Sabrina Cronier

Nathalie Gros

M Howard Tattum

Graham S Jackson

Anthony R Clarke

John Collinge

Jonathan D F Wadsworth
email: j.d.wadsworth@prion.ucl.ac.uk

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Keywords: prion, prion protein, scrapie, thermolysin, transmissible spongiform encephalopathy, variant Creutzfeldt-Jakob disease CJD, Creutzfeldt-Jakob disease, PK, proteinase K, PrP, prion protein, PrPP C , cellular PrP isoform, PrP Sc , pathogenic PrP isoforms, vCJD, variant Creutzfeldt-Jakob disease

Disease-related prion protein, PrP Sc , is classically distinguished from its normal cellular precursor, PrP C , by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease resistant fragments of PrP Sc using proteinase K (PK), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrP Sc in its full-length form. Here we show that thermolysin can degrade PrP C while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In variant Creutzfeldt-Jakob disease (vCJD), the human counterpart of bovine spongiform encephalopathy (BSE), up to 90 % of total PrP present in brain resists degradation with thermolysin whereas only ~15 % of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases.

INTRODUCTION

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), kuru and variant CJD (vCJD) in humans [1][START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Wadsworth | Update on human prion disease[END_REF]. Their central feature is the post-translational conversion of host-encoded, cellular prion protein (PrP C ), to an abnormal isoform, designated PrP Sc [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. Human prion diseases are biologically unique in that the disease process can be triggered through inherited germline mutations in the human prion protein gene (PRNP), infection (by inoculation, or in some cases by dietary exposure) with prion-infected tissue or by rare sporadic events that generate PrP Sc [1][START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Wadsworth | Update on human prion disease[END_REF][START_REF] Collinge | Molecular neurology of prion disease[END_REF]. According to the proteinonly hypothesis [START_REF] Griffith | Self Replication and Scrapie[END_REF], an abnormal PrP isoform is the principal, if not the sole, component of the transmissible prion, with prion propagation occurring through PrPP Sc acting to replicate itself with high fidelity by recruiting endogenous PrP C [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Weissmann | The state of the prion[END_REF][START_REF] Collinge | A general model of prion strains and their pathogenicity[END_REF]. Within the framework of the protein-only hypothesis of prion propagation, the distinct clinical and neuropathological phenotypes that distinguish prion strains are thought to be determined by the propagation of PrP Sc isoforms with divergent physicochemical properties [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Collinge | A general model of prion strains and their pathogenicity[END_REF][START_REF] Bessen | Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy[END_REF][START_REF] Telling | Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity[END_REF][START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD[END_REF][START_REF] Parchi | Molecular Basis of Phenotypic Variability in Sporadic Creutzfeldt-Jakob Disease[END_REF][START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF].

PrP Sc is extracted from affected tissue as highly aggregated, detergent insoluble, material that is not amenable to high-resolution structural techniques. However, Fourier transform infrared spectroscopic methods show that PrP Sc , in distinction to PrP C , has a high β-sheet content [START_REF] Pan | Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins[END_REF][START_REF] Caughey | Strain-dependent differences in β-sheet conformations of abnormal prion protein[END_REF]. Biochemically, PrP Sc can be distinguished from PrP C by its partial resistance to proteolysis and its marked insolubility in detergents (for review see [1,[START_REF] Riesner | Biochemistry and structure of PrP(C) and PrP(Sc)[END_REF]). Under conditions in which PrP C exists as a detergent soluble monomer and is completely degraded by the non-specific protease proteinase K (PK), PrPP Sc exists in an aggregated form with the C-terminal two thirds of the protein showing marked resistance to proteolytic degradation leading to the generation of amino terminally truncated fragments of di-, mono-and non-glycosylated PrP [1,[START_REF] Riesner | Biochemistry and structure of PrP(C) and PrP(Sc)[END_REF].

Although the molecular diagnosis of prion disease has historically relied upon the detection of PrP Sc using PK, it has become apparent that PK-sensitive pathological isoforms of PrP may have a significant role in prion disease pathogenesis [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF][START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Tremblay | Mutant PrP(Sc) Conformers Induced by a Synthetic Peptide and Several Prion Strains[END_REF][START_REF] Safar | Diagnosis of human prion disease[END_REF][START_REF] Nazor | Immunodetection of diseaseassociated mutant PrP, which accelerates disease in GSS transgenic mice[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF][START_REF] Thackray | Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay[END_REF]. In particular in inherited prion disease, PrP Sc isoforms may be generated with unique physicochemical properties, reflected by sensitivity to PK digestion and PrP Sc /prion infectivity ratios that can be very different from the PrPP Sc types propagated in sporadic and acquired forms of human prion disease (for review see [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Wadsworth | Update on human prion disease[END_REF][START_REF] Hill | Distinct glycoform ratios of protease resistant prion protein associated with PRNP point mutations[END_REF]). Accordingly, the development of new diagnostic tests that do not rely on PK digestion is required, and in this context, a conformation-dependent immunoassay [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF] shows high diagnostic sensitivity in human prion disease [START_REF] Safar | Diagnosis of human prion disease[END_REF]. More recently, Owen and colleagues reported thermolysin as a complementary tool to PK. Thermolysin destroys PrP C in ovine or bovine brain while leaving PrP Sc in its full-length form, thereby allowing the amino-terminal domain of PrP Sc to be exploited for improved methods of prion disease diagnosis [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF] or prion strain discrimination [START_REF] Owen | Molecular profiling of ovine prion diseases using thermolysin-resistant PrPSc and endogenous C2 PrP fragments[END_REF]. In this study, we now extend these findings and show that thermolysin preserves both PK-sensitive and PK-resistant disease-related isoforms of PrP while concomitantly destroying PrP C in both rodent and human brain. Using mouse RML prions, we were able to investigate prion infectivity associated with PK-sensitive PrP isoforms. It is anticipated that these methods will facilitate detailed biochemical characterization of PK-sensitive isoforms of disease-related PrP associated with multiple prion strain/host combinations.

MATERIALS AND METHODS

Prion infected tissues

Storage and biochemical analysis of human brain samples was performed with consent from relatives and with approval from the Local Research Ethics Committee of the Institute of Neurology/National Hospital for Neurology and Neurosurgery. All procedures were carried out in a microbiological containment level III facility with strict adherence to safety protocols. Ten percent (w/v) brain homogenates from patients with neuropathologically confirmed variant Creutzfeldt-Jakob disease (vCJD) or from control samples of normal human brain were prepared in Dulbecco's phosphate buffered saline (D-PBS) lacking Ca 2+ or Mg 2+ ions by serial passage through needles of decreasing diameter or the use of tissue grinders (Anachem, Luton, UK) [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD[END_REF][START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay[END_REF]. Brains from two hundred terminal CD-1 mice infected with the RML prion strain [START_REF] Chandler | Encephalopathy in mice produced by inoculation with scrapie brain material[END_REF] were homogenized in D-PBS lacking Ca 2+ or Mg 2+ ions using tissue grinders and pooled to produce 970 ml of 10 % (w/v) RML brain homogenate (designated I6200). Two batches of eighteen brains from uninfected CD-1 mice were homogenized to produce two pools of ~90 ml of 10 % (w/v) normal CD-1 brain homogenate (designated I7219 and I8402). Similar procedures were used to generate 10 % (w/v) brain homogenates from hamsters terminally infected with the Sc237 prion strain or from normal uninfected hamsters. Pooled homogenates were dispensed as aliquots and maintained at -70 °C until use.

