(Received for publication, August 13, 1996, and in revised form, December 24, 1996)
From the Department of Molecular Biology, the Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel
Cells infected with prions contain both prion
protein isoforms cellular prion protein (PrPC) and scrapie
prion protein (PrPSc). PrPSc is formed
posttranslationally through the pathological refolding of
PrPC. In scrapie-infected ScN2a cells, the metabolism of
both PrP isoforms involves cholesterol-dependent pathways.
We show here that both PrPC and PrPSc are
attached to Triton X-100-insoluble, low-density complexes or
"rafts." These complexes are sensitive to saponin and thus probably
contain cholesterol. This finding suggests that the transformation PrPC PrPSc occurs within rafts. It also
reveals the existence of rafts in late compartments of the endocytic
pathway, where most PrPSc resides. When Triton X-100
lysates of cells were incubated at 37 °C prior to density analysis,
PrPC was still found in buoyant complexes, although it now
failed to sediment at high speed. This property was shared by another glycophosphatidyl inositol protein, Thy-1, and also by the raft resident GM1. In one ScN2a clone and in the brain of a Syrian hamster
with scrapie, Triton X-100 extraction at 37 °C permitted resolution
of PrPC and PrPSc into two distinct peaks of
different densities. This suggests that there are two populations of
PrP-containing rafts and may permit isolation of
PrPC-specific rafts from those containing
PrPSc. Our findings reinforce the contention that rafts are
involved in various aspects of PrP metabolism and in the "life
cycle" of prions.
Prions are unique proteinaceous pathogens that cause a series of
fatal encephalopathies such as Creutzfeldt-Jakob disease of humans,
scrapie of sheep, and bovine spongiform encephalopathy (1). Prions seem
to propagate in the host by posttranslationally (2, 3) refolding a
normal host protein, the cellular prion protein
(PrPC),1 to an aberrant
conformation (4, 5). The only known component of prions is the
misfolded isoform of PrPC, the scrapie prion protein
(PrPSc) (6, 7). Current evidence argues that direct
interaction of PrPSc with PrPC is a
prerequisite for the transformation PrPC + PrPSc 2PrPSc (8, 9). PrPC is a
phosphoinositol glycolipid (GPI)-anchored glycoprotein present on the
surface of neurons and other cells (10, 11). The PrP isoforms appear to
be chemically identical (12) but differ in their conformation (4);
PrPC contains ~40%
-helix and is devoid of
-sheet,
whereas PrPSc has more than 40%
-sheet (4, 13-16). The
two PrP isoforms differ considerably in their properties;
PrPC is readily soluble in most detergents and is
completely degraded by proteases, whereas PrPSc is
insoluble in detergents, possesses a protease-resistant core termed
PrP27-30, and polymerizes into amyloidic structures called prion rods
(17, 18). Since no isoform-specific PrP antibody has yet been
developed, the disparate properties of PrPC and
PrPSc serve as the sole ways to differentiate
experimentally between these proteins.
The subcellular sites where PrPSc is formed, and the trafficking pathways leading to these sites, remain largely unknown. Scrapie-infected mouse neuroblastoma ScN2a cells synthesize both PrPC and PrPSc, whereas only PrPC is found in uninfected cells (2, 19, 20). Like other GPI proteins, most PrPC is found in cholesterol-rich, detergent-resistant microdomains of the plasma membrane (21-23). The PrP isoforms also localize to caveolae-like domains isolated without the use of detergents (70). Plasma membrane PrPC seems to recycle to the interior of the cell in about 1 h (24). Two mutually exclusive posttranslational fates await PrPC in ScN2a cells. Although most PrPC molecules turn over with a t1/2 of ~6 h by a two-step degradation pathway (22, 25, 26), a small minority of PrPC molecules (~5%) escape degradation, acquire a protease-resistant core, and become PrPSc (2, 3, 27). Whether PrPSc is formed on the plasma membrane or during the internalization of PrPC is unknown (27-30). PrPSc is further N-terminally trimmed in an acidic compartment (26, 30) and accumulates primarily in lysosomes (20, 26, 31, 32). Interfering with cholesterol-dependent structures or pathways in ScN2a cells inhibits both metabolic fates of PrPC (22). Depriving ScN2a cells of cholesterol inhibited the formation of PrPSc and also retarded the degradation of PrPC. Dissociating PrPC from detergent-insoluble complexes by replacing the GPI anchor of PrPC with the transmembrane and cytoplasmic regions of mouse CD4 almost completely prevented the formation of PrPSc (22).
