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Effect of Glycosylphosphatidylinositol Anchor-dependent and -independent Prion Protein Association with Model Raft Membranes on Conversion to the Protease-resistant Isoform

2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês

10.1074/jbc.m210840200

ISSN

1083-351X

Autores

Gerald S. Baron, Byron Caughey,

Tópico(s)

Neurological diseases and metabolism

Resumo

Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI+) and GPI anchor-deficient (GPI−) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34–94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI+ PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI+ PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI− PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res. Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI+) and GPI anchor-deficient (GPI−) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34–94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI+ PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI+ PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI− PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res. prion protein glycosylphosphatidylinositol detergent-resistant membrane protease-sensitive PrP protease-resistant PrP sphingolipid-cholesterol-rich liposome GPI anchor containing GPI anchor-deficient by deletion of GPI signal sequence phosphatidylinositol-specific phospholipase C GPI anchor-deficient by PI-PLC cleavage citrate-buffered saline polyethylene glycol proteinase K wild-type phosphatidylcholine brain sphingomyelin brain cerebrosides cholesterol N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphatidylserine peptide N-glycosidase F Prion protein (PrP)1 is a glycoprotein usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor (1Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (913) Google Scholar). Like other GPI-anchored proteins, PrP is enriched in sphingolipid- and cholesterol-rich membrane microdomains known as detergent-resistant membranes (DRMs), or rafts (2Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.W.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar). Several lines of evidence from biochemical and molecular biological approaches suggest raft association is required for conversion of the normal protease-sensitive isoform (PrP-sen) to the transmissible spongiform encephalopathy-associated protease-resistant isoform (PrP-res) in a cell culture model of infection (2Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.W.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 3Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (518) Google Scholar, 4Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar, 5Naslavsky N. Stein R. Yanai A. Friedlander G. Taraboulos A. J. Biol. Chem. 1997; 272: 6324-6331Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 6Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 7Mangé A. Nishida N. Milhavet O. McMahon H.E.M. Casanova D. Lehmann S. J. Virol. 2000; 74: 3135-3140Crossref PubMed Scopus (116) Google Scholar). Although cell-free studies using purified PrP molecules have provided new insights into binding and conversion of PrP-sen by PrP-res (reviewed in Ref. 8Caughey B. Raymond G.J. Callahan M.A. Wong C. Baron G.S. Xiong L.W. Adv. Protein Chem. 2001; 57: 139-169Crossref PubMed Scopus (55) Google Scholar), few studies have considered the membrane-associated nature of PrP (9DeArmond S.J. Qiu Y. Sanchez H. Spilman P.R. Ninchak-Casey A. Alonso D. Daggett V. J. Neuropathol. Exp. Neurol. 1999; 58: 1000-1009Crossref PubMed Scopus (101) Google Scholar, 10Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 11Zuegg J. Gready J.E. Glycobiology. 2000; 10: 959-974Crossref PubMed Scopus (82) Google Scholar, 12Mahfoud R. Garmy N. Maresca M. Yahi N. Puigserver A. Fantini J. J. Biol. Chem. 2002; 277: 11292-11296Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 13Sanghera N. Pinheiro T.J.T. J. Mol. Biol. 2002; 315: 1241-1256Crossref PubMed Scopus (166) Google Scholar) and the influence of this association on PrP-sen/PrP-res interactions (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Given the complex composition of cellular raft membranes, which contain molecules other than PrP-sen that might influence interactions with PrP-res, investigations into the effect of PrP-sen association with rafts on these interactions would benefit from the use of a defined system that replicated raft membranes in the absence of other raft-associated molecules. One candidate system involves the use of sphingolipid-cholesterol-rich raft-like liposomes (SCRLs) containing phosphatidylcholine, sphingomyelin, brain cerebrosides, and cholesterol. SCRLs have been shown to resemble rafts in several respects, including major lipid composition and low buoyant density, which permits their isolation by floatation through density gradients (15Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar). GPI-anchored