Proc. Natl. Acad. Sci. USAVol. 83, pp. 2310-2314, April 1986Biochemistry

Separation and properties of cellular and scrapie prion proteins(brain subcellular fractionation/membrane protein immunoblotting/detergent solubilization/amyloid rod formation/slow infections)

RUDOLF K. MEYER, MICHAEL P. MCKINLEY, KAREN A. BOWMAN, MICHAEL B. BRAUNFELD,RONALD A. BARRY, AND STANLEY B. PRUSINER*Departments of Neurology and of Biochemistry and Biophysics, University of California, San Francisco, CA 94143

Communicated by Robley C. Williams, November 26, 1985

ABSTRACT Purified preparations of scrapie prions con-tain a sialoglycoprotein of Mr 27,000-30,000, designated PrP27-30, which is derived from the scrapie prion protein [Mr,33,000-35,000 (PrP 33-35sC)] by limited proteolysis. Underthese same conditions of proteolysis, a cellular protein of thesame size (PrP 33-35C) is completely degraded. Subcellularfractionation of hamster brain showed that both PrP 33-35scand PrP 33-35c were found only in membrane fractions. NaCl,EDTA, and osmotic shock failed to release the prion proteinsfrom microsomal membranes. Electron microscopy of thesemicrosomal fractions showed membrane vesicles but not prionamyloid rods. Detergent treatment of scrapie-infected mem-branes solubilized PrP 33-35c, while PrP 33-35sk aggregatedinto amyloid rods; the concentration ofPrP 33-35C was similarto that recovered from analogous fractions prepared fromuninfected control brains. The apparent amphipathic charac-ter of the PrP 33-35SC may explain the association of scrapieinfectivity with both membranes and amyloid rilaments.

Significant progress in purification of the scrapie agent wasmade after a more rapid and economical bioassay wasdeveloped using incubation time interval measurements (1,2). The infective particles causing scrapie were isolated tonear homogeneity by multiple detergent extractions, protein-ase K digestion, and sucrose gradient sedimentation (3).Amyloid rods composed largely of a glycoprotein with anapparent molecular weight of 27,000-30,000 (PrP 27-30) werefound in purified fractions (4-7). Observations by othersappear to have confirmed the existence of scrapie agentproteins and their glycosylation (8, 9). Attempts to identify ascrapie-specific nucleic acid within these purified fractionshave been unsuccessful to date. The unusual properties andapparent small size of the scrapie agent have led to intro-duction of the term "prion" to denote this class of slowinfectious pathogens (10).Immunoblots of brain homogenates from scrapie-infected

hamsters have demonstrated prion proteins (PrP) with a Mrof 33,000-35,000 (PrP 33-35sc) (11). An immunoreactiveprotein of similar Mr (PrP 33-35C) was also found in brainhomogenates prepared from uninfected healthy controls.Proteinase K digestion of infected fractions was observed toconvert PrP 33-35Sc to PrP 27-30, whereas the cellular proteinPrP 33-35C was completely degraded.

Experimental studies reported in this communication showthat PrP 33-35C and PrP 33-35Sc have characteristics ofmembrane-bound proteins. After detergent solubilization ofthe membranes, PrP 33-35Sc polymerized into amyloid rods,providing evidence for its amphipathic character. BecausePrP 33-35c did not polymerize into rods, it could be separatedfrom PrP 33-35sc. The levels ofPrP 33-35c in scrapie-infectedbrains were similar to those found in uninfected healthybrains, indicating that both PrP 33-35C and PrP 33-35sc are

synthesized during scrapie infection. Since the molecularbasis for the differences in properties of PrP 33-35C and PrP33-35sc remains to be established, these proteins must bedefined operationally instead of structurally at present. Thearrangement of PrP within cellular membranes is unknown,but recent studies have shown that aggregates of theseproteins assemble into amyloid filaments within the extra-cellular space of the infected brain to form amyloid plaques(12).

MATERIALS AND METHODSMaterials. All chemicals were of the highest grades com-

mercially available. The BCA-protein assay used to measureprotein concentration was obtained from Pierce. 125I-labeledgoat anti-rabbit antibodies were purchased from New En-gland Nuclear.

Source of Scrapie Prions and Bioassay. A hamster-adaptedisolate of the scrapie agent was passaged and prepared asdescribed (1, 13).