Most, if not all, GPI-anchored proteins become largely insoluble in cold TX-100 while traversing the Golgi stacks (22, 23). In a seminal article, Brown and Rose (21) demonstrated that GPI-anchored proteins owe this insolubility to their association with membrane complexes enriched in cholesterol, sphingolipids, and glycolipids. Such membrane microdomains or "rafts" of specialized lipid composition had previously been hypothesized by Simons and van Meer (33) to explain the sorting of lipids in polarized epithelial cells. In these cells, GPI anchors act as a sorting signal that functions in concert with these membrane subdomains (21, 34-37). In addition to containing GPI proteins, detergent-insoluble membrane complexes are also enriched in several cytoplasmic proteins, including nonreceptor-type tyrosine kinases (38, 39). Caveolae, which are cholesterol-dependent invaginations of the plasma membrane present in many cell types, appear to constitute a specialized subset of all cellular rafts. Caveolae are involved in the potocytosis of folate, among other putative functions (36, 40), and are equipped with components of the machinery of vesicular fusion (41). In cells containing caveolae, detergent-insoluble complexes are also enriched in caveolin/VIP-21, a component of the striated coat of caveolae (37, 42-44). The existence of caveolae and caveolin in N2a cells remains a matter of debate (23, 45, 46). However, these cells do contain rafts, and since we were unable to observe caveolae in ScN2a using transmission electron microscopy in thin sections,2 we have limited the experiments described here to detergent-insoluble rafts. In this article, we further characterize the relationship of the PrP isoforms with rafts.
The normal physiological configuration of rafts prior to cell lysis is
still unknown, but they may reflect the lipid microenvironment experienced by their associated proteins in cellular membranes. As
criteria for the attachment of macromolecules to rafts, we have used
here operational definitions generated by the article by Brown and Rose
(21) as well as through the work of other groups. Namely, the PrP
isoforms are assumed to be attached to rafts if they fulfill all three
of the following criteria: (i) their solubility in TX-100, as assayed
by high speed sedimentation, is temperature-dependent,
i.e. the protein sediments at 4 °C but stays in
supernatant if the lysates are warmed to 37 °C; (ii) they float up
density gradients following extraction in cold Triton X-100; and (iii)
their buoyancy is reduced when saponin is added to the extract (47)
(indicating the importance of cholesterol in the attachment of GPI to
these complexes) or when TX-100 is replaced with n-octyl
-D-glucopyranoside (NOG) in the lysis of the cells (21).
Throughout this study, we used the ganglioside GM1 as a marker of rafts
(48).
We report here that the pathological prion protein isoform
PrPSc is attached to rafts in ScN2a cells. Since the
metabolic precursor PrPC also resides in these complexes,
the transformation PrPC PrPSc thus appears
to occur within rafts. When cells were lysed in TX-100 at 37 °C
instead of the "canonical" 4 °C (21), PrPC was
"solubilized" as judged from its sedimentation properties, but it
still floated up density gradients. This property was shared by another
GPI protein, Thy-1, which was extracted from EL-4 T-lymphoma cells. In
one ScN2a clone, as well as in the brain of a Syrian hamster with
scrapie, TX-100 extraction at 37 °C permitted resolution of
PrPC and PrPSc into two distinct peaks with
different densities. This result suggests that there are two
populations of PrP-containing rafts and may permit the isolation of
PrPC-specific rafts from those containing
PrPSc. Our findings reinforce the contention that rafts are
intimately involved in PrP metabolism and in the "life cycle" of
prions. They also reveal the presence of detergent-insoluble membrane complexes in intracellular compartments, probably secondary lysosomes, in which a major portion of PrPSc resides.
Reagents for cell culture were purchased from Biological Industries (Beit Haemek, Israel). Tissue culture plates were from Miniplast (Ein Shemer, Israel). G418 was from Life Technologies, Inc. Peroxidase-coupled cholera toxin subunit B (CTXB-POD; 227041), NOG (494459), and Biotin-X-NHS (water-soluble; 203189) were from Calbiochem. Phosphatidylinositol-specific phospholipase C (PIPLC; P8804) was from Sigma. Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Protein A-Sepharose was from Pharmacia Biotech Inc. All other reagents were from Sigma.
Cells and PrP PreparationsMouse N2a cells were originally obtained from the American Type Culture Collection (Rockville, MD). Mouse lymphoma EL-4 cells expressing Thy-1 were from Dr. M. Baniyash (Hebrew University, Jerusalem, Israel). ScN2a is the persistently infected N2a clone described by Butler et al. (19). N2a-c3 and ScN2a-MHM2 express the MHM2-PrP chimeric gene driven by the pSPOX vector (49). N2a-c10 and ScN2a-c10 cells express the same chimeric PrP gene driven by the commercial expression vector pCI-neo (Promega, Madison, WI). All the cells were grown at 37 °C in Dulbecco's modified Eagle's medium 16 supplemented with 10% fetal bovine serum. Cells expressing the chimeric MHM2-PrP gene were maintained in 1 mg/ml G418. Syrian hamster prion rods purified by the sucrose gradient method (50) were a kind gift from Dr. S. B. Prusiner (University of California, San Francisco). Enriched preparations of MHM2-PrP carrying the 3F4 epitope were prepared from N2a-c3 cells using ion metal affinity chromatography as modified from the method of Pan et al. (51).
