It is not clear, however, whether the mechanism involved in copper-stimulated PrPC endocytosis is identical to that responsible for constitutive endocytosis of PrPC [5,32,33]. The mechanism of PrPC internalization is currently debated, and caveolae, rafts and clathrin-coated pits (Boxes 1,2 and Figure 2) have been shown to be involved (reviewed in Refs [3,23]). In one of the first studies of internalization, Harris and coworkers demonstrated that the chicken homolog of mammalian PrPC constitutively cycles between the cell surface and an endocytic compartment with a transit time of approximately 60 min (reviewed in Ref. [3]). This and other studies demonstrated that the N-terminal region of the protein is essential for both localization in coated pits and internalization [3,32]. A basic amino acid motif present in this region was subsequently shown to confer the ability to enter clathrin-coated pits [34]. This finding led to the proposal that PrPC binds through this N-terminal region to a transmembrane protein containing a localization signal for coated pits, causing it to enter the clathrindependent internalization pathway [34]. A candidate transmembrane protein is the laminin receptor, although apparently this receptor could be responsible for only 25–50% of all protein internalization (reviewed in Ref. [14]). Thus, PrPC internalization is likely either to involve interactions with other factors, possibly through a different domain of the protein (reviewed in Ref. [14]), or to occur via a non-clathrin-mediated pathway [14,35–37] (Box 2). Studies of the localization of PrPC in different types of cell indicate that it clusters in caveolae or caveolae-like domains (CLDs) [36,37]. In Chinese hamster ovary cells, which express caveolin-1, PrPC is enriched both in caveolae at the trans-Golgi Network and the plasma membrane, and in interconnecting chains of endocytic caveolae, but it is apparently absent in clathrin-coated pits and vesicles [35]. Thus, the initial recruitment of PrPC to pre-endocytic membranes might be a complex event that occurs by more than one mechanism (Figure 2). It is possible that PrPC is internalized by clathrin-coated vesicles by default and that caveolae or CLDs (Box 2) offer alternative internalization pathways in particular cells or under some conditions. These different mechanisms could provide a range of possibilities for protein conversion and pathological spread.

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Box 2. Multiple pathways for endocytosis Endocytosis of cell-surface components is achieved by different mechanisms, which are principally defined by their requirements for, or independence from, the membrane coat protein clathrin [70,71]. † Clathrin-mediated endocytosis represents a selective and efficient cellular mechanism of uptake and is important for various biological processes ranging from nutrient uptake to receptor signaling and synaptic vesicle recycling [70,71]. The assembly of clathrin and the heterotetrameric adaptor complex AP-2, which binds to both clathrin and cytoplasmic tyrosine-based signals on transmembrane proteins into progressively curved lattices, provides the driving force for generating coated pits and coated vesicles [71]. Clathrin-independent mechanisms are less well characterized; the best-understood system involves caveolin. † Caveolae are small flask-shaped invaginations at the cell surface that are decorated by a caveolin-1 coat and have been implicated in different processes, including endocytosis, transcytosis and regulating signaling cascades [72]. Because they contain specific lipids and

Role of the endocytic pathway in PrPC–PrPSc transconformation Accumulating evidence suggests that the endocytic pathway could be involved in the conversion of PrPC to PrPSc [24,29]. Formation of PrPSc is inhibited by lowering the temperature, which blocks the endocytosis and internalization of PrPC [24]. In addition, PrPSc is trimmed at its N terminus in an acidic compartment immediately after its synthesis and seems to accumulate in late endosomes [20,21,29]. Furthermore, because the expression of a dominant-negative version of the small GTPase Rab4, which inhibits plasma membrane recycling, increases the production of PrPSc in infected cells, it has been proposed

proteins typical of detergent-resistant membranes (Box 1), caveolae can be considered to be a specialized type of raft. It has been proposed that caveolae arise from the coalescence of small rafts driven by the oligomerization of caveolin-1, which forms the caveolar specialized coat [72]. † CLD is a broader definition of caveolae that is used to describe structures that show caveolar characteristics in terms of morphology and/or composition but do not possess the caveolin-1 coat. † GPI-anchored protein internalization pathways. Most GPI-anchored proteins are constitutively internalized through a pathway that is independent of clathrin and dynamin and dependent on rafts [73] (Box 1). By contrast, crosslinked GPI-anchored proteins are internalized via caveolae, which also represent a mechanism of endocytosis for different pathogens [58,73]. Finally, when interacting with transmembrane proteins possessing a signal sequence for internalization by coated pits, GPI-anchored proteins have been shown to enter the clathrin-dependent internalization pathway [73].