Titration of RML prions in CD-1 mice

All procedures were carried out in a microbiological containment level III facility with strict adherence to safety protocols. Care of mice was according to institutional guidelines. Ten percent (w/v) RML brain homogenate (I6200) was serially diluted 10 -1 to 10 -8 using 1 % (w/v) normal CD-1 brain homogenate as dilutant. Aliquots of each dilution were inoculated either intracerebrally (30 μl) or intraperitoneally (100 μl) into groups of 6 CD-1 mice as described previously [START_REF] Asante | BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein[END_REF][START_REF] Wadsworth | Human Prion Protein with Valine 129 Prevents Expression of Variant CJD Phenotype[END_REF][START_REF] Asante | Dissociation of pathological and molecular phenotype of variant Creutzfeldt-Jakob disease in transgenic human prion protein 129 heterozygous mice[END_REF]. Mice were examined daily and were killed if exhibiting signs of distress or once a diagnosis of clinical prion disease was established [START_REF] Carlson | Linkage of prion protein and scrapie incubation time genes[END_REF]. Infectious prion titre (LD 50 ) was calculated using the Reed-Müench formula [START_REF] Reed | A simple method of estimating fifty per cent endpoints[END_REF].

Enzymatic digestion

Thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus rokko was obtained freeze-dried from Sigma-Aldrich, St Louis, MO, USA. The specific enzymatic activity is 50-100 units / mg protein where 1 unit liberates 1 μmol of tyrosine / min at pH 7.5 and 37 °C using casein as substrate. Proteinase K (EC 3.4.21.14) from Tritirachium album limber was obtained freeze-dried from Merck Biosciences, Ltd, Nottingham, UK. The specific enzymatic activity is approximately 30 Anson units / g, where 1 Anson unit is the amount of enzyme that liberates 1 mmol of Folin positive amino acids / min at pH 7.5 and 35 °C using haemoglobin as substrate. Stock solutions of 1 mg/ml thermolysin or PK were prepared in water and aliquots stored frozen at -70 °C. Aliquots of 10 % (w/v) brain homogenates in D-PBS were digested for variable time periods with thermolysin at a final protease concentration of 100 μg/ml at 70 °C or 37 °C or with PK at a final concentration of 50 μg/ml (mouse brain) or 100 μg/ml (human brain) at 37 °C. Aliquots of the digests were snap frozen for infectivity studies, or processed immediately for analysis by either immunoblotting or ELISA. Enzymatic deglycosylation of PrP prior to immunoblotting was accomplished by incubating 20-μl aliquots of SDS and heat denatured brain homogenate with 1000 units of recombinant PNGase F (New England Biolabs Ltd, Hitchin, UK) in buffer containing 1 % Nonidet P-40 for 2 h at 37 °C according to the manufacturers instructions. Samples were precipitated with 100 % acetone for 1 h at -20 °C and centrifuged at 13 200 r.p.m. (16 100 g) for 30 min in a microfuge. Pellets were re-suspended in 1 x SDS sample buffer and analyzed by immunoblotting.

PrP immunoblotting

Aliquots of brain homogenate were mixed with an equal volume of 2 x SDS sample buffer (125 mM Tris/HCl, pH 6.8, 20 % (v/v) glycerol, 4 % (w/v) SDS, 4 % (v/v) 2-mercaptoethanol, 0.02 % (w/v) bromophenol blue containing 8 mM 4-(2-aminoethyl)benzenesulphonyl fluoride) and immediately transfered to a 100 °C heating block for 10 min. Samples were analyzed by electrophoresis and immunoblotting as described previously [START_REF] Hill | Distinct glycoform ratios of protease resistant prion protein associated with PRNP point mutations[END_REF][START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay[END_REF]. Blots were blocked in PBS containing 0.05 % (v/v) Tween-20 (PBST) and 5 % non-fat milk powder and probed with anti-PrP monoclonal antibodies ICSM 35 (D-Gen Ltd, London, UK), 3F4 (Signet Laboratories Inc, Dedham, MA, USA) or SAF32 (Spibio, Montigny le Bretonneux, France) at 0.2 μg/ml final antibody concentration in PBST. Blots were developed using anti-mouse IgG-alkaline phosphatase conjugated secondary antibody and chemiluminescent substrate CDP-Star (Tropix Inc, Bedford, MA, USA), and visualized on Biomax MR film (Kodak; Anachem Ltd, Luton, UK) as described [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay[END_REF]. Densitometric analysis of PrP was performed by using SCION IMAGE analysis software.

Detergent solubility studies

Twenty microlitre aliquots of 10 % (w/v) brain homogenate were treated with 0.5 μl benzonase (Benzon nuclease, purity 1; Merck KGaA, Darmstadt, Germany). Samples were subsequently adjusted with 80 μl PBS and 100 μl PBS containing 4 % (w/v) sodium lauroylsarcosine (Calbiochem; Merck Biosciences Ltd, Nottingham, UK) and incubated for 30 min at 37 °C with constant agitation. Samples were then centrifuged at 13 200 r.p.m. (16 100 g) for 30 min in a microfuge to generate soluble (supernatant) or insoluble (pellet) fractions. Soluble protein in the supernatant was precipitated with 1 ml cold methanol (-20 °C) and recovered by centrifugation at 13 200 r.p.m. (16 100 g) for 30 min in a microfuge. The original detergent-insoluble pellets and methanol precipitated supernatant protein pellets were resuspended to 40 μl with PBS containing 0.1 % (w/v) sodium lauroylsarcosine and 10 μl aliquots were either left untreated or digested with thermolysin (100 μg/ml final protease concentration, 30 min, 70 °C) or proteinase K (50 μg/ml final protease concentration, 1 h, 37 °C). Samples were analyzed by electrophoresis and immunoblotting as described above.

ELISA detection of PrP

Methods were adapted from previously published procedures [START_REF] Wadsworth | Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein[END_REF]. Brain homogenates were treated with thermolysin (100 μg/ml final protease concentration, 70 °C or 37 °C) or proteinase K (50 or 100 μg/ml final protease concentration, 37 °C) for a range of incubation times. Subsequently 10 μl aliquots of these samples, or untreated brain homogenate and temperature controls, were adjusted with 10 μl 4 % (w/v) sodium dodecyl sulphate (SDS) and heated at 100 °C for 10 min. Samples were centrifuged at 800 r.p.m. (100 g) for 30 s before adjustment with 600 μl 50 mM Tris pH 8.4 containing 2 % (v/v) Triton X-100, 2 % (w/v) sodium lauroylsarcosine (Calbiochem; Merck Biosciences Ltd, Nottingham, UK) and 2 % (w/v) bovine serum albumin (Fraction V, protease free) (Sigma-Aldrich, St Louis, MO, USA). Fifty microlitre aliquots were transferred into the wells of microtiter plates (Greiner, microlon 96W, Greiner Bio-One Ltd, Stonehouse, UK) containing 250 ng per well immobilized anti-PrP monoclonal antibody ICSM18 (D-Gen Ltd, London, UK). After incubation with constant agitation for 1 h at 37 °C, wells were washed with 3 × 300 μl PBST using an automated microplate washer followed by the addition of 100 μl PBS containing 1 % (v/v) Tween-20 and 1 μg/ml biotinylated anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, London, UK). Following incubation with constant agitation for 1 h at 37 °C, wells were washed (as above) followed by the addition of 100 μl PBS containing 1 % (v/v) Tween-20 and a 1 : 10 000 fold dilution of streptavidin-horseradish peroxidase conjugate (Dako UK Ltd, Ely, UK). After incubation with constant agitation for 30 min at 37 °C, wells were washed with 4 × 300 μl PBST. Wells were developed with 100 μl QuantaBlu working solution (Pierce, Rockford, IL, USA) and the reactions stopped by addition of 100