proteins reconstituted into SCRLs or related sphingolipid-rich liposomes acquire properties of their cell-associated counterparts, most notably insolubility in cold Triton X-100 (15Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar, 16Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Hence, SCRLs serve as a reasonable approximation of the natural membrane environment of PrP in the absence of other raft-associated molecules. Our previous work examined the effect of PrP-sen association with rafts on interactions with PrP-res using raft membranes prepared from neuroblastoma cells (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). These experiments showed that raft-bound PrP-sen resisted conversion to PrP-res until PrP-sen was released from rafts by phospholipase digestion or the PrP-res was inserted into contiguous membranes. To examine the effect of PrP-sen membrane association on its interactions with PrP-res under more defined conditions and to determine if membrane association itself inhibits conversion of PrP-sen by exogenous PrP-res, we have employed a model system using PrP-sen reconstituted into SCRLs. While developing this system, two groups recently reported a novel property of recombinant PrP-sen expressed in Escherichia coli: binding to model membranes of various compositions (10Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 13Sanghera N. Pinheiro T.J.T. J. Mol. Biol. 2002; 315: 1241-1256Crossref PubMed Scopus (166) Google Scholar). We had also observed this phenomenon in our system using GPI anchor-deficient PrP-sen expressed in mammalian cell lines and have further characterized the nature of this binding activity using various forms of PrP-sen that may more closely represent the native state of the molecule, particularly with respect to the addition of N-linked glycans. Furthermore, we have directly tested the effect of the two methods of PrP-sen association with membranes (i.e. GPI anchor-dependent and -independent) on its interactions with exogenous PrP-res molecules as assayed by its ability to serve as a substrate for conversion to the protease-resistant state under cell-free conditions. Our results indicate that the method of PrP-sen association with model membranes has strikingly different effects on its ability to interact with PrP-res. Hamster PrP-sen was derived from mouse fibroblast cell lines expressing either full-length (GPI+ PrP-sen) or GPI anchor-deficient (GPI− PrP-sen) wild-type (wt) PrP-sen (17Chesebro B. Wehrly K. Caughey B. Nishio J. Ernst D. Race R. Dev. Biol. Stand. 1993; 80: 131-140PubMed Google Scholar, 18Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar). The corresponding N-terminal hamster PrP-sen deletion mutants were derived from cell lines created by Lawson and co-workers (19Lawson V.A. Priola S.A. Wehrly K. Chesebro B. J. Biol. Chem. 2001; 276: 35265-35271Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Wild-type GPI+ mouse PrP-sen was isolated from a mouse neuroblastoma cell line described elsewhere (20Wong C. Xiong L.-W. Horiuchi M. Raymond L. Wehrly K. Chesebro B. Caughey B. EMBO J. 2001; 20: 377-386Crossref PubMed Scopus (211) Google Scholar). PrP-sen molecules were immunoprecipitated from cells metabolically labeled with [35S]methionine as described previously (21Caughey B. Horiuchi M. Demaimay R. Raymond G.J. Methods Enzymol. 1999; 309: 122-133Crossref PubMed Scopus (31) Google Scholar). Mouse PrP-sen was immunoprecipitated using rabbit antiserum (R30) against a PrP synthetic peptide (amino acids 89–103) (22Caughey B. Raymond G.J. Ernst D. Race R.E. J. Virol. 1991; 65: 6597-6603Crossref PubMed Google Scholar). Hamster PrP-sen was immunoprecipitated with 3F4 monoclonal antibody (23Kascsak R.J. Rubenstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar) or rabbit polyclonal antiserum (R20) raised against a C-terminal (amino acids 218–232) PrP synthetic peptide (22Caughey B. Raymond G.J. Ernst D. Race R.E. J. Virol. 1991; 65: 6597-6603Crossref PubMed Google Scholar). The R20 antiserum was used specifically to isolate PrP-sen molecules (both wt and mutants) for experiments involving the N-terminal deletion mutants, because one mutant (Δ124) lacks the 3F4 epitope. Cell culture supernatants containing phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from metabolically labeled cells expressing GPI+ PrP-sen were prepared as described previously (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Where indicated, PI-PLC-released hamster PrP-sen (referred to as GPIPI-PLC PrP-sen) was immunoprecipitated from the supernatants as described elsewhere with 3F4 or R20 (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). For some experiments, PI-PLC culture supernatant proteins were deglycosylated with PNGase F (New England BioLabs) as per the manufacturer's instructions prior to PrP-sen immunoprecipitation. Sphingolipid-cholesterol-rich liposomes (SCRLs) were