that PrPSc formation does not require cell-surface recycling and occurs in an intracellular compartment [25]. Notably, a recent report shows that both PrPC and PrPSc are released into the medium of infected cells in association with exosomes, indicating that one fate of the endosomal and lysosomal compartment containing PrPSc is exocytic fusion (Figure 1). Furthermore, PrPSc exosomes are infectious, suggesting that they might be involved in transferring infectivity from cell to cell [38]. Whether these organelles participate in the conversion of PrPC to PrPSc remains, however, to be demonstrated. Both CLDs and clathrin-coated pits have been proposed to be involved in PrPC–PrPSc transconformation (see below) [34–37], but PrPC Raft Clathrin

(i)

Caveolin

(ii)

Transmembrane receptor

Clathrincoated pit

Caveola

Dynamin Clathrin-coated vesicle Recycling endosome

Rab5 Early endosome

(iii)

Rab4

Non-clathrin, non-caveolin endocytosis

Caveosome ? Late endosome

Lysosome TRENDS in Cell Biology

Figure 2. Pathways of PrPC internalization. At the plasma membrane, PrPC can be constitutively internalized and its endocytosis can be increased by extracellular copper ions (not shown). A chief pathway of PrPC internalization in neuronal cells seems to depend on clathrin-mediated endocytosis (i). Caveolin-related endocytosis and trafficking have been implicated in PrPC transport in Chinese hamster ovary and glial cells (ii). Rab5-positive endosomes and recycling endosomes involving Rab4 have also been implicated in the endocytic transport of PrPC. Finally, non-clathrin and non-caveolin but raft-dependent endocytosis has been proposed to participate in the internalization and conversion of prion protein (iii). www.sciencedirect.com

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until the internalization pathway of PrPC is clarified it will be difficult to establish the involvement of one or the other pathway in transconformation. Interestingly, PG14, a mutant form of PrPC that contains nine additional octarepeats and is associated with familial prion diseases, does not undergo coppermediated endocytosis [39]. This observation indicates that the neurodegeneration associated with some forms of prion diseases might arise from impairment of endocytosis in response to metal ions caused by mutations in the N-terminal part of the protein. Indeed, N-terminal deletion mutants of PrPC, which are not internalized after stimulation with copper ions, cause neurological symptoms in PrPC knockout mice [32]. Another consideration is that impairment of endocytosis might have be involved in the pathogenesis of prion diseases because it could fail to switch off signaling through PrPC [8,9]. This hypothesis is supported by the recent finding that crosslinking of PrPC triggers neuronal apoptosis in mice [13]. Whether the endocytic pathway is involved in prion conversion and whether it has a role in regulating PrP function and/or the pathogenic effects of prions need further investigation. These issues seem to be particularly important in light of recent findings showing that misfolded and aggregated proteins that are responsible for other neurodegenerative disorders, including Huntington’s disease and corea acanthocytosis, cause impairment in the early steps of endocytosis in yeast and mammalian cells, leading to cell death [40]. These neurodegenerative diseases could therefore share a common pathogenetic pathway. Hitchhiking with rafts In addition to its intracellular location, the precise membrane subdomain (or ‘raft’) with which PrP associates seems to be important in the conversion process. Rafts have been proposed to have a central role in many cellular processes [41], including membrane sorting and trafficking [42], cell polarization [41,43] and signal transduction [44] (Box 1). Like other GPI-anchored proteins, PrPC is associated with rafts. The association of GPI-anchored proteins with rafts is generally a dynamic phenomenon, and the residency time of these proteins in rafts correlates well with their different routing through both the endocytic [45] and the exocytic [46] pathways. The association with rafts occurs earlier in the secretory pathway for PrPC than for other GPI-anchored proteins; thus, it might be necessary for the correct folding of PrPC in the ER ([47]; and see below). Nature of the interaction between PrPC and lipid rafts Differing from that of other GPI-anchored proteins, the raft association of PrPC is mediated by both its GPI anchor [37,48] and the N-terminal region of its ectodomain [49]. Furthermore, several independent studies have shown that the interaction of PrPC with model membranes, including sphingolipid–cholesterol-rich raft-like liposomes, can occur in a GPI-independent manner [49–52]. The observation that the raft association of PrPC is not important for its exocytic transport and sorting to the www.sciencedirect.com