Sodium phosphotungstic acid precipitation

Methods for the use of sodium phosphotungstic acid were adapted from Safar and colleagues [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF] essentially as described previously [START_REF] Wadsworth | Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay[END_REF]. Briefly, 100 µl aliquots of 10 % (w/v) brain homogenate were treated with 1 μl benzonase and were subsequently either digested with thermolysin (100 μg/ml final protease concentration) for 90 min at 37 °C or left untreated. Thermolysin digestion was stopped by addition of EDTA (10 mM final concentration). Samples were subsequently adjusted with 100 μl PBS containing 4 % (w/v) sodium lauroylsarcosine, incubated for 30 min at 37 °C with constant agitation then further adjusted with 16.3 µl of a stock solution containing 4 % (w/v) sodium phosphotungstic acid (NaPTA) (lacking magnesium chloride) prepared in water pH 7.4 to give a final concentration in the sample of 0.3 % (w/v). Samples were incubated at 37 ºC for 30 min with constant agitation before centrifugation at 13 200 r.p.m. (16 100 g) for 30 min in a microfuge. After careful isolation of the supernatant, pellets were resuspended to their original volume with PBS. Aliquots were processed immediately for analysis by ELISA.

Scrapie cell assay

High-sensitivity cell culture assays for RML prion infectivity were performed as described by Klohn et al [START_REF] Klohn | A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions[END_REF]. Briefly, PK1 cells, a highly scrapie-susceptible N2a subclone, were exposed for 3 days in 96-well plates to serial dilutions (10 -3 , 10 -4 ) of 10 % (w/v) RML brain homogenate either untreated or following digestion with thermolysin (100 μg/ml final protease concentration at 37 °C or 70 °C) or proteinase K (50 μg/ml final protease concentration at 37 °C) for a range of incubation times. A serial dilution of untreated 10 % (w/v) RML brain homogenate (3.10 -5 to 10 -7 ) of known infectivity titre was performed in parallel. Subsequently cells were split and passaged appropriately for the Scrapie Cell Assay [START_REF] Klohn | A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions[END_REF] and the infectivity titre of each sample was deduced from the reference preparation.

Statistical Analysis

All experiments were conducted at least three times. Figures show representative data and report the mean ± standard error of the mean (SEM) or standard deviation (SD).

RESULTS

Characterization of RML prion-infected brain homogenate

In order to standardize a large series of experiments we generated a 970 ml stock of 10 % (w/v) RML prion-infected brain homogenate from terminally affected CD-1 mice and titrated this by intracerebral or intraperitoneal inoculation in CD-1 mice (Table 1). Using the Reed-Müench formula, 10 % (w/v) RML brain homogenate (I6200) has an infectious prion titre of 10 1a). In the absence of protease digestion a mixture of full-length and truncated PrP species is observed. After PK digestion the characteristic pattern of amino-terminally truncated fragments of di-, mono-and non-glycosylated PrP is observed with a predominance of mono-glycosylated PrP (Figure 1a).

Thermolysin degrades mouse PrP C while preserving full-length PrP Sc

In initial experiments we adapted the methods of Owen and colleagues [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF] to determine conditions in which thermolysin could efficiently degrade mouse PrP C . We found that digestion of 10 % (w/v) normal CD-1 brain homogenate with 100 μg/ml thermolysin for 30 min at 70 °C efficiently degraded PrP C (Figure 1b). In agreement with Owen et al [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF], silver stain analysis of thermolysin digested samples revealed that the majority of proteins in brain homogenate were destroyed by this treatment (data not shown). In sharp contrast, application of the same thermolysin digestion conditions to 10 % (w/v) RML prion-infected brain homogenate produced no apparent change in the PrP fragment pattern and only a modest reduction in overall signal intensity (Figure 1b). These data (which accord with the findings of Owen et al using ovine or bovine brain homogenates [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF]) indicate that thermolysin can efficiently degrade mouse PrP C while leaving PrP Sc intact. The presence of full-length PrP in thermolysin digested RML brain homogenate was confirmed through the use of an N-terminal specific anti-PrP monoclonal antibody and by deglycosylation. The SAF32 monoclonal anti-PrP antibody, with an epitope spanning residues 79-92 of hamster PrP [START_REF] Vincent | Phorbol esterregulated cleavage of normal prion protein in HEK293 human cells and murine neurons[END_REF], detected all three full-length PrP glycoforms in thermolysin digested samples (Figure 1d). After deglycosylation, thermolysin-resistant PrP migrated as two bands (Figure 1b) with apparent molecular weights corresponding to either full-length PrP or an endogenously truncated C2 fragment that has been characterized previously [START_REF] Owen | Molecular profiling of ovine prion diseases using thermolysin-resistant PrPSc and endogenous C2 PrP fragments[END_REF][START_REF] Chen | Truncated forms of the human prion protein in normal brain and in prion diseases[END_REF][START_REF] Jiménez-Huete | Endogenous proteolytic cleavage of normal and disease-associated isoforms of the human prion protein in neural and non-neural tissues[END_REF]. Densitometry showed that ~25 % of total PrP remaining after 30 min digestion with thermolysin is in full-length form. Importantly, short time periods of digestion with 100 μg/ml thermolysin at 70 °C appear to be required for isolating full-length PrP Sc from RML brain homogenate. Prolonged digestion (90 min) with thermolysin at 70 °C leads to the generation of truncated PrP with a migration pattern equivalent to that observed after digestion with PK (Figure 1c). As thermolysin has no preferred cleavage sites in the amino-terminal region of PrP [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF] the generation of truncated PrP may be attributable to thermolysin acting at a non-preferred scissile bond or to the activity of a minor contaminating protease. Truncation of disease-related PrP by this activity appears to be prion strain specific [START_REF] Owen | Molecular profiling of ovine prion diseases using thermolysin-resistant PrPSc and endogenous C2 PrP fragments[END_REF] and these data indicate that conditions for digestion with thermolysin should be optimized empirically for different prion strain/host combinations.