AntibodiesRabbit antiserum R073 reacts with Syrian hamster (SHa) PrP and mouse (Mo) PrP, as well as with MHM2-PrP, and its specificity in Western blotting has been described elsewhere (20, 52). 3F4 is a monoclonal antibody raised against SHaPrP27-30 (53). Its epitope, which includes Met109 and Met112 (54), is present on SHaPrP as well as on MHM2-PrP, but is absent from MoPrP. Thus this mAb does not recognize the wild type MoPrP endogenous to N2a cells but does react with the products of the chimeric MHM2-PrP genes (49). Both antibodies were used at a dilution of 1:5000 (of the serum or the ascitic fluid). Thy-1 mAb G7 supernatant was a kind gift from Dr. M. Baniyash.
Flotation AssaysFlotation of detergent-insoluble complexes was performed as described by Brown and Rose (21) with some modifications, as follows ("the standard flotation"). Confluent cells growing in two 10-cm plates (about 3 × 107 cells) were incubated with 400 µl of lysis buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM ETDA, 1% Triton X-100) on ice in the cold room for 30 min. This ratio of lysis buffer volume and cell number was kept constant throughout the experiments described here. In some cases, other detergents were used, or the lysates were further incubated at 37 °C, as specified in each experiment. All the subsequent steps were performed at 4 °C. Lysates were adjusted to 35% Nycodenz by adding an equal volume of ice-cold 70% Nycodenz prepared in TNE (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA) and loaded at the bottom of Beckman Instruments TLS-55 ultracentrifuge tubes. A 8-35% Nycodenz linear step gradient in TNE was then overlaid above the lysate (200 µl each of 25, 22.5, 20, 18, 15, 12, and 8% Nycodenz), and the tubes were spun at 55,000 rpm for 4 h at 4 °C in a TLS-55 rotor (gav, 200,000 × g). Fractions were collected from the top of the tube. We found that Nycodenz advantageously replaced sucrose (21) in the flotation assay, permitting us to reduce the centrifugation time needed to reach density equilibrium from 18 to 4 h.
To eliminate PrPC from fractions we used proteinase K. The fractions were made 1% with NOG (to disrupt rafts and their possible protective effects on PrPC (21, 23)) and incubated with 20 µg/ml proteinase K (37 °C, 1 h). The reaction was stopped with 1 mM phenylmethylsulfonyl fluoride prior to Western analysis. To isolate PrPC from PrPSc in fractions, we took advantage of the differential solubility of these proteins in Sarkosyl (18). Fractions were diluted three times in TN (150 mM NaCl, 10 mM Tris, pH 7.8) to reduce their density, made 1% with Sarkosyl, and incubated on ice for 30 min to promote the solubilization of rafts and the aggregation of PrPSc. The fractions were then spun at 45,000 rpm for 2 h at 4 °C in a Beckman TLA-45 rotor (gav, 109,000 × g), and the supernatants (enriched in PrPC) were collected for further analysis.
In some cases, the cells were labeled with CTXB-POD prior to lysis and flotation. The procedure was performed in the cold room on ice. Cells growing on a 10-cm plate were rinsed three times with ice-cold phosphate-buffered saline and then incubated with ice-cold phosphate-buffered saline containing 5 µg/ml CTXB-POD (45 min on a rocker) prior to their lysis in TX-100 lysis buffer. The cells were then rinsed three times in ice-cold phosphate-buffered saline to remove unbound toxoid and then directly lysed as described before. In some cases, the toxoid conjugate was added directly to cell lysates rather than to whole cells, as described in "Results."
Detection of Cell Surface Thy-1Cell surface biotinylation of EL-4 cells was performed as follows. Cells growing in a T75 flask were resuspended in ice-cold buffer A (150 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 20 mM Hepes, pH 7.4), rinsed three times in the same buffer, and then incubated for 40 min on ice in buffer A containing NHS-X-biotin (water-soluble; 500 µg/ml). The cells were then rinsed three times in cold buffer A supplemented with 50 mM NH4Cl to quench the biotinylation reagent and then lysed as described in the text. To immunoprecipitate Thy-1 from fractions of Nycodenz gradients, the fractions were made 1% with Sarkosyl, mixed with 1 ml of G7 mAb hybridoma supernatant, and incubated for 18 h at 4 °C. Protein A-Sepharose was then added, and the mixture was incubated for an additional 30 min at room temperature. The beads were rinsed five times in TNS (100 mM NaCl, 1% Sarkosyl, 10 mM Tris-HCl, pH 7.5) and then boiled in SDS sample buffer prior to analysis by SDS-polyacrylamide gel electrophoresis.
Western and Dot ImmunoblotsProteins in cell lysates were resolved in 12% polyacrylamide gels (55) and electrotransferred to BioTrace polyvinylidene difluoride membranes (Gelman Instrument Co.) in a Tris/glycine buffer (56) containing Sarkosyl (48 mM Tris base, 39 mM glycine, 20% methanol, 0.001% Sarkosyl). The membranes were blocked with 5% low fat milk in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) prior to incubation with antibodies. POD-conjugated secondary antibodies were used, and the blots were developed by chemoluminescence. To detect CTXB-POD in fractions, 10-µl aliquots were dotted on a nitrocellulose membrane, which was then developed with chemoluminescence reagents.