Preparation of the Subcellular Fractions. Weanling ham-sters (LVG/LAK) were inoculated intracerebrally with 107ID50 units of the scrapie agent. The brains were collectedfrom hamsters sacrificed 60 days after infection and fromage-matched uninfected animals. The brains were suspendedin 0.32 M sucrose (10%, wt/vol) and homogenized with sixbursts of 10 sec each by using a Polytron homogenizer set atmedium speed. The homogenates were centrifuged in aBeckman 50.2 Ti rotor. Pellets were resuspended in 0.32 Msucrose solution and the volumes were adjusted to that of thesupernatant. All solutions were kept on ice and all centrifu-gations were performed at 4°C. The fractions were adjustedto 10 mg of protein per ml with 0.32 M sucrose solutions.One-milliliter samples were centrifuged in the Beckman 50 Tirotor at 38,000 rpm for 1 hr at 4°C. Pellets were resuspendedand treated as described in Results. For digestion experi-ments, N-lauroylsarcosine (sarkosyl) was added from a 10%stock solution in 25 mM Tris'HCl/0.1 M NaCl, pH 7.4, tosome of the samples. Control samples were diluted by thesame amount of Tris buffer. Digestions were initiated byaddition ofan aliquot of proteinase K (2 mg/ml) in Tris bufferto give a final concentration of 0.1 mg/ml. After 30 min atroom temperature, the digestions were terminated by addi-tion of an aliquot of 0.1 M phenylmethylsulfonyl fluoride inethanol to yield a final concentration of 10mM and by heatingfor 5 min in a boiling water bath.

Additional purification of microsomal fractions was ac-complished by sucrose-gradient centrifugation (14).PrP 27-30 Antiserum and Immunoblotting. The prepara-

tion, characterization, and affinity purification of the PrP

Abbreviation: PrP, prion protein(s).*To whom reprint requests should be addressed at: Department ofNeurology, HSE-781, University of California, San Francisco, CA94143.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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27-30 antiserum is described elsewhere (12, 15, 16). Immu-noblotting was performed as described (16, 17).

Electron Microscopy. Thin sections of microsomes wereprepared from pellets fixed in 3% glutaraldehyde in 0.05 Mphosphate buffer, postfixed in 1% osmium, and embedded inaraldite. Samples were taken from the supernatants andpellets obtained by cell fractionation and extraction and werestained with uranyl formate as described (6). To some of thesamples, an equal volume with scrapie PrP at 0.1 mg/mlaggregated into rods was added. Scrapie prion rods werepurified as described (6). The microsomes and rods weremixed in a Vortex prior to staining and analysis. All gridswere examined in a JEOL 100B electron microscope set at 80keV (1 eV = 1.602 x 10-19 J).

RESULTS

Subcellular Fractionation. The subcellular distribution ofPrP 33-35sc and PrP 33-35c was determined in brain fractionsobtained from scrapie-infected and age-matched uninfectedcontrol hamsters. The brains were suspended in 0.32 Msucrose solution and homogenized with a Polytron. Theresulting 10% (wt/vol) homogenate was fractionated bydifferential centrifugation. Aliquots of pellets and superna-tant fractions were assayed for prion-related proteins byimmunoblots using affinity-purified PrP 27-30 antiserum(Table 1) (12, 13, 15). PrP 33-35 was found in all the pelletsfrom scrapie-infected and from age-matched uninfected ham-ster brains, but it was absent from the high-speed superna-tant. The concentration of PrP 33-35, which is the sum of PrP33_35C plus PrP 33-35sc, in scrapie-infected brain was in-creased 5- to 10-fold 60 days after intracerebral inoculationcompared to uninfected brains. The presence of PrP 33-35Scin infected animals was inferred from measurements of PrP27-30. Both PrP 33-35SC and PrP 27-30 were correlated withscrapie infectivity (Table 1). No infectivity was found inparallel fractions derived from uninfected animals; the dis-tribution of PrP 33-35C was similar to that observed for PrP27-30 (Table 1)-i.e., PrP 33-35C was found in all fractionscontaining membranes but not in the 100,000 x g superna-tant. Based on these results, a simplified fractionationscheme was adopted for subsequent studies. Brain homog-enates were centrifuged at 10,000 x g for 30 min to removemost of the debris, nuclei, cytoskeletal components, andmitochondria. After a second centrifugation at 100,000 x gfor 1 hr, a pellet and a high-speed supernatant were obtained.

Table 1. Subcellular fractionation of scrapie-infected hamsterbrains by differential centrifugation

Subcellular log titer, IDM unitsfraction* PrP 33-35t PrP 27-30 per ml + SEM

Homogenate + + 8.3 ± 0.16Pellet

(1000 x g) + + 8.5 ± 0.11Supernatant

(1000 x g) + + 8.1 ± 0.30Pellet

(10,000 x g) + + 7.9 + 0.18Pellet

(100,000 x g) + + 7.5 + 0.22Supernatant

(100,000 X g) - - 4.7 ± 0.29

*Brain homogenates were fractionated by differential centrifugationat 40C for 60 min. Aliquots of pellets and supernatant fractions wereassayed for infectivity and prion-related proteins.tPrP 33-35 represents the sum of PrP 33-35c plus PrP 33-35sC. Thepresence (+) or absence (-) of PrP detected by immunoblotting isnoted.