Thermolysin-resistant PrP is mainly insoluble and comprises both PK-sensitive and PK-resistant PrP

In addition to protease resistance, disease-related isoforms of PrP are defined by their aggregation state and insolubility in non-denaturing detergents. Therefore, we examined the detergent solubility of thermolysin-resistant PrP. Aliquots of brain homogenate were incubated with 2 % (w/v) sodium lauroylsarcosine and then centrifuged to generate detergent-soluble supernatants and detergent-insoluble pellets. Aliquots of these fractions were subsequently digested with thermolysin or PK or left untreated. Immunoblot analysis of PrP present in the supernatant and pellet fractions showed that the majority of thermolysin-resistant PrP from RML brain is insoluble (Figure 2a). In distinction, under the same conditions, PrP C from normal CD-1 brain was entirely soluble and was recovered only in the supernatant fraction (Figure 2b). These findings show that thermolysin-resistant PrP in RML brain homogenate has the properties of disease-related PrP.

Notably, in the foregoing experiments, we often observed an apparent increase in PrP signal strength on immunoblots after digestion with PK (see for example, Figure 1a). A c c e p t e d M a n u s c r i p t assess the proportions of total PrP in RML brain homogenate that are resistant to digestion with either thermolysin or PK we used an ELISA protocol that provides a sensitive means for immunodetection of denatured PrP regardless of whether this is derived from PrP C or PrP Sc [START_REF] Wadsworth | Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein[END_REF] (and our unpublished data). After 30 min of digestion of RML brain homogenate with 100 μg/ml thermolysin at 70 °C (conditions that leave PrP Sc intact and efficiently degrade PrP C ) ∼55 % of total PrP was preserved (Figure 3). In contrast, after 30 min digestion with 50 μg/ml PK at 37 °C only ~20 % of total PrP remained (Figure 3). After 5 min digestion with these protease concentrations at these temperatures, PrP C was completely degraded in normal CD-1 brain homogenate (Figure 3). At this time point, in RML brain, there was an even higher differential between PrP remaining after thermolysin (~85 %) or PK (~20 %) digestions. To investigate the reproducibility of this finding, instead of using pooled RML brain homogenate we examined 10 % (w/v) brain homogenates prepared from four individual terminal RML infected CD-1 mice. After 5 min digestion, the mean percentages of thermolysin-resistant PrP and PK-resistant PrP (± SD) were highly reproducible, giving respective values of 91 ± 6 % and 23 ± 3 %. Notably, prolonged digestion (90 min) with thermolysin leads to further degradation of disease-related PrP reaching levels comparable to those seen after digestion with PK (Figure 3). These data can be correlated with immunoblot analysis of thermolysin digests which show a change from a mixture of full-length and truncated PrP species at 30 min to only truncated PrP after 90 min (compare Figure 1 panels b andc). Thus the conditions defined here show that for this prion strain/host combination, disease-related PrP in RML brain homogenate is degraded at different rates by thermolysin or PK, with the majority of diseaserelated thermolysin-resistant PrP being rapidly degraded by PK under standard digestion conditions.

The majority of PK-sensitive disease-related isoforms of PrP do not contribute to RML prion infectivity

Our data show that digestion of 10 % (w/v) RML-infected brain homogenate with 100 μg/ml thermolysin for 30 min at 70 °C leads to efficient degradation of PrP C while preserving both PK-resistant and PKsensitive disease-related isoforms of PrP. To determine whether the PK-sensitive PrP species contribute to RML prion infectivity, we measured prion infectivity in PK and thermolysin digested brain homogenate using the scrapie cell assay [START_REF] Klohn | A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions[END_REF], and compared this with PrP content determined in parallel by ELISA and immunoblotting. As control experiments, we first investigated the effects of temperature alone on prion infectivity in non-digested RML brain homogenate. However, we found that incubation of brain homogenate at 70 °C rapidly destroyed up to ∼90 % of RML prion infectivity (Figure 4a). Clearly exposure to 70 °C is incompatible with conducting meaningful RML prion infectivity studies and this finding is in close agreement with the temperatures shown to produce substantial inactivation of other rodent-adapted prion strains [START_REF] Somerville | Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications[END_REF]. Accordingly, we investigated the effect of incubating RML brain homogenate at 37 °C. At this temperature reproducible fluctuation in prion infectivity was observed over a 90 min time course (Figure 4b). Overall however, the loss of infectivity at 37 °C was more modest and acceptable than seen at 70 °C, with ~65 % of starting infectivity remaining after 90 min incubation (Figure 4b). Importantly, as shown in figure 5, the majority of PrP C from normal CD-1 brain was destroyed after 5 min incubation with 100 μg/ml thermolysin at 37 °C with ~10 % PrP remaining after digestion for 90 min (see also Figure 6). In normal CD-1 brain homogenate, PrP C level is around ~20 % of total PrP present in untreated RML brain homogenate (Figure 3, compare PrP content at t = 0). Therefore, assuming PrP C expression level is similar in uninfected and RML-infected CD-1 brains, PrP C would correspond to only ~2 % of total PrP in RML brain homogenate after digestion with thermolysin for 90 min at 37 °C.

Based upon the foregoing data we performed comparative experiments on RML brain homogenate with thermolysin and PK at 37 °C. After digestion with thermolysin for 90 min, ~85 % of total PrP was preserved (Figure 4c and6). In sharp contrast only ~20 % of total PrP was preserved in RML brain homogenate after equivalent digestion with PK at 37 °C (Figure 4c). RML brain homogenate (comprising both the full-length and truncated PrP isoforms) was precipitated by sodium phosphotungstic acid whereas no PrP C from thermolysin-digested normal CD-1 brain was recovered by this treatment (Figure 6). These data show that disease-related PrP comprises up to ~85 % of total PrP present in RML mouse brain homogenate, however the majority (∼75 %) of these disease-related PrP isoforms are readily sensitive to rapid digestion by PK. This finding agrees well with previous studies of RML-infected mouse brain which also showed that only a minority of total disease-related PrP is PKresistant [START_REF] Safar | Prion clearance in bigenic mice[END_REF][START_REF] Thackray | Mouse-adapted ovine scrapie strains are characterized by different conformers of PrP Sc[END_REF]. Strikingly, contrary to the marked disparity in PrP content, infectivity titres in both PK and thermolysin treated samples remained closely similar throughout the time course of digestion (Figure 4c). After 90 min digestion ~20 % of starting infectivity was present in both samples. These data indicate that the majority of PK-sensitive thermolysin-resistant disease-related PrP isoforms do not contribute significantly to RML prion infectivity in cell culture. Immunoblot analysis (Figure 5) showed that while thermolysin produced no apparent change in the PrP fragment pattern over the digestion time course, PK rapidly produced the characteristic pattern of amino-terminally truncated PrP fragments. The close similarity of prion infectivity seen in both thermolysin digested and PK-digested samples, suggests that removal of the N-terminus of PrP with PK does not adversely affect RML prion infectivity, at least as determined by scrapie cell assay.

Thermolysin digestion of PrP associated with human and hamster prion strains

In a further series of experiments we investigated the ability of thermolysin to degrade PrP C while preserving disease-related isoforms of PrP associated with other prion strains. Under conditions that efficiently degrade hamster and human PrP C (Figure 7a,b), preservation of intact PrP Sc was observed in brain homogenate from patients with vCJD or terminally clinically-affected hamsters propagating the Sc237 prion strain (Figure 7a,b). The presence of full-length PrP Sc in these samples was confirmed by immunoblotting with monoclonal antibody SAF32 (Figure 7c) or deglycosylation with PNGase F (data not shown). These findings, combined with the data of Owen and colleagues [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF], establish that thermolysin can be used to isolate full-length PrP Sc from multiple prion strain/host combinations.