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plasma membrane [53], in contrast to that of other GPIanchored proteins [42], suggests that this association could have an alternative role. Indeed, recent data from our laboratory [47] suggest that rafts have a role in PrPC maturation and folding. Specifically, PrPC and its main precursor associate with cholesterol-enriched rafts during synthesis in the ER. This association is required for correct protein folding, because perturbation by cholesterol depletion leads to misfolding [47]. As in the infectious process, cholesterol and sphingolipids seem to have different roles in PrPC maturation because only depletion of cholesterol slows down protein maturation and causes its misfolding. There are two explanations for the specific effect of cholesterol depletion on PrPC misfolding: either depletion of cholesterol leads to a perturbation in association of the immature PrPC form with specific detergent-resistant membranes in the ER, or cholesterol itself has a direct role in the PrPC folding by functioning as a lipochaperone in the ER [54,55]. Role of lipid rafts in PrPSc formation Many recent studies report the involvement of rafts in different types of pathology [56,57]. Furthermore, several groups of pathogens and parasites hijack lipid rafts during infection [56,58]. Like other pathogens, prions might therefore use rafts to enter the cells and possibly to initiate and/or propagate the PrPC–PrPSc transconformation process that facilitates prion amplification. A role for rafts in the formation of PrPSc is inferred from the finding that both PrPC and PrPSc are present in rafts extracted from infected cells and from mouse brain [29,48,52,59–61]. The PrPC- and PrPSc-associated rafts seem to have distinct characteristics, however, because they can be separated from each other by solubilization and flotation on density gradients [61]; this finding suggests either that the types of raft associated with each isoform differ (Figure 3) or that the membrane association of each isoform has distinct characteristics. Rafts have been further implicated in PrPC–PrPSc transconformation because removing PrPC from rafts by exchanging its GPI anchor for a transmembrane domain prevents the formation of PrPSc [37,48]. Conversely, impairing raft association with drugs that reduce intracellular levels of cholesterol decreases the formation of PrPSc in infected cells [48]. A chief issue is how rafts control the formation of PrPSc. Although the exact mechanisms are unknown, we can envisage three different models. First, rafts could be involved in targeting PrPC to the specific compartment in which PrPSc transconformation occurs (Figure 4a). We have previously shown in transfected cells that impairing the association of PrPC with rafts does not affect exocytic trafficking of PrPC to the plasma membrane [53]. By contrast, preliminary data (D. Sarnataro, unpublished) suggest that impairing PrPC raft association by cholesterol depletion slows down endocytotic trafficking of PrPC, suggesting that rafts could have a role in regulating the biosynthesis of PrPSc during endocytosis (see above). Second, rafts might contain machinery that is indispensable for PrPSc formation, such as proteins that facilitate

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(a)

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PrPC–PrPSc interaction in same rafts

Birth of PrPSc-specific rafts after PrPSc formation

(b)

PrPC and PrPSc interact with different rafts

Interaction induces raft coalescence and PrPSc formation

PrPC PrPSc PrPC-specific raft PrPSc-specific raft Non-raft membrane TRENDS in Cell Biology