prepared essentially as described previously (15Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar) with exceptions as noted below. All lipids were obtained from Avanti Polar Lipids. SCRLs contained bovine liver phosphatidylcholine (PC)/brain sphingomyelin (SM)/brain cerebrosides (CB)/cholesterol (Chol) in a 2:1:1:2 molar ratio. For some experiments, modified SCRLs were prepared consisting of PC/CB/Chol (1:1:1), PC/SM/Chol (1:1:1), PC/SM/CB (2:1:1), SM/CB/Chol (1:1:2), or PC/Chol (1:1). For each type of modified SCRL, the total moles of lipid used in the preparation as well as the molar ratio of phospholipid:sphingolipid:cholesterol was matched to that contained in SCRLs where possible. SCRLs were usually prepared in citrate-buffered saline (CBS, 10 mm citrate, 137 mm NaCl, pH 6.0) except for the experiments with modified SCRLs (Tricine-buffered saline, pH 7.8) and the reconstitution experiments to evaluate the effect of pH where saline buffered with acetate (pH 5.0), phosphate (pH 7.0), or Tricine (pH 7.8) was also used. After dialysis, SCRLs were either used for reconstitution or binding studies as outlined below. PrP-sen was reconstituted into SCRLs using a protocol adapted from Schroeder et al. (15Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar). [35S]PrP-sen was adjusted to contain 137 mmNaCl and 0.8% Sarkosyl just before addition to SCRLs that had been freeze-thawed three times. The samples were rapidly mixed and dialyzed immediately in Slide-a-lyzer cassettes (Pierce) against the buffer in which the SCRLs were prepared (usually CBS). The cassettes were manually agitated approximately hourly for the first few hours of dialysis to keep the liposomes well dispersed. The final concentration of Sarkosyl in the initial PrP-sen/SCRL mixture was ≤0.08%. Large scale preparations usually contained about 200 ng of [35S]PrP-sen (∼1–2 × 106 cpm) and 2 mg (3.2 × 10−6 mol) of lipids. After overnight dialysis, the PrP/liposome mixture was adjusted to 10% Optiprep (Invitrogen) in the appropriate buffered saline. The sample was then overlaid with a linear step gradient of Optiprep in buffered saline consisting of either five steps (1%, 2.5%, 4%, 5.5%, and 7%) or three steps (1%, 4%, and 7%). Large scale gradients contained 300 μl (for five steps) or 500 μl (for three steps) per step for the overlaid fraction and were centrifuged in a Beckman SW50.1 rotor at 21,000 rpm for 90 min at 4 °C in polycarbonate tubes. To prepare Triton X-100-treated SCRLs containing GPI+ PrP-sen, the dialyzed PrP-sen/SCRL mixture was first diluted with 1 ml of CBS and centrifuged in a Beckman SW50.1 rotor at 40,000 rpm for 60 min at 4 °C to pellet the liposomes. The pellet was then resuspended in 300 μl of 1% Triton X-100 in CBS and incubated for 20 min on ice prior to adjustment to 10% Optiprep and centrifugation as above. After centrifugation, the SCRL lipid band was collected and stored on ice until use. Relative to the input cpm, typically ∼40–60% of the GPI− or GPI+ PrP-sen was associated with the SCRLs (without Triton X-100 treatment) and ∼20–30% (of GPI+ PrP-sen) was recovered in the SCRL fractions after Triton X-100 extraction. Small scale preparations (scaled down 1/10 for amount of PrP-sen and lipids) were used to evaluate the effect of lipid composition and pH on incorporation efficiency. Small scale gradients for analytical purposes contained 30 μl (for five steps) or 50 μl (for three steps) per step for the overlaid fraction and were centrifuged in a Beckman TLS-55 rotor at 25,000 rpm for 90 min at 4 °C in polycarbonate tubes. Six fractions were collected from these gradients starting from the top: five gradient fractions of 30 μl each and one fraction corresponding to the bottom (i.e. the volume of sample loaded in 10% Optiprep). For five-step gradients, the lipid band formed at the 1%/2.5% Optiprep interface and the majority of the lipid was collected in fractions 1 and 2. For three-step gradients, the lipid band formed at the 1%/4% interface and the majority of the lipid was collected in fraction 2. The fractions were mixed with 20 μg of thyroglobulin, and proteins were precipitated with 4 volumes of cold methanol. After centrifugation at 21,000 × g for 20 min, methanol pellets were resuspended in SDS-PAGE sample buffer, boiled for 5 min, and analyzed on Novex pre-cast acrylamide gels. Radioactive proteins were visualized and quantitated using a Storm PhosphorImager instrument (Amersham Biosciences). SCRLs were first washed in CBS and concentrated by centrifugation as described above. For experiments for testing the pH dependence of the binding, SCRLs were washed and resuspended in buffered saline of the appropriate pH (i.e. acetate (pH 5.0), citrate (pH 6.0), HEPES (pH 7.0), or Tricine (pH 7.8) each at 10 mm). To bind PrP-sen to SCRLs in the absence of detergent, [35S]PrP-sen (10,000–15,000 cpm or ∼1–2 ng) was added to SCRLs (∼67 μg of lipid) in binding buffer consisting of a