While the presence of PK-sensitive disease-related PrP has been previously documented in many prion strain/host combinations, including prion-infected hamster brain [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF][START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF], prion-infected mouse brain [START_REF] Safar | Prion clearance in bigenic mice[END_REF][START_REF] Thackray | Mouse-adapted ovine scrapie strains are characterized by different conformers of PrP Sc[END_REF], scrapie infected sheep brain [START_REF] Thackray | Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay[END_REF] and patients with sporadic CJD [START_REF] Safar | Diagnosis of human prion disease[END_REF], this has not been reported in vCJD brain. Accordingly, using ELISA, we quantified disease-related PrP in 10 % (w/v) brain homogenates from four patients with vCJD (Figure 8). Remarkably, up to ~90 % of total PrP was resistant to digestion with thermolysin under conditions that efficiently degrade PrP C in normal human brain (Figure 8). In contrast, only ~10-20 % of total PrP in vCJD brain resisted digestion with PK. This ratio was highly reproducible in all four patients with mean percentages of thermolysin-resistant PrP and PK-resistant PrP (± SD) of 83 ± 13 % and 19 ± 6 %, respectively.

DISCUSSION

It is becoming increasingly clear that the pathogenesis of both human and animal prion diseases involves the propagation of protease-sensitive disease-related isoforms of PrP [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF][START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Tremblay | Mutant PrP(Sc) Conformers Induced by a Synthetic Peptide and Several Prion Strains[END_REF][START_REF] Safar | Diagnosis of human prion disease[END_REF][START_REF] Nazor | Immunodetection of diseaseassociated mutant PrP, which accelerates disease in GSS transgenic mice[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF][START_REF] Thackray | Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay[END_REF]. Indeed, in multiple prion strain/host combinations it now appears that the majority of disease-related PrP may be destroyed by PK under conditions that are typically employed to detect prototypical PrP Sc [START_REF] Safar | Diagnosis of human prion disease[END_REF][START_REF] Thackray | Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay[END_REF]. PK-sensitive diseaserelated forms of PrP have been demonstrated by conformation dependent immunoassay [START_REF] Safar | Eight prion strains PrP Sc molecules with different conformations[END_REF][START_REF] Safar | Diagnosis of human prion disease[END_REF][START_REF] Thackray | Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay[END_REF][START_REF] Safar | Prion clearance in bigenic mice[END_REF][START_REF] Thackray | Mouse-adapted ovine scrapie strains are characterized by different conformers of PrP Sc[END_REF] and by immunoblotting following biochemical purification [START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF], cold PK digestion [START_REF] Tremblay | Mutant PrP(Sc) Conformers Induced by a Synthetic Peptide and Several Prion Strains[END_REF] or immunological capture [START_REF] Nazor | Immunodetection of diseaseassociated mutant PrP, which accelerates disease in GSS transgenic mice[END_REF].

To date little is known about the physicochemical properties of PK-sensitive disease-related PrP and it remains unclear whether PK-sensitive and PK-resistant disease-related PrP species are [START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF].

In the present study we now report a simple method for detecting PK-sensitive disease-related PrP by showing that limited digestion with thermolysin not only preserves disease-related PrP in full-length form [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF] but also preserves a disease-related PrP fraction that is readily degraded by PK. In RML brain homogenate, under optimized conditions of proteolysis which destroy PrP C , ~85 % of total PrP resists degradation with thermolysin and is precipitated by NaPTA, whereas only ~20 % of total PrP resists degradation with PK. Using the scrapie cell assay, that quantifies RML prion infectivity with a greater accuracy than conventional mouse bioassay [START_REF] Klohn | A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions[END_REF], we were able to quantify prion infectivity in these protease digested samples. We found that the thermolysin-resistant PrP fraction, that preserves the majority of PK-sensitive disease-related PrP isoforms, contained the same level of infectivity as the PKresistant PrP fraction equivalent to ∼20 % of starting prion infectivity. Thus while the remaining RML prion infectivity appears roughly proportional to the concentration of PK-resistant PrP, there is clear uncoupling of disease-related PrP concentration and prion infectivity in thermolysin digested samples. We conclude from these data that the majority of PK-sensitive disease-related PrP isoforms are noninfectious, at least when measured by scrapie cell assay.

The finding that both PK and thermolysin digested brain homogenates contain only ∼20 % of starting prion infectivity despite thermolysin digested samples containing ~85 % of total homogenate PrP can be interpreted in several ways. Because direct inactivation of thermolysin-resistant PrP by the protease seems unlikely given the stability of observed PrP fragment size, two other possibilities seem more plausible, (i) that ~80 % of RML infectivity is associated with a minor population of PrP conformers that are equally sensitive to degradation by PK and thermolysin, or, (ii) that a non-PrP component of infectious prions that modifies specific prion infectivity is more efficiently degraded by thermolysin than PK. Of these two possibilities the former is more strongly supported by the findings of other researchers. Caughey and colleagues have demonstrated that hamster 263K prions have different sized PrP aggregates possessing markedly different specific prion infectivities [START_REF] Silveira | The most infectious prion protein particles[END_REF]. Small PrP aggregates comprising 14-28 PrP molecules which represent only a minor proportion of total hamster PrP have specific infectivities ~70 fold higher than large PrP aggregates [START_REF] Silveira | The most infectious prion protein particles[END_REF]. A similar physical distribution of infectious PrP aggregates associated with the RML prion strain would provide a reasonable physical basis for our findings. Given that small PrP aggregates appear to be more readily accessible to proteolytic degradation [START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF], both thermolysin and PK may equivalently destroy a subset of PrP species corresponding to the most infectious prions that account for up to 80 % of total infectivity.

The recent data of Soto and colleagues have established that PK-sensitive disease-related hamster PrP isolated from the upper fractions of sucrose density gradients has the ability to convert PrP C into protease resistant PrP using the PMCA assay [START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF]. Importantly, because PK-sensitive disease-related hamster PrP was isolated on the basis of density [START_REF] Pastrana | Isolation and Characterization of a Proteinase K-Sensitive PrP(Sc) Fraction[END_REF] these preparations are unlikely to contain the full ensemble of PK-sensitive PrP species. While there are of course caveats when comparing different prion strains and different methods, our data suggest that PK-sensitive PrP species are heterogeneous and that the majority may be non-infectious.

There is increasing evidence that an abnormal PrP species, distinct from PrP Sc , may be responsible for neurotoxicity in prion disease [START_REF] Collinge | A general model of prion strains and their pathogenicity[END_REF]. Clearly, purification of thermolysin-resistant PK-sensitive diseaserelated PrP is now required for detailed evaluation of its specific infectivity in mouse bioassay and possible contributions to neurotoxicity or other roles in prion disease pathogenesis.