The pellet contained mainly membrane vesicles and frag-ments, as judged by electron microscopy and describedbelow; thus, it was designated a microsomal fraction. Frac-tionation of scrapie-infected brains by this abbreviated pro-tocol yielded a 10,000 x g pellet, a microsomal pellet and a100,000 x g supernatant fraction containing 107.8, 107-3, and104 2 ID50 units per ml of prions, respectively. Additionalpurification of microsomes on sucrose gradients (14) disso-ciated neither the PrP nor the scrapie infectivity frommembranes (data not shown).Attempts to Release PrP from Membranes. To assess

whether or not PrP are membrane proteins, several proce-dures were examined for their ability to solubilize the prionproteins. Aliquots of microsomal pellets were suspended in25 mM Tris HCl (pH 7.4) containing 0.5 NaCl, 0.1 M NaCl,or 5 mM EDTA. Another aliquot was suspended in distilledwater. After incubation for 30 min at 20°C, the samples werecentrifuged again at 100,000 x g. The pellets were resus-pended and analyzed by immunoblotting (Fig. 1). In fractionsobtained from uninfected animals, PrP 33-35c could not beextracted by salt or EDTA (Fig. LA); thus, no immunoreac-tive proteins were found in the supernatant fractions. Os-motic shock with distilled water released

Proc. Natl. Acad. Sci. USA 83 (1986)

immunoblotting of microsome fractions (Fig. 1). This protein(designated P42) was present in normal and scrapie-infectedfractions in almost equal concentrations and showed proteasesensitivity only after detergent treatment. P42 has never beenfound in purified fractions of prions, and its properties aredissimilar to those of both PrP 33-35sc and PrP 33-35c. Atpresent, we have no evidence that the primary structure ofP42 is related to that ofthe PrP; in fact, antisera raised againsta synthetic peptide (PrP-P1) encoding the NH2-terminal 13amino acids of PrP 27-30 do not react with P42 (data notshown). Conversely, PrP-P1 antisera do react with both PrP33-35c and PrP 33-355c, arguing that these proteins are related(18). About 50% of P42 was released by osmotic shock (Fig.1, lanes 7 and 8).

Separation of Ceilular and Scrapie PrP. Proteinase Kdigestion of infected fractions converted PrP 33-35sc to PrP27-30, whereas PrP 33-35c was completely degraded (Fig. 2A,lanes 1-4) (11). To test the influence of detergent onproteolytic digestion, microsomal fractions were washed in0.1 M NaCl/25 mM Tris*HCl, pH 7.4, by centrifugation andresuspended in 2% sarkosyl (lanes 1 and 2). The proteolyticconversion of PrP 33-35sC to PrP 27-30 and the degradation ofPrP 33-35c by proteinase K were unaltered when the deter-gent extraction was omitted (lanes 3 and 4). Undigested

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samples were also subjected to the same sarkosyl extraction(lanes 5-8).The distribution of PrP after ultracentrifugation of deter-

gent-extracted microsomes was examined (Fig. 2B). Thesupernatant fluid and pellet were carefully separated (19) andthe supernatant fraction was centrifuged again. The initialpellet and the final supernatant fraction were then analyzedfor PrP by immunoblotting. PrP 33-35C was found in thesarkosyl supernatant fraction prepared from uninfected con-trol brains (lane 1). The protein was digested upon exposureto proteinase K (lane 2). No immunoreactive proteins werefound in the pellet (lanes 3 and 4). A protease-sensitive PrP33-35 was found in the supernatant fraction prepared fromscrapie-infected brains (lanes 5 and 7). From the proteasesensitivity of the protein, we presume that it represents PrP33-35C. PrP 33-35 appears to be extracted quantitatively bydetergent treatment, since its concentration in the superna-tant fraction is similar to that found in normal brain prepa-rations (lane 1). The pellet of the scrapie-infected extractcontained PrP 33-35sc, which was converted to PrP 27-30upon proteinase K digestion (lanes 6 and 8).

Prion Rods Form During Microsome Solubilization. Whenthe microsomes prepared from normal or scrapie-infectedhamster brains were examined by electron microscopy, onlymembrane vesicles were found (Fig. 3). No rod-shapedstructures like those found in purified prion preparationswere seen either by thin section or by negative staining. Wealso searched for rods in microsome fractions prepared bysucrose-gradient sedimentation but failed to find any rods(data not shown).