PrPSc-specific dot immunoblots were performed as described (52). Briefly, aliquots from each fraction were dotted on a nitrocellulose membrane. The membrane was thoroughly air dried, rewetted with TBST, and incubated with proteinase K (50 µg/ml, 37 °C, 1 h). The membranes were then rinsed in water and sequentially incubated with 1 mM phenylmethylsulfonyl fluoride in TBST (30 min, room temperature) to stop the proteolysis and with guanidine thiocyanate (GdnSCN) (3 M, 5 min, room temperature) to expose PrPSc epitopes. The membranes were then blocked with 5% milk and further treated for PrP immunodetection as described for Western blots.
Like other GPI-anchored proteins (21), PrPC clusters on the cell surface (22, 31, 45) and is insoluble in cold TX-100, as judged by its sedimentation properties (22, 23). Because glycolipid/cholesterol rafts and cholesterol-dependent subcellular pathways appear to play such a crucial role in the metabolism of PrP and thus in the biogenesis of prions (22), we sought to better characterize the relationship of the PrP isoforms to these complexes.
We first examined the solubility of PrPC from N2a cells in
cold TX-100, using a flotation assay adapted from the method of Brown
and Rose (21). N2a cells (Fig. 1A) were lysed
in 1% TX-100 lysis buffer on ice in the cold room, brought to 35%
Nycodenz, overlaid with lower density Nycodenz cushions, and then spun
for 4 h at 4 °C at 55,000 rpm in a TLS-55 rotor (the standard
flotation assay). Western analysis verified that PrPC
migrated up the gradient as expected from a resident of rafts (Fig.
1A). Identical results were obtained with N2a-c3 and N2a-c10 cells that overexpress the chimeric MHM2-PrP gene (not shown). In
contrast, most cellular proteins remained in the bottom fractions (8-11) of the gradient, as judged from silver staining of the gel (not
shown).
We compared the distribution of PrPC in the flotation gradient with that of another resident of rafts, the cell surface ganglioside GM1. This glycolipid is enriched in caveolae of A431 cells (48) and partitions with low density, TX-100-insoluble complexes in lymphocytes (57). To detect cell surface GM1 in N2a cells, we used its ligand CTXB. N2a cells were incubated with CTXB-POD (5 µg/ml) at 0 °C for 45 min, and then the cells were rinsed, lysed in TX-100, and subjected to the standard flotation assay. CTXB-POD was detected in fractions using a simple dot blot. CTXB migrated to fractions identical to those reached by PrPC (Fig. 1B), as expected from a resident of plasma membrane rafts. Interestingly, the migration of PrPC in the Nycodenz gradient was unaltered in cells preincubated with the toxoid (not shown). This observation is in accord with results of Fra et al. (57), who have previously shown that CTXB induces the capping of GM1 but fails to induce the cocapping of the GPI-anchored Thy-1 in T hybridoma cells, suggesting that, although experiencing a similar membrane microenvironment, these two residents of rafts are not necessarily linked.
We presume that PrPC is attached to rafts through its GPI moiety. Lehmann and Harris (58) have recently shown, however, that recombinant PrP molecules carrying one of several pathogenic mutations that cause familial Creutzfeldt-Jakob disease in humans may have membrane attachment sites additional to the GPI anchor (when expressed in Chinese hamster ovary cells). The results of these investigators prompted us to verify whether PrPC in N2a cells (which do not contain any of these pathological mutations) is bound to membranes exclusively through its GPI anchor, or whether there is a subpopulation of PrPC molecules that have an alternative or additional mode of membrane attachment. To check this, we biotinylated N2a-c10 cells with membrane-impermeant NHS-X-biotin and then incubated them with PIPLC (0.1 units/ml, 37 °C, 2 h). To prevent the synthesis of new PrPC molecules during the enzymatic treatment, we added cycloheximide (20 µg/ml) to the culture medium during the incubation with PIPLC. The cells were then rinsed and lysed, and PrP was immunoprecipitated using the PrP antiserum R073, which recognizes both wild type and MHM2-PrP expressed in these cells. This treatment removed all detectable PrPC molecules from the cell surface (Fig. 1C). We thus conclude that these molecules are attached to the cell surface solely through their GPI anchor.