Recently, Safar and colleagues reported that 80-90 % of disease-related PrP in sporadic CJD brain is destroyed by PK and that detection of this fraction by conformation dependent immunoassay enhances diagnostic sensitivity [START_REF] Safar | Diagnosis of human prion disease[END_REF]. Our finding that up to 90 % of total PrP in vCJD brain homogenate resists digestion with thermolysin under conditions that destroy human PrP C verifies the proposal [START_REF] Owen | Use of thermolysin in the diagnosis of prion diseases[END_REF] that thermolysin may be useful in the development of high sensitivity diagnostic tests. Thermolysin may also be useful in identifying unusual pathogenic PrP isoforms in isolates where undetectable or barely detectable levels of PK-resistant PrP are observed, for example in certain inherited prion diseases [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Kovacs | Mutations of the prion protein gene phenotypic spectrum[END_REF][START_REF] Mead | Prion disease genetics[END_REF] and in atypical prion disease of ruminants [START_REF] Le Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Baron | Atypical transmissible spongiform encephalopathies (TSEs) in ruminants[END_REF] have reported the potential value of thermolysin for prion strain discrimination based upon variable generation of truncated PrP fragments [START_REF] Owen | Molecular profiling of ovine prion diseases using thermolysin-resistant PrPSc and endogenous C2 PrP fragments[END_REF]. Our data obtained with two distinct prion strains (mouse RML and human vCJD prions) suggest that measuring the ratio of PK-resistant disease-related PrP to thermolysin-resistant disease-related PrP may also be informative in this regard.

Figure 2 Thermolysin-resistant PrP from RML brain is mainly insoluble

Aliquots of 10 % RML brain homogenate (a) or untreated 10 % normal CD-1 brain homogenate (b) were diluted 10-fold in the presence of 2 % (w/v) sodium lauroylsarcosine and incubated for 30 min prior to centrifugation at 16 100 x g for 30 min. PrP recovered in detergent-insoluble pellets (P) or detergent-soluble supernatants (SN) was analyzed before (-) or after digestion with thermolysin (TL) (100 μg/ml, 70 °C, 30 min) or proteinase K (PK) (50 μg/ml, 37 °C, 1 h) by immunoblotting with anti-PrP monoclonal antibody ICSM35. 10 % w/v homogenates from normal CD-1 brain (CD-1, white bars) or RML brain (RML, black bars) were either untreated, digested with thermolysin (TL) (100 μg/ml, 37 °C, 90 min), precipitated by sodium phosphotungstic acid (NaPTA) or sequentially digested with thermolysin (100 μg/ml, 37 °C, 90 min) followed by NaPTA precipitation. NaPTA pellets were resuspended to their original starting volume in PBS. PrP concentration in all samples was measured by ELISA and is expressed as a percentage of total PrP present in the respective untreated samples. Data show the mean of 3 independent experiments ± SEM.

Figure 8 Predominance of thermolysin-resistant PrP in vCJD brain

PrP in 10 % brain homogenate from four patients with vCJD was measured by ELISA in the absence of protease digestion (70 °C, 1 h temperature control) (Total, white bars) or after digestion with thermolysin (100 μg/ml, 1 h at 70 °C) (TL-res, black bars) or proteinase K (100 μg/ml, 1 h at 37 °C) (PK-res, grey bars). PrP concentration is expressed as a percentage of total PrP and data show the mean of four replicates of each sample. The SD was in all cases less than 10 %. The proportion of PK-sensitive PrP species present after thermolysin digestion was estimated by the ratio (TL-res -PK-res)/ TL-res and was ∼80 % in samples from all four vCJD patients.

4

  Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t μl QuantaBlu stop solution (Pierce, Rockford, IL, USA). Fluorescence (excitation 325 nm; emission 425 nm) was measured on a Tecan spectra image microplate reader.

Therefore to reliably 6 Biochemical

 6 Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235

  All thermolysin-resistant PrP in 7 Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t

8

  Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t conformationally distinct or simply comprise different sized aggregates of essentially the same PrP conformers

  . More recently, Owen et al 9 Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t

Figure 1

 1 Figure 1 Proteinase K and thermolysin digestion of mouse PrP

Figure 3

 3 Figure 3 Measurement of PK-sensitive PrP in RML brain by ELISA

Figure 4

 4 Figure 4 RML prion infectivity and PrP content following digestion with thermolysin or PK

Figure 5

 5 Figure 5 Immunoblot analysis of mouse PrP following digestion with thermolysin or PK at 37 °C10 % w/v normal CD-1 (CD-1) or RML (RML) brain homogenates were digested at 37 °C with thermolysin (TL 37 °C) (100 μg/ml) or proteinase K (PK 37 °C) (50 μg/ml) for a range of

Figure 6

 6 Figure 6 Thermolysin-resistant PrP from RML brain is precipitated by sodium phosphotungstic acid

Figure 7

 7 Figure 7 Thermolysin digestion of human and hamster brain homogenate

Biochemical

  Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235 A c c e p t e d M a n u s c r i p t

  8.3 intracerebral LD 50 / ml and 10 6.6 intraperitoneal LD 50 / ml. Examination of 10 % (w/v) RML brain homogenate by immunoblotting before or after standard PK digestion (50 μg/ml PK for 1 h at 37 °C) showed the expected pattern of PrP bands 5 Biochemical Journal Immediate Publication. Published on 06 Aug 2008 as manuscript BJ20081235

	A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081235

(Figure

Table 1 . Titration of RML prions in CD-1 mice. i.c inoculation i.p inoculation ‡

 1 Mice were inoculated intraperitoneally (i.p) with 100 μl of each dilution. § n 0 , number of inoculated mice; n, number of affected mice. ║ Incubation periods are reported for clinically affected animals in days; where n ≥ 3 the mean ± SD is reported.

	Dilution * n/n 0	§	Mean survival time ± SD (days) ║	n/n 0	§	Mean survival time ± SD (days) ║
	10 -1	6/6		134 ± 2	6/6		182 ± 12
	10 -2	5/5		152 ± 7	6/6		184 ± 13
	10 -3	6/6		154 ± 3	6/6		200 ± 5
	10 -4	6/6		162 ± 12	6/6		203 ± 8
	10 -5	6/6		167 ± 4	6/6		209 ± 7
	10 -6	4/4		211 ± 17	1/4		208
	10 -7	2/6		208, 222	0/6		-
	10 -8	1/6		287	0/6		-

* Dilution of 10 % RML brain homogenate I6200 in 1 % normal CD-1 brain homogenate.

Mice were inoculated intracerebrally (i.c) with 30 μl of each dilution. ‡

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGMENTS

We thank all patients and their families for generously consenting to use of human tissues in this research, and the neuropathologists who have kindly helped in providing these tissues. We thank Ray Young for preparation of the figures. This work was funded by the UK Medical Research Council.

DECLARATION OF INTEREST

J.C. is a Director and J.C., G.S.J., A.R.C. and J.D.F.W. are shareholders and consultants of D-Gen Limited, an academic spin-out company working in the field of prion disease diagnosis, decontamination, and therapeutics. D-Gen markets the ICSM35 and ICSM18 antibodies used in this study.