final concentration of 50 mm buffer of appropriate pH (usually citrate), 137 mm NaCl. To bind35S-labeled PI-PLC-released/secreted proteins to SCRLs in the absence of detergent, PI-PLC culture supernatant (1/25 to 1/50 equivalents released from a T-25 flask of cells) was mixed with SCRLs (∼67 μg of lipid) in 50 mm CBS (final concentration). Binding reactions were incubated at 37 °C for 1–2 h unless indicated otherwise and were mixed at ∼15-min intervals to keep the liposomes well dispersed. The reactions were then chilled on ice, adjusted to 10% Optiprep, and processed on small scale analytical gradients as described above. In some cases, binding was quantitated by liquid scintillation counting of the gradient fractions and included boiling the empty centrifuge tube in a volume of SDS-PAGE sample buffer (30 μl) equal to the volume of the "bottom" fraction to ensure recovery of any residual protein. These results are expressed as the mean percentage of counts recovered in the low density fractions (1Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (913) Google Scholar, 2Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.W.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 3Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (518) Google Scholar, 4Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar)versus total counts recovered (sum of the six gradient fractions and protein recovered after boiling the empty tube in SDS-PAGE sample buffer). Where indicated, gradient fractions were deglycosylated with PNGase F prior to methanol precipitation or immunoprecipitation with 3F4 antibody and SDS-PAGE as above. Binding reactions were scaled up to contain ∼200 ng of [35S]GPI− PrP-sen and 2 mg of lipid and fractionated on a 600-μl gradient (200 μl each of 1%, 4%, and 7% Optiprep) in a TLS-55 rotor as above to prepare sufficient PrP-containing SCRLs for use in cell-free conversion reactions. Protein-containing SCRLs were harvested from Optiprep gradients, washed in buffered saline, and centrifuged in an ultracentrifuge as described above to recover the SCRLs. Liposome pellets were usually resuspended directly in extraction buffer consisting of either 0.1 m sodium carbonate (pH 11.5), 1 or 3 m NaCl in 10 mm citrate buffer (pH 6.0), or 1% Triton X-100 in CBS. In some cases, an SCRL pellet was resuspended in CBS and mixed with one volume of 2% Triton X-100 in CBS or 2m NaCl in citrate buffer. Extractions with NaCl or sodium carbonate were incubated on ice for 30–60 min. Extractions with Triton X-100 were either incubated on ice or at 37 °C for 20 min. Samples were then adjusted to contain 10% Optiprep and processed on small scale analytical gradients as above. Cell-free conversion reactions were performed essentially as described previously in 50 mm citrate (pH 6.0), 137 mm NaCl, and 5 mm MgCl2 (conversion buffer) supplemented with 100 μg/ml heparan sulfate and protease inhibitors (0.1 mmphenylmethylsulfonyl fluoride, 0.7 μg/ml pepstatin A, 1 μg/ml aprotinin) with exceptions noted below (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Crude brain microsomes were prepared either from mice infected with 87V mouse-adapted scrapie or hamsters infected with 263K hamster-adapted scrapie and used as a source of PrP-res (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Microsomes from normal uninfected animals were used as a control. [35S]PrP-sen-containing SCRLs were washed and resuspended in CBS prior to addition to conversion reactions (∼10,000–15,000 cpm/reaction). Reactions containing the different forms of GPI− PrP-sen were adjusted to contain equivalent amounts of input radioactivity and simply involved mixing the PrP-sen-containing SCRLs or purified PrP-sen with microsomes in conversion buffer with heparan sulfate and protease inhibitors. Also, the [35S]GPI− PrP-sen used in all these reactions was derived from a single preparation of protein divided into three parts: one part was added directly to the reactions (free PrP-sen), a second part was used to prepare PrP-sen bound to SCRLs in the presence of Sarkosyl, and the remaining portion was used to prepare PrP-sen bound to SCRLs in the absence of Sarkosyl. Reactions using GPI+ PrP-sen-containing SCRLs were set up as described for [35S]DRMs in our previous study, although we modified our protocol to include a dilution step (addition of 50 μl of conversion buffer) after the 5-min incubation at room temperature with polyethylene glycol (PEG) or conversion buffer to allow efficient recovery of membranes from PEG-treated reactions in the subsequent pelleting step (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Conversion reactions were incubated at 37 °C for either 2 days or 1 day (in the case of some of the GPI− PrP-sen reactions) and processed as described previously with incubation at 37 °C for 20 min in 50 mmTris-HCl (pH 8.0), 0.5% Triton X-100, 0.5% sodium deoxycholate, and 137 mm NaCl (1× extraction buffer) followed by proteinase K (PK) digestion for 1 h with 33 μg/ml PK (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). In some cases, the samples were deglycosylated after PK digestion but before methanol precipitation by treatment with PNGase F (New England BioLabs) as per the manufacturer's instructions. Immunoblot detection of PrP was performed as described by Horiuchi et al. (24Horiuchi M. Chabry J. Caughey B. EMBO J. 1999; 18: 3193-3203Crossref PubMed Scopus (207) Google Scholar) using a mouse/human recombinant monoclonal antibody Fab (R1, 0.36 μg/ml) that binds hamster PrP between residues 220 and 231 (25Williamson R.A. Peretz D. Pinilla C. Ball H. Bastidas R.B. Rozenshteyn R. Houghten R.A. Prusiner S.B. Burton D.R. J. Virol. 