Having shown that PrPC is attached to buoyant TX-100-insoluble fractions in N2a cells, we turned to the pathological PrP isoform PrPSc. Because PrPSc is inherently insoluble in detergents (18), it is not possible to determine whether it is attached to TX-100-insoluble complexes simply by assessing its sedimentation properties following extraction of cells with TX-100. It was thus hitherto unclear whether PrPSc, an abnormal GPI protein that accumulates primarily intracellularly (20, 31), associates with detergent-insoluble complexes like most normal GPI-anchored proteins. Furthermore, although PrPSc possesses a GPI tail (11), it is not known whether this moiety serves at all to anchor this pathological protein to cellular membranes. To determine whether PrPSc is attached to detergent-insoluble complexes, TX-100 lysates of ScN2a cells (not shown), as well as of ScN2a-MHM2 cells that overexpress the chimeric gene MHM2-PrP (Fig. 1D), were subjected to a Nycodenz flotation assay. Gradient fractions were first incubated with proteinase K to degrade PrPC prior to analysis by SDS-polyacrylamide gel electrophoresis. Like PrPC, protease-resistant PrP migrated to the upper part of the gradient (Fig. 1D), as would be expected from a resident of rafts. Identical results were obtained with ScN2a cells (not shown). When ScN2a-MHM2 cells were incubated with CTXB-POD prior to the lysis, the toxoid migrated to fractions identical to those reached by PrPSc (Fig. 1E).
Prion-infected ScN2a cells contain both PrPSc and PrPC. Although ScN2a cells do not exhibit any obvious cytopathic effect, they still display a variety of abnormalities (59, 60). Thus, there is no a priori reason to assume that the flotation properties of PrPC in these cells must be identical to those of PrPC in uninfected cells. We therefore sought to determine the specific flotation characteristics of PrPC in ScN2a-MHM2 cells. To this end we analyzed the distribution of Sarkosyl-soluble PrP in the flotation gradient. Fractions were diluted three times in TN, made 1% with Sarkosyl, and incubated on ice for 30 min, and then the insoluble PrP was removed by a 2-h spin at 45,000 rpm at 4 °C in a TLA-45 rotor. Since most PrPSc is insoluble in Sarkosyl (18), the supernatant is highly enriched in PrPC. As shown in Fig. 1F, the Sarkosyl-soluble PrP was also found in the lighter fractions of the gradient and was as buoyant as was PrPC in uninfected cells (Fig. 1A). To ascertain that the Sarkosyl-soluble PrP is indeed primarily PrPC, we incubated it with proteinase K (20 µg/ml, 37 °C, 1 h) and found that this PrP species is indeed completely digested under these conditions (not shown).
Exogenous PrPC and PrPSc Added to TX-100 Cell Lysate Do Not Migrate with Buoyant FractionsWe wished to
further ascertain that the observed buoyancy of the PrP isoforms in the
flotation assay was not due to the spontaneous association of these
proteins to TX-100-insoluble structures in vitro during the
lysis but rather reflected their attachment to insoluble complexes
prior to the detergent extraction. GPI proteins are indeed known for
their affinity to cholesterol- and sphingolipid-containing liposomes
(in the absence of detergent) (61) as well as to the plasma membranes
of whole cells. We therefore added exogenous PrPC and
PrPSc to the lysate of cells prior to the flotation and
determined their buoyancy. For PrPC, we used an enriched
fraction of recombinant MHM2-PrPC prepared from N2a-c3
cells by ion metal affinity chromatography. This chimeric PrP carries
the mAb 3F4 epitope and can thus be differentiated from wild type MoPrP
endogenous to N2a cells. Purified MHM2-PrPC was incubated
with the TX-100 lysate of N2a cells on ice for 30 min, and the lysate
was then subjected to the standard flotation assay. In contrast to
endogenous PrPC, these exogeneously added PrPC
molecules failed to float up the gradient (Fig.
2A).
As a control for exogenous PrPSc, we used purified prion rods obtained from Syrian hamsters. These PrPSc-containing infectious aggregates are extensively delipidated during their purification (50). To determine whether purified prion rods attach to buoyant complexes in cell lysates, we incubated purified prion rods with the TX-100 lysate of ScN2a cells on ice for 30 min, and we then proceeded with the standard flotation procedure. The rods were detected in the fractions using the PrPSc-specific dot-immunoblotting procedure based on the sequential treatment of the membrane with proteinase K and GdnSCN (52). To differentiate exogenous SHaPrPSc in the rods from mouse PrPSc endogenous to ScN2a, we again used the mAb 3F4. As seen in Fig. 2B, these rods did not float up the gradient as did endogenous PrPSc but instead sedimented to the bottom of the lysate fraction. An identical experiment, performed in the absence of exogenous PrPSc, verified the specificity of the 3F4 antibody for SHaPrP in the dot blot immunoassay (Fig. 2C). These results show that the buoyancy of endogenous PrPC and PrPSc in TX-100 cell extracts indeed stems from the physiological association of these proteins with cellular structures prior to the cell lysis.
In contrast to the results with exogenous PrP, when CTXB-POD was added to the lysate of N2a cells and incubated for 30 min (on ice) prior to the centrifugation, most of it did float to the same location as when incubated with whole cells (Fig. 2E). However, if the toxoid was resuspended in TX-100 lysis buffer without cell lysate, it remained at the bottom of the gradient (Fig. 2D). This shows that the toxoid can recognize its ligand within TX-100 cell lysates.