Richard Lundmark
email: richard.lundmark@medchem.umu.se

Gary J Doherty

Yvonne Vallis

Brian J Peter

Harvey T Mcmahon

Curvature sensitive activation of Arf family small G-proteins ! " ARF FAMILY GTP LOADING IS ACTIVATED BY, AND GENERATES, POSITIVE MEMBRANE CURVATURE

Keywords: Curvature sensitive activation of Arf family small G-proteins Clathrin-mediated endocytosis, COP, amphipathic helix, small G-protein, membrane curvature sensing, membrane curvature generation Biochemical

come     Arf family GTP loading is activated by, and generates, positive membrane curvature

INTRODUCTION

Small G-proteins cycle between a GTP-bound active state and a GDP-bound inactive state [START_REF] Gillingham | The small G proteins of the Arf family and their regulators[END_REF][START_REF] Souza-Schorey | ARF proteins: roles in membrane traffic and beyond[END_REF]. In the active state, these proteins are able to recruit a wide variety of effector molecules that participate in various cellular processes. The GTP-loaded status is regulated positively by guanine nucleotide exchange factors (GEFs) and negatively by GTPase activating proteins (GAPs). A common theme for small G-proteins is that they localize to membranes, and it is here that they execute their functions. Most members of the Ras superfamily of small G-proteins acquire a C-terminal lipid modification (geranylgeranylation or farnesylation) that anchors these proteins to the membrane. Arf (ADP-ribosylation factor) and Arl (Arf-like) family proteins have a myristoylated (C14:0) N-terminal helix that is thought to facilitate membrane binding. The myristoyl moiety is attached to the second glycine following removal of the first methionine.

The six mammalian Arfs can be categorized into three subclasses based on sequence similarities. Class I Arfs (Arf1-3) are involved in the recruitment of membrane budding machineries while the roles of Class II Arfs (Arf4 and Arf5) remain unclear. Arf6 alone constitutes the third class and this protein has been shown to regulate trafficking in the endocytic system, actin polymerization and cell migration. Structural details of Arf1 and Arf6 have been described through crystallographic interrogation of the GDP-and GTP-bound proteins [START_REF] Pasqualato | The structural GDP/GTP cycle of human Arf6[END_REF][START_REF] Amor | Structure of the human ADP-ribosylation factor 1 complexed with GDP[END_REF][START_REF] Menetrey | Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity[END_REF]. Surprisingly, the rearrangements induced by GTP loading are relatively minor but must be sufficient to provide information for the specific recruitment of effectors. In addition, the precise localization of the Arfs has been suggested to allow for specific effector recruitment [START_REF] Peters | Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments[END_REF]. The exposure of the N-terminal helix is thought to be closely linked to the GDP/GTP cycle of Arfs. Here binding of GTP causes a shift in the intermolecular switch region which suggestively displaces the helix that is normally buried in a hydrophobic pocket in the GDP-bound state.

Arf1 and Arf6 are each vital for particular membrane trafficking events, although it seems that they act via slightly different mechanisms. Arf1 is absolutely required for the recruitment of COPI, AP-1 and GGA coat proteins to the Golgi. Furthermore, Arf1 has been suggested to recruit AP-3 and AP-4 [START_REF] Robinson | Adaptable adaptors for coated vesicles[END_REF]. However, the precise mechanism of action of Arf1 at the budding vesicle remains elusive. It has been shown that, in addition to recruiting coat proteins, Arf1 couples the generation of vesicles to the incorporation of cargo molecules [START_REF] Bremser | Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors[END_REF][START_REF] Lanoix | GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles[END_REF]. It has previously been demonstrated that when cargo is present, the association between Arf1 and AP-1 is stabilized and ArfGAP1 activity is inhibited [START_REF] Meyer | Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis[END_REF].

Arf6 localizes to the plasma membrane and endosomal system where it is known to regulate both clathrin-dependent and -independent endocytic pathways as well as endomembrane recycling mechanisms. This is not thought to be facilitated via direct recruitment of coat proteins but rather through the generation of distinct membrane domains [START_REF] Donaldson | Arfs, phosphoinositides and membrane traffic[END_REF]. Arf6 activates phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K). Arf6 and PIP5K act synergistically to generate membrane domains enriched in phosphatidylinositol (4,5) bisphosphate (PI(4,5)P 2 ), thereby helping to recruit the protein budding machineries that rely on this phospholipid. Generation of membrane curvature is a prerequisite for the budding of vesicles from a flat membrane. Recent evidence suggests that Arf effectors and Arfs themselves might be directly involved in membrane remodeling. The recruitment of the COPII budding machinery to the endoplasmic reticulum (ER) is dependent upon Sar1, a small G-protein that is closely related to the Arfs. Sar1 was recently shown to induce membrane curvature [START_REF] Lee | Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle[END_REF]. In addition, overexpression of regulatory GAPs (centaurins) or GEFs (EFA6) for Arfs have been found to result in the tubulation of cellular membranes [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF][START_REF] Brown | Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic[END_REF]. This, coupled with our previous observations of amphipathic helix-mediated membrane curvature generation [15], led us to study the role of Arfs as membrane curvature regulators. Here we have used purified proteins to study the role of Arfs in generating membrane curvature and how this ability is influenced by, and influences, GTP loading.

EXPERIMENTAL cDNA construct preparation

cDNA constructs encoding human Arf1 and Arf6 were obtained from the Guthrie cDNA resource center. Full-length Arf1 (residues 1-181), Arf1 delta N-terminus (residues 18-181) and full-length Arf6 (residues 1-175) were subsequently cloned into the pBac4x1 vector for baculovirus expression or the pET15b vector for bacterial expression. The Arf6 K3E mutation was generated by site-directed mutagenesis. The plasmid encoding N-myristoyltransferase was kindly provided by Sean Munro.

Protein expression and protein purification

His-tagged myristoylated Arf1, Arf6 and Arf6 (K3E) were purified from baculovirus-infected Sf9 cells using nickel-agarose beads and gel filtration. Arf1 was expressed in Escherichia coli BL21 strain with or without a plasmid encoding N-myristoyltransferase (a kind gift from Sean Munro) and purified according to reference [START_REF] Franco | Myristoylation of ADPribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels[END_REF].

Liposome binding and tubulation

Generation of liposomes from total brain lipids (FOLCH fraction I) (Sigma Aldrich) or synthetic lipids (Avanti Polar Lipids), and specification of liposome diameter (made by sequential filtration) were performed as previously described [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF]. The sequential filtration results in slightly lower concentrations of smaller diameter versus larger diameter liposomes being present in assays. Liposome binding assays were performed as previously described [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF]. Briefly, proteins were incubated together with 1mg/ml liposomes followed by centrifugation and analysis of the pellet and supernatant by SDS-PAGE and Coomassie staining. In vitro liposome tubulation assays were performed and analyzed as previously described [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF].

Real-time fluorescent GTP-loading assay

GTP-loading of Arfs was measured as described in reference [START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF]. Briefly, purified GDP-loaded Arfs (0.5µM) were incubated at 37ºC in the presence or absence of liposomes (final concentration of 0.1mg/ml Folch lipids) and HKM buffer (50mM HEPES pH7.2, 120mM KAc, 1mM MgCl 2 ) and loading was induced by sequential addition of 1mM GTP and 2mM EDTA. Tryptophan fluorescence was measured at 340nm following excitation at 297.5nm in a FluoroMax-2 fluorimeter (Horiba Jobin Yvon Ltd.). Data was normalized by setting the fluorescence value before induction of GTP-loading to zero and the value obtained after 1000s for the 50nm liposomes and Arf1/Arf6, respectively as the maximum value in each experimental set. All data were processed identically and are presented as % of the maximum value to demonstrate differences in GTP loading.