Figure 3. Models of the association of PrPC and PrPSc with rafts. Some data suggest that PrPC and PrPSc associate with different types of raft – an observation that can be explained by two speculative models. (a) In the first, PrPSc interacts with PrPC inside rafts in which PrPC is normally located. Once bound to PrPC, PrPSc can destabilize this membrane environment, promoting both PrPC–PrPSc conversion and the birth of PrPSc-specific rafts. (b) In the second, PrPC and PrPSc associate with different rafts. The interaction between PrPC and PrPSc occurs after raft coalescence, when transconformation occurs to induce PrPSc-specific raft formation.

its conversion (Figure 4b). Third, rafts could provide a favorable environment for transconformation by facilitating close encounters between the substrate (PrPC) and the seed (PrPSc), by concentrating these molecules within confined stretches of the plasma membrane or by aligning them in a way that promotes their interaction (Figure 4c). The role of rafts as a ‘meeting place’ between PrPC and PrPSc is supported by the work of Baron et al. [60], who have shown that when purified rafts are used as a source of PrPC and brain microsomes from scrapie-infected mice are used as a source of PrPSc, transconformation does not occur in the absence of fusion of the PrPSc- and PrPCcontaining membranes [60]. These data suggest that the conversion process occurs when the two protagonists of the reaction are inserted into contiguous membranes. Role of lipid rafts in PrPC folding An alternative role for rafts is to enable the direct interaction of sphingolipids and/or cholesterol with PrPC and/or with PrPSc, which could affect the conformational stability of PrPC (Figure 4d). In this model, different raftresident lipids might function as molecular chaperones to facilitate the unfolding of one or more a-helices of PrPC and/or their refolding into b-sheets. Alternatively, a change in the local environment (in terms of enrichment in specific lipids and proteins) could mediate this process. The importance of the membrane environment in the conversion reaction has been underscored by several www.sciencedirect.com

studies in which specific lipids have been shown to have direct roles as chaperones in protein folding [54,55]. In particular, the binding of monosialogangloside GM1 to b-amyloid results in a conformational transition of b-amyloid to a b-sheet structure, supporting the idea that gangliosides might be important in modulating the amyloidogenic properties of b-amyloid [62]. In addition, processing of the amyloid precursor protein (APP) is regulated by its access to a- and/or b-secretases, which seems to depend on the dynamic interaction of APP with rafts [63,64]. Recombinant PrP can be refolded into an a-helical structure designated ‘a-PrP’ or into a b-sheet-enriched isoform known as ‘b-PrP’. Because the shift from an a-helix-enriched structure to a b-sheet-enriched form is the most evident result of PrPC–PrPSc transconformation, the binding of a-PrP and b-PrP to model lipid membranes has been recently investigated to determine the role of lipid rafts in this conformational transition [65]. a-PrP was found to bind with decreasing affinity to palmitoyloleoylphosphatidylglycerol, dipalmitoylphosphatidylcholine and raft membranes, suggesting that at steady state most PrPC is in non-raft membranes [65]. Other studies have shown, however, that binding to raft membranes results in the stabilization of a-helical structures, whereas interactions with negatively charged lipid (non-raft) membranes increases the content of b-sheet [51]. Thus, these data indicate that the binding

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(a) Trafficking vehicle

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(b) Transconformation machinery cointainer

Proteasome (c) PrPC and PrPSc meeting place PM ER

(i) Golgi

Lysosome

Recycling endosome

(ii)

Early Late endosome endosome (d) Membrane environment leading to transconformation PrPC PrPSc Misfolded PrP PrPC-specific raft PrPSc-specific raft PrP hybrid raft Non-raft membrane Protein X or lipochaperone TRENDS in Cell Biology

Figure 4. Potential roles of raft association in prion formation. Rafts could be implicated in different aspects of prion conversion as indicated by the models shown. (a) Rafts could be the vehicle of prion transport to an intracellular compartment, such as the plasma membrane (PM), endolysosomes, caveolae or ER, in which the transconformation occurs. (b) Rafts could contain the factors (protein X or lipid chaperones) comprising the transconformation machinery. (c) Rafts could be a platform on which PrPC accumulation occurs, functioning to promote the encounter between PrPC and PrPSc and to enhance the prion conversion reaction (i). Alternatively, rafts could be a point of accumulation for both PrPC and PrPSc, and the coalescence of these two specific prion rafts could initiate prion conversion (ii). (d) Specific raft domains could be involved in stabilizing the conformation of PrPC so that, when PrPC exits the rafts, it is misfolded and can interact better with PrPSc to undergo transconformation. This event could lead to the formation of specific PrPSc rafts (in accordance with the model in Figure 3b). Alternatively, fibrillization, not transconformation, might occur in PrPSc-specific rafts.