Saponin and NOG Reduce the Buoyancy of PrPC and PrPSc in Flotation GradientsTo determine whether the
buoyant properties of PrPC and PrPSc in cold
TX-100 depend on cholesterol, we added saponin (1%) to the TX-100 cell
lysates and incubated them for 30 min on ice prior to the
centrifugation (47). This protocol greatly reduced the buoyancy of both
PrPC from N2a cells and PrPSc from ScN2a-MHM2
cells (Fig. 3, A and B,
respectively). Thus, the buoyancy of both PrP isoforms in TX-100 indeed
depends on cholesterol. When NOG was substituted for TX-100 in the
extraction of the cells, the buoyancy of both PrPC (Fig.
3C) and PrPSc (Fig. 3D) was also
markedly reduced.
Taken together, our data demonstrate that both PrP isoforms associate
with complexes that satisfy our criteria for rafts. This in turn
suggests that the transformation PrPC PrPSc
takes place within rafts. Since most PrPSc is
intracellular, these findings also reveal the existence of intracellular rafts in scrapie-infected cells.
Since the primary subcellular localizations of PrPC and PrPSc in neuroblastoma cells are different, we surmised that the rafts to which they are attached may be of different lipid or protein compositions, so that perhaps experimental conditions may be devised to differentiate between these putatively disparate PrP-containing complexes. In particular, we sought to determine whether PrPSc and PrPC might behave differently in a flotation assay when lysed in TX-100 at temperatures higher than the "canonical" 4 °C.
We first studied PrPC in uninfected cells.
PrPC, like other GPI proteins, fails to sediment at high
speed following lysis in TX-100 at 37 °C (22, 23). A simple
sedimentation experiment performed with TX-100 lysates of N2a cells
confirmed this property (Fig. 4A). It also
confirmed that the addition of saponin to cold TX-100 lysates greatly
increased the solubility of PrPC (Fig. 4A, lanes
5 and 6). We next sought to determine whether PrPC would still migrate up a Nycodenz density gradient if
the TX-100 lysate was incubated at 37 °C prior to the
centrifugation. N2a cells were lysed on ice in cold TX-100 lysis buffer
as before. The lysate was then further incubated at 37 °C for 1 h, cooled back on ice, and then subjected to the standard flotation
procedure. To our surprise, we found that the incubation at 37 °C
did not prevent the migration of most PrPC molecules toward
the lighter fractions of the Nycodenz gradients (Fig. 4B).
This property was also shared by cell surface GM1, another resident of
rafts, as judged by the migration of CTXB-POD in the standard flotation
assay following lysis of N2a cells at 37 °C (Fig. 4C). In
some experiments PrPC floated to even lighter fractions
than when the lysis was performed at 4 °C (not shown). The results
of the flotation assay thus contrasted sharply with the increased
apparent solubility of PrPC in TX-100 at 37 °C, as
judged by the sedimentation method (compare with Fig. 4A, lanes
1-4; Ref. 22).
To see whether other GPI proteins also remain insoluble and buoyant after lysis at 37 °C, we examined the flotation properties of Thy-1 extracted from EL-4 T-lymphoma cells with TX-100 at 37 °C (Fig. 4, D and E). Because the mAb G7 does not recognize Thy-1 on Western blots, we resorted to cell surface biotinylation followed by immunoprecipitation to detect Thy-1. EL-4 cells were biotinylated using a membrane-impermeant NHS-X-biotin. They were then lysed in TX-100, and one-half of the lysate was warmed to 37 °C for 1 h and then transferred back to ice (Fig. 4E), whereas the other half stayed on ice for the whole period (Fig. 4D). The lysates were subsequently subjected to a standard flotation procedure, and Thy-1 was immunoprecipitated from the gradient fractions using the mAb G7. The immunoprecipitates were then analyzed in Western blots developed with streptavidin-POD. As seen in Fig. 4E, Thy-1 indeed floated up the gradient even when the lysates had been subjected to an incubation at 37 °C, a property that is thus not exclusive to PrPC in N2a cells.
PrPC and PrPSc Localize to Complexes of Different Densities in 37 °C Lysates of Some ScN2a ClonesHaving shown that most PrPC still localizes to
light fractions in density gradients even when the cells are lysed at
37 °C, we turned our attention to the behavior of PrPSc
in lysates at this temperature. When the TX-100 lysate of ScN2a cells
was incubated at 37 °C for 1 h prior to the standard flotation assay, the protease-resistant PrP floated to fractions 3-7, similar to
the distribution of PrPC in N2a cells (compare Figs.
5A and 4B). Thus,
PrPSc extracted from ScN2a cells stayed in
detergent-insoluble buoyant rafts even following lysis at the higher
temperature, therefore behaving like PrPC in this
particular experiment. In some experiments (not shown), the peak of
PrPSc was slightly heavier than that of PrPC,
but this difference was not very accentuated in these cells.