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FIG. 2. Microsomes from scrapie-infected hamster brains con-tain both PrP 33-35SC and PrP 33_35C. (A) Immunoblot of PrP.Samples in lanes 1-4 were digested for 30 min with proteinase K at100 jg/ml. No proteinase K was added to the samples of lanes 5-8.Odd-numbered lanes, samples obtained from scrapie-infected ham-sters; even-numbered lanes, samples obtained from uninfectedanimals. Sarkosyl was added at a final concentration of2% (wt/vol)to aliquots of the microsomal fractions (lanes 1, 2, 5, and 6). (B)Microsomal fractions were extracted and centrifuged as described inthe text. The samples were analyzed for PrP by immunoblotting.Supernatant and pellet obtained by prolonged centrifugation weretested. Lanes: 1-4, samples were obtained from uninfected animals;5-8, samples were obtained from scrapie-infected hamsters. Lanes1 and 5, supernatant, no proteinase K added; lanes 2 and 6, pellet,no proteinase K added; lanes 3 and 7, supernatant digested byproteinase K; lanes 4 and 8, pellet digested by proteinase K.Digestions were for 30 min at 25°C. No proteinase K-resistant proteinis detectable in the supernatant fraction after ultracentrifugation(lane 7). The amount of protease-sensitive PrP 33-35 in the super-natant fluid after centrifugation is similar to that found in fractionsfrom uninfected animals (compare lane 1 with lane 5).

FIG. 3. Microsomes from scrapie-infected hamster brains do notcontain prion rods. Microsomes were collected by centrifugation for1 hr at 100,000 x g. The pellets were either processed for thinsections (A and C) or were negatively stained with uranyl formate (Band D). (A and B) Samples from normal animals; (C and D) samplesfrom scrapie-infected hamsters. (Bars = 100 nm.)

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Addition of purified prions containing 108 ID50 units andnumerous rods to a microsomal prion fraction containing anequivalent number of ID50 units shows that the rods wouldhave been easily recognized among the microsomal mem-branes had they been there (Fig. 4A). No rods were found insarkosyl-extracted fractions isolated from uninfected controlbrains (Fig. 4B). However, rods were found in the detergent-extracted pellets prepared from microsomes of scrapie-infected brains (Fig. 4 C and D). It is this pellet in which PrP33-35sc was found (Fig. 2B, lanes 6 and 8). The formation ofprion amyloid rods is not dependent on extraction withanionic detergents such as sarkosyl, because rods have alsobeen found after extraction with the nonionic detergentoctylglucoside (data not shown).

DISCUSSIONTo test the membrane association of PrP, extraction exper-iments were performed under conditions known to releaseperipheral or integral membrane proteins (20). The micro-somal fractions were used, because they were free of nuclei,mitochondria, and cytoskeletal elements. Extraction of theisolated microsomes with NaCl or EDTA failed to release thePrP (Fig. 1). Osmotic shock with distilled water also failed torelease most of these proteins. In contrast, detergent extrac-tion of the membranes solubilized PrP 33-35c, while PrP33-35sc aggregated into rods (Figs. 2B and 4). These obser-

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FIG. 4. Detergent solubilization of scrapie-infected brainmicrosomes produces prion rods. (A) Prion rods added to scrapie-infected microsomes. (B) Sarkosyl at a final concentration of 2%(wt/vol) added to normal microsomes; no rod-shaped structureswere found. (C and D) Sarkosyl added to scrapie microsomesproduced prion rods. All specimens were negatively stained withuranyl formate. (Bars = 100 nm.)

vations suggest that both PrP 33-35C and PrP 33-35SC aremembrane-bound proteins that may span the membranebilayer (20, 21). Our finding that PrP 33-35Sc is converted toPrP 27-30 whether or not microsomal membranes wereextracted with detergent argues against the notion that largeamounts ofPrP 33-35sc are trapped within membrane vesicles(Fig. 2A). The translated cDNA sequence of the PrP showstwo helices composed of amino acids with hydrophobic sidechains (11). One helix is near the NH2 terminus of PrP 27-30and the other is at the COOH terminus. Sequencing ofclonedgenomic PrP DNA and a 2.1-kilobase PrP cDNA demon-strated a signal sequence at the apparent NH2 terminus ofPrP33-35 proteins (K. Basler, M. Scott, B. Oesch, M.P.M., D.Westaway, S.B.P., and C. Weissmann, unpublished obser-vations).The experimental results described here are consistent