of PrP to raft-like membranes should induce the formation of an a-helical structure [51]. b-PrP, by contrast, was found to bind to all three types of membrane [65,66]. Whereas binding of b-PrP to raft membranes resulted in substantial unfolding of b-PrP and a delay in the process of fibril formation, b-PrP binding to negatively charged palmitoyloleoylphosphatidylglycerol (non-raft) membranes had a disruptive effect on the integrity of the bilayer, suggesting overall that the b-PrP isoform is destabilized by a raft environment [65]. Thus, the current evidence suggests that rafts have a protective role in the transconformation process. Binding of PrPC to raft-like membranes could induce folding of its unstructured N-terminal domain, thereby stabilizing the ‘normal’ PrP conformation. This ‘protective’ interaction would be destabilized when exogenous PrPSc is inserted in the vicinity of PrPC in the raft environment. Similarly, it has been suggested that PrPC is more susceptible to the conformational transition during its internalization because it has to leave the rafts to enter the coated pits [34]. This hypothesis is supported by the data of Fantini and colleagues [50], who have proposed that PrPC can maintain a non-pathological conformation by interacting with lipid rafts through a sphingolipid-binding domain (a V3-like domain) that is not functional in the E200K PrP mutant that undergoes PrP transconformation in familial www.sciencedirect.com

Creutzfeldt–Jacob disease. Notably, a similar sphingolipid-binding motif has been identified in the gp120 HIV glycoprotein and in the b-amyloid peptide implicated in Alzheimer’s disease, suggesting that lipid rafts might have a role in the pathogenesis of these different diseases. Concluding remarks In this review, we have examined the intracellular routing of PrPC and have discussed the possible intracellular sites of PrPC to PrPSc conversion. Although several reports have stressed the role of intracellular trafficking in PrP conversion, the intracellular conversion site remains unknown. We believe that analyzing the different steps involved in the intracellular transport of prion proteins and identifying the cellular site involved in their conformational changes will help to elucidate both the function of prion proteins and the mechanisms underlying prion formation. In particular, we have discussed the role of the ER and PrPC internalization in PrPC–PrPSc transconformation. We consider that the ER might have a principal role in the conversion of mutant PrP in genetic forms of disease, whereas transport of PrPC to the plasma membrane and its subsequent internalization seem to be required for conversion of PrPC in the infectious forms of disease. Furthermore, the precise membrane domains (rafts) with

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which PrPC and PrPSc are associated also seem to be important in the conversion process. Although the molecular determinants that control the interaction of PrP molecules with rafts need to be defined better, there are clear indications that rafts are important for stabilizing the conformation of PrPC and that only after PrPC exits these domains can encounters between PrPC and PrPSc promote pathological conversion. This point is particularly significant because rafts also seem to have a key role in the pathogenesis of other neurodegenerative diseases – in particular, Alzheimer’s disease. A more detailed understanding of lipid raft composition and the application of new methods, such as singleparticle tracking, fluorescence recovery after photobleaching and fluorescence resonance energy transfer, aimed at analyzing the specific location of PrP in living cells and revealing the molecular interactions with its putative partners and/or the structural changes that it undergoes should provide a better understanding of prion localization and function. In turn, this will lead to a fuller understanding of the pathogenesis of prion diseases and possibly to the development of new drugs for prevention and therapy.

Acknowledgements We thank Chris Bowler for critically reading the manuscript. Prion research in the Zurzolo laboratory is supported by European Union grants (HPRN-CT-2000–00077 and QLK-CT-2002–81628) and the WeizmannPasteur Foundation.

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