RESULTS

In the Arf-GDP crystal structure, amino acids 2-10 form a helical structure buried in a hydrophobic pocket in the core of the protein (PDB: 1HUR). However, upon the structural rearrangements induced by GTP binding, this helix is thought to be displaced from its groove. It was previously shown that myristoylation of the N-terminal helix is absolutely required for membrane binding in the GDP-bound state and that point mutations in this helix decreased binding to membranes [START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF][START_REF] Randazzo | The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid-and GTP-sensitive switch[END_REF]. Helical wheel presentations of the N-terminal amino acids of Arf1 and Arf6 shows that, when the myristoylation of the first glycine is taken into account, the helices have an amphipathic nature (Figure 1A). Sequence comparison of the N-termini of Arfs and Arls shows that the amphipathic properties of the first 13 amino acids are conserved in many family members (Figure 1B). We analyzed the binding of purified Arf1 to artificially generated liposomes in the presence of GTP or GDP. When Arf1 was purified from insect cells or bacteria co-expressing N-myristoyltransferase, which enables myristoylation, robust membrane binding was observed. This was true for both GTP-and GDP-loaded Arf1 although more efficient binding was observed when GTP was included in the assay. However, if Arf1 was purified without myristoylation or without the N-terminus, no significant binding could be detected (Figure 1C). These results are in agreement with previous studies and demonstrate that our proteins are functional, behaving as expected [START_REF] Franco | Myristoylation of ADPribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels[END_REF][START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF]. Similar results were obtained for Arf6, but Arf6 bound even more strongly to membranes in the absence of GTP, likely due to binding sites on the protein core [ [START_REF] Paleotti | The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes[END_REF][START_REF] Amor | Structures of yeast ARF2 and ARL1: distinct roles for the N terminus in the structure and function of ARF family GTPases[END_REF] and data not shown].

The association of Arfs with membranes even in the GDP-bound state suggested that membrane binding of the myristoylated protein does not necessarily involve the exposure of the amphipathic helix. Thus, the helix itself might not be absolutely required for binding, but instead may have a more elaborate role. We reasoned that following GTP-dependent release of the amphipathic helix, it might insert into the lipid bilayer and subsequently impose positive membrane curvature by acting as a wedge [START_REF] Campelo | The hydrophobic insertion mechanism of membrane curvature generation by proteins[END_REF]. To study this, liposomes were incubated together with myristoylated Arf1 or Arf6 and GTP or GDP and analyzed using electron microscopy (Figure 1D). We found that the normal spherical morphology of the liposomes always persisted after incubation with either GDP-loaded Arf1 or Arf6. However after incubation with either GTP-loaded Arf1 or Arf6 we consistently observed a drastic change in the shape of many of the liposomes. Frequently, straight tubular structures with an apparent diameter of 45±5nm were observed (Figure 1D; arrowheads). In addition smaller 13-17nm diameter tubular structures were detected (Figure 1D; asterisks). We have observed similar structures on rare occasions with the N-BAR domain of amphiphysin (data not shown). The nature of such small structures is uncertain but might result from single layers of lipids being forced into tubular micelles. These results suggest that Arfs generate high membrane curvature in a GTP-dependent manner through the insertion of a helix in the lipid bilayer.

In theory, the insertion of a helix into the membrane would be favored by positive membrane curvature since the lipid headgroups apposing the helix would be more separate from one another. This would be reflected by increased membrane binding as liposome diameter decreases. To test this, we generated liposomes with low curvature (800nm in diameter) and high curvature (50nm in diameter) and assayed binding of Arf1-GDP and Arf6-GTP to these liposomes (see Figure 2A andB; insets [and see discussion]). The results show that both Arf1 and Arf6 show a small increase in binding in the presence of higher curvature liposomes (Figure 2A and B; insets). Curvature sensitivity in this assay may reflect an increased amount of GTP loading, since amphipathic helix insertion occurs in response to GTP binding (Figure 1D), which would reduce protein off-rates from membranes. We therefore studied GTP loading in the presence of liposomes of different diameters. The structural rearrangements in Arfs upon GTP binding can be detected by an increase in tryptophan fluorescence [22]. This can be used to analyze GTP-loading in real time [START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF]. No increase in tryptophan fluorescence was observed upon the binding of GDP-loaded Arf1/Arf6 to membranes (data not shown). This strongly suggests that the amphipathic helix does not change conformation upon lipid binding per se, consistent with the observation that this helix is buried in the core of the GDP-bound Arf [15]. By incubating Arf1 with liposomes of different diameters in the presence of GTP, we were able to observe that Arf1 was loaded faster with GTP, and to a greater extent, in the context of higher liposome curvature (Figure 2A). As was previously observed, GTP-loading was very slow in the absence of membranes [START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF][START_REF] Weiss | Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor[END_REF]. Similar results were obtained for the loading of Arf6 (Figure 2B). Thus, curvature sensitive GTP-loading may be a general property of Arf family proteins. Liposomes of different diameter did not on their own yield any significant change in fluorescence (Figure 2C and data not shown). The increase in the extent of GTP loading in the context of higher membrane curvature likely reflects a change in the steady state distribution of Arfs, with more being found bound to more highly curved membranes.

Our results suggest that the N-terminus of Arfs is responsible for both curvature sensing and generation. We therefore created a point mutant in the helix to disrupt a positively charged residue that would be expected to align with the negatively charged lipid headgroups once the helix is inserted in the membrane (Arf6 K3E). We used Arf6 as a model since this protein showed greater curvature sensitivity than Arf1. We chose to mutate this particular residue since, according to the structure, this mutation should not affect myristoylation or localization of the helix in the GDP-bound state (having no interaction with other residues in the crystal structure), while being sufficiently N-terminal to be readily accessible for membrane insertion. When the Arf6 K3E mutant was assayed for its ability to load GTP, we found that this protein loads GTP at the same rate as wild type Arf6 in the absence of liposomes (Figure 2C). However, when liposomes of differing diameters were added to the reaction we found that Arf6 K3E showed a slight increase in the GTP-loading rate, but this increase was independent of liposome diameter. This strongly suggests that the introduced mutation renders the helix unable to properly insert in the membrane and therefore Arf6 K3E cannot respond to membrane curvature. We next examined if the K3E mutation affected membrane curvature generation. Liposomes that had been incubated together with wild type Arf6 or Arf6 K3E were analyzed by electron microscopy. Indeed we found that, in contrast to wild type Arf6, Arf6 K3E was unable to tubulate liposomes (Figure 2D). To resolve if GTP loading might result in a tubular localization of Arf6 in vivo, we expressed constitutively GTP-associated Arf6 (Arf6 Q67L-myc). This protein was indeed found on tubular structures in cells, which is consistent with curvature sensitive membrane binding of this protein.

Taken together, our data are consistent with a model in which Arf family proteins function as membrane curvature sensors and further facilitate membrane budding through direct GTPdependent membrane remodeling (Figure 2E). In the GDP-bound state, myristoyl-enabled membrane binding of Arfs is insufficient due to a relatively high off rate. This can be overcome by the local high positive membrane curvature seen for example at a budding vesicle. In addition, GEFs function to induce local GTP-loading of Arfs that subsequently generate curvature via insertion of the helix, thereby creating a positive feedback loop. Once the helix