In contrast to these results with ScN2a cells, well separated PrPC and PrPSc fractions were observed when the transfected ScN2a-MHM2 subclone was used in similar solubilization experiments. When TX-100 lysates of ScN2a-MHM2 cells were incubated at 37 °C and then subjected to the standard flotation assay, protease-resistant PrPSc migrated to the middle of the gradient (Fig. 5B). In contrast, Sarkosyl-soluble PrP (made up primarily of PrPC; Ref. 18) migrated to the top of the gradient (Fig. 5C). Thus, these lysis conditions did generate fractions of clearly different buoyancy for PrPC and PrPSc when applied to ScN2a-MHM2 cells.
We sought to determine whether the enhanced separation of the PrPC and PrPSc peaks in ScN2a-MHM2 cells could be due to the overexpression of chimeric MHM2-PrP in these cells. To verify this, we used another subclone of ScN2a (ScN2a-c10), which expresses MHM2-PrP at yet higher levels than ScN2a-MHM2 cells (but driven by another expression vector). In contrast to the results with ScN2a-MHM2 cells, PrPSc extracted from this clone at 37 °C (Fig. 5D) reproducibly comigrated with PrPC (Fig. 5E) to the lighter fractions of the gradient. Thus, the separation of the PrP isoforms in the flotation gradient is not a result of chimeric PrP overexpression in these cells but rather seems to depend on clonal differences between the cells. We surmise that details in the lipid composition of cellular membranes (and in particular of rafts) may vary in these two transfected lines and determine disparate buoyant characteristics of PrP rafts.
PrPC and PrPSc from SHa Brain Also Partially Separate following Lysis at 37 °CHaving devised lysis conditions that permit the separation of PrPC and PrPSc rafts in ScN2a-MHM2 cells, we moved on to determine the flotation characteristics of the PrP isoforms in the brain following lysis with TX-100 at 37 °C. Since PrP is found in very diverse cells in the brain, in which lipid composition can be expected to vary widely, it was not obvious that the PrP isoforms would separate in these conditions. To explore the flotation characteristics of the PrP isoforms, we used the brain of a SHa clinically sick with Sc237 scrapie. To keep the lysis conditions as close as possible to the standard flotation assay, we added a small piece of this brain (~1 mg of wet tissue) to the TX-100 lysate of N2a cells. The lysate was then warmed to 37 °C for 1 h, returned to ice, and then subjected to the standard flotation assay. We then used Western blots (developed with 3F4 to detect SHaPrP above the MoPrP background) to determine the distribution of proteinase K-resistant PrPSc and of Sarkosyl-soluble PrP in the gradient. A major portion of brain PrPC was buoyant (Fig. 5F) and separated well from the PrPSc peak (Fig. 5G). Similar results were obtained when brain samples were lysed directly in TX-100 lysis buffer without mixing with lysates of cultured cells (not shown).
Taken together, these results suggest that in some cells PrPSc molecules distribute to a population of rafts that differ in both solubility and density following detergent extraction at 37 °C. Alternatively, it is possible that the extractability of PrPSc from its carrier rafts differs from that displayed by PrPC, perhaps due to a different anchoring mode. Further studies will be needed to discern between these and other possibilities.
The finding that most PrPSc cofractionates with rafts
in ScN2a clones has several important implications. First, since the
metabolic precursor PrPC also localizes to rafts (22), it
is probable that the transformation PrPC PrPSc occurs within this differentiated lipid environment.
This conclusion is in line with our previous data that
cholesterol-dependent pathways, as well as attachment of
PrPC to rafts, are essential for the efficient conversion
of PrPC into PrPSc (22). How would these
membrane domains facilitate the formation of PrPSc? It is
possible that rafts favor the interaction of PrPC with
existing PrPSc "seed" molecules, maybe by concentrating
PrP molecules within confined stretches of the plasma membrane or
aligning them in a manner propitious for their interaction. Another
possibility is that rafts contain some indispensable machinery engaged
in the formation of PrPSc, such as protein X (62) or other
possible facilitators of PrPSc formation. Analysis of rafts
from ScN2a cells or rodent brains could thus conceivably enable us to
discover such putative cofactors engaged in the formation of
PrPSc. It is possible that such rafts could yield a
favorable environment to study the formation of PrPSc
in vitro (9). It will be interesting to see, for instance, whether
protease-resistant PrP can be formed in rafts isolated from ScN2a
cells. Finally, it is also possible that rafts serve merely as vehicles
that target PrPC to as yet unidentified subcellular sites
hospitable to the conversion to PrPSc.