1998; 72: 9413-9418Crossref PubMed Google Scholar) and was a generous gift from Drs. Anthony Williamson and Dennis Burton (The Scripps Research Institute). Bound R1 antibody was detected using alkaline phosphatase-conjugated goat anti-human IgG secondary antibody (Sigma (A8542)) at a 1:10000 dilution. We adapted a previously described technique (15Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar) to allow reconstitution of PrP-sen into SCRLs. Hamster PrP-sen was expressed in fibroblasts as either full-length GPI-anchored protein (GPI+) or a GPI anchor-deficient (GPI−) form lacking the GPI anchor addition sequence. Purified PrP-sen was mixed with SCRLs in the presence of a low concentration (≤0.08%) of Sarkosyl and immediately dialyzed against buffered saline. SCRLs and SCRL-bound proteins were then isolated by floatation through a density gradient. GPI+ PrP-sen stably associated with SCRLs in the low density fractions (1Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (913) Google Scholar, 2Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.W.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 3Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (518) Google Scholar, 4Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar) of the gradient (Fig. 1A). Surprisingly, control experiments with GPI− PrP-sen revealed that this derivative also avidly associated with SCRLs (Fig.1B). When fractionated on gradients in the absence of SCRLs, all of the PrP-sen (both GPI+ and GPI−) was found in the bottom fraction (fraction 6) indicating that floatation of PrP-sen in the gradient is dependent upon SCRLs (data not shown). To verify the SCRL association was not due to trapping of PrP-sen inside the liposomes, we incubated the PrP-containing SCRLs with proteinase K (PK). As shown in Fig. 2, the vast majority (∼90%) of SCRL-bound PrP-sen was susceptible to PK digestion without detergent-mediated disruption of the liposomes, indicating that the bulk of the protein was surface-accessible. These data show that PrP-sen exhibits an alternative, GPI anchor-independent method of associating with model membranes, a conclusion consistent with other recent studies (10Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 13Sanghera N. Pinheiro T.J.T. J. Mol. Biol. 2002; 315: 1241-1256Crossref PubMed Scopus (166) Google Scholar).Figure 2PrP-sen reconstituted into SCRLs is surface-localized. SCRLs containing reconstituted [35S]PrP-sen as described in Fig. 1 were digested with PK (10 μg/ml) in the presence or absence of detergent (1% Triton X-100) at 37 °C for 50 min. Results are representative of two independent experiments, each performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The nature of the binding interactions of the PrP-sen molecules with SCRLs was characterized by subjecting the PrP-bound SCRLs to various extraction conditions. Treatments with either high salt (1 m NaCl (Fig.3A) or 3 m NaCl (data not shown)) or 0.1 m sodium carbonate (pH 11.5) (Fig.3B), which extract peripheral membrane proteins, failed to remove either type of PrP-sen molecule from SCRLs. A significant fraction (∼40–50%) of GPI+ PrP-sen resisted extraction with Triton X-100 at 4 °C (Fig. 3C, lanes 1and 2 versus lane 6) but not 37 °C (Fig. 3D, lanes 1 and 2 versus lane 6), resembling the behavior of GPI-anchored proteins in rafts (14Baron G.S. Wehrly K. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar, 26Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar). Treatment of the cold-Triton X-100-extracted SCRLs with PI-PLC to cleave the GPI anchors caused the release of a majority (∼60%) of the formerly GPI-anchored PrP-sen molecules from the SCRLs upon re-extraction with cold Triton (data not shown). However, GPI− PrP-sen was readily extracted with cold Triton X-100 without PI-PLC treatment (Fig. 3C,lanes 7 and 8 versus lane 12), suggesting that this GPI anchor-independent method of attachment was mediated by hydrophobic protein-lipid interactions. A very small proportion of GPI− PrP-sen apparently resisted extraction by cold Triton X-100, possibly due to use of an insufficient amount of detergent or inversion o

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