with earlier studies describing the association of scrapieinfectivity with membrane fractions (22-24). These observa-tions gave rise to the scrapie membrane hypothesis and thenotion that separation of infectivity from membranes was notpossible (25-27). Subsequently, characterization of deter-gent-extracted fractions demonstrated an extreme number ofmolecular forms ofthe scrapie agent and suggested that thesemight arise from hydrophobic interactions (28-30).That PrP 33-35sc was not solubilized by detergent can be

explained by aggregation ofthis protein into amyloid rods (6).Indeed, electron micrographs of pellets obtained from deter-gent-extracted membranes revealed rods similar to thosefound in purified prion preparations (Fig. 4) (3, 6, 16, 31).Rods were not found in unextracted samples (Fig. 3). Eitherthe rods are hidden within the membranes or they form aftersolubilization of the membranes. The latter possibility seemsmost likely since no rods were detected in thin sections ofthemicrosomal membranes (Fig. 3). Solubilization of mem-branes may allow PrP 33-35sc molecules to make closecontact with each other and to polymerize into amyloid rods.We estimate that in vivo =1% of Prp 33-35sc exists asextracellular filamentous polymers, which have been identi-fied within amyloid plaques in brains of scrapie-infectedhamsters (12). Presumably, the remaining PrP 33-35sc mole-cules are membrane bound.We separated PrP 33-35c from PrP 33-35Sc by ultracentrif-

ugation after detergent solubilization of microsomes, sincePrP 33-35C does not polymerize into rods (Fig. 2B). Thereappears to be little or no difference between the amount ofPrP 33-35C in the brains of scrapie-infected and controlhamsters (Table 2). All the additional immunoreactive pro-tein of Mr 33,000-35,000 found in scrapie-infected hamster

Table 2. Cellular and scrapie PrP in hamstersProperty PrP 33-35c PrP 33-35Sc

Uninfected brain Present AbsentScrapie brain Level unchanged AccumulatesConcentration*

Proc. Natl. Acad. Sci. USA 83 (1986)

brains appears to be PrP 33-35Sc. An important and distinc-tive structural feature of the PrP 33-35sc is its proteaseresistance (32); it is converted to PrP 27-30 during proteolyticdigestion. More than 60 amino acids are removed from theNH2 terminus and possibly 20 residues are removed from theCOOH terminus (11); PrP 27-30 is relatively resistant tofurther degradation (32). Proteolytic digestion of scrapie-infected microsomes generated PrP 27-30 without furtherdegradation both before and after extraction with detergent(Fig. 2A). This observation argues against the protection ofprotease-sensitive domains as a result of PrP 33-35sc poly-merization into prion rods. We have attempted to render PrP33-35C resistant to proteolytic digestion by concentrating it asmuch as 100-fold prior to proteolysis; however, this proce-dure was unsuccessful at inducing protease resistance. Theseresults suggest that polymerization and protease resistancecannot be explained by concentration alone. Specificglycosylation ofPrP 33-35sc might render the core (PrP 27-30)resistant to proteolytic degradation (33).The role of PrP 33-35C in cellular metabolism and the

structure that prevents its polymerization into rods areunknown. The molecular basis for the different properties ofPrP 33-35c and PrP 33-35sc is unknown. Whether thesedifferences arise from alterations in amino acid sequence,post-translational modifications, or protein conformationremains to be established. Indeed, this difference betweenPrP 33-35c and PrP 33-35sc may be quite small, as in the caseof sickle cell hemoglobin where a single amino acid changeresults in diminished oxygen binding and polymerization intofilaments (34). Perhaps PrP 33-35c has some as yet undefinedreceptor function on the external surface of cells. In contrastto PrP 33-35c, PrP 33-35sc behaves like an amphipathicprotein, which explains its association with and its possibleintegration into membranes as well as its aggregation intorods (Table 2). Within a single amphipathic protein, bothhydrophobic and hydrophilic properties are found. Recentstudies have shown that brain prion proteins are synthesizedprimarily in neurons (35). Our findings suggest that neuronaldegeneration does not occur during scrapie infection bydecreasing the levels ofPrP 33-35C; instead, the accumulationof a similar but slightly different protein, PrP 33-35sc, mayplay a pivotal role in the pathogenesis of scrapie.

We thank Dr. Detlev Riesner for his valuable discussions andcritical reading ofthe manuscript, as well as L. Gallagher for editorialassistance. This work was supported by research grants from theNational Institutes of Health (AG02132 and NS14069) as well as bygifts from R. J. Reynolds Industries, Inc., and Sherman FairchildFoundation. R.K.M. was supported by a research grant from theSwiss National Foundation (83.302.0.84).

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