The exact subcellular compartments in which PrPC is degraded and PrPSc forms and the trafficking pathways leading to these sites have not been characterized. Several lines of evidence suggest that an internalization step may be involved in the formation of PrPSc in ScN2a cells. First, when ScN2a cells are metabolically radiolabeled and chased at 18 °C, even for long periods, radiolabeled PrPC does reach the plasma membrane (PM), but PrPSc is not formed. However, if the cells are warmed to 37 °C for 30 min and then transferred back to 18 °C, protease-resistant PrPSc now forms at the lower temperature (27). The existence of an 18 °C block that is relieved by a short exposure at 37 °C suggests the involvement of the endocytic pathway. Second, nascent PrPSc, most of which is formed from PM PrPC, is trimmed in an acidic compartment shortly after its formation and thus has to be first endocytosed (27-30). However, it is still possible that some or most PrPSc is formed on the PM. Indeed, small amounts of PrPSc seem to be present on the cell surface,3 and they could function as seed molecules for the reaction. A central question is, thus: What internalization pathway, if any, is involved in the formation of PrPSc? Because PrP is attached to glycolipid rafts, the emerging nonclathrin endocytic pathways (63; reviewed in Ref. 64) are attractive candidates for such internalization mechanisms. Both caveolae and some GPI proteins are internalized through mechanisms that depend on actin and protein kinases (65-67). These may involve vesicles that contain various members of the general fusion machinery, such as NSF (41), as well as additional specific proteins, which may be homologous to the apical transport vesicle-specific annexin XIIIb (68).
In contrast to the localization of PrPC to rafts, Shyng et al. (46) have recently provided evidence that PrPC is enriched in clathrin-coated pits in N2a cells. Whether their finding presents a real contradiction to the localization of most PrPC in rafts is not clear. First, the data of Shyng et al. (46) pertain only to a small minority of cell surface PrPC molecules that localize to clathrin-coated pits. Second, association with coated pits and attachment to rafts may not be mutually exclusive. Whether clathrin-coated pits could contain some raftlike subdomains is not known. Based on the inhibition of PrPC endocytosis by hypertonic sucrose, these authors also suggested that PrPC internalizes via clathrin-coated pits. However, whether this drastic treatment specifically inhibits coated pit endocytosis without affecting other internalization pathways is unknown. In addition, because the experiments described here were designed to address only the steady state solubilization properties of PrP, we would have been unable to detect possible transient events such as, for instance, a temporary removal of PrP from rafts. For instance, Rijnboutt et al. (69) have recently shown that although most (GPI-anchored) folate receptor is detergent-insoluble in KB cells, it is the soluble minority that is endocytosed and thus presumably participated in the potocytosis of folate. Clearly, more work is needed to decipher the trafficking pathways used by PM PrPC.
That PrPC and PrPSc rafts could be separated from each other in some neuroblastoma lines as well as in the brain has both theoretical and practical implications. There are at least two potential mechanisms that could lead to such a separation. One possibility is that most PrPSc molecules indeed reside in rafts of different lipid and protein compositions that are denser, after TX-100 extraction, than those that carry PrPC. This is certainly plausible, since the primary localization of these proteins is entirely different. Whereas PrPC is found mainly on the cell surface, PrPSc is primarily intracellular and accumulates mainly (but not only) in secondary lysosomes of infected cells (20, 31). Our 37 °C extraction procedure paves the way for the isolation and characterization of both types of rafts.
It is also possible that the reason for the segregation of PrPC and PrPSc in different fractions of the flotation gradient is that at 37 °C these proteins are extracted from the membranes with different efficiencies. The mode of association of PrPSc with membranes has never been elucidated. Although our results suggest that most cellular PrPSc is attached to saponin-sensitive buoyant rafts, probably through its GPI moiety, the details of this association are unknown. It is possible, for instance, that PrPSc exists as aggregates that attach to cellular membranes only through a small fraction of the GPI moieties present in the aggregate. Such aggregates would be more tenuously attached to membranes and therefore extracted from them more easily, thus becoming less buoyant after extraction at 37 °C. Additional work will be needed to determine the mode of attachment of PrPSc to membranes. Finally, because the Sc237 strain of scrapie used to inoculate this Syrian hamster induces sizable amounts of extacellular PrPSc plaques, it is possible that some of the PrPSc detected in Fig. 5G is a priori extracellular and not bound to membranes.
What is the reason for the apparent contradiction between results obtained with the sedimentation and the flotation assays when lysates are incubated at 37 °C? It is possible that the insoluble complexes generated by the lysis of cells in TX-100 at 37 °C may be smaller than those produced at 4 °C but may retain a similar density. This would decrease their sedimentability (Fig. 4A) while maintaining their buoyancy in Nycodenz gradients (Fig. 4, B and E). Further studies will be needed to clarify this point.
Since most PrPSc is intracellular, the finding that it is attached to rafts reveals the existence of TX-100-insoluble rafts in intracellular organelles, probably in late compartments of the endocytic pathway. Whether this is a pathological property of prion-infected cells or a general attribute of many cell types remains to be determined. Various investigators have shown that the internalization pathway of GPI proteins intersects that of proteins that are internalized via clathrin-coated pits. Thus, rafts could find their way to the late endocytic pathway. That lysosomes contain cholesterol was shown previously using filipin binding techniques.
In summary, the results presented in this report support a model in which the various aspects of PrP metabolism take place within cholesterol and glycolipid rafts. The exact subcellular compartments involved and their molecular components remain to be characterized.
We are grateful to Dr. Ruth Gabizon for many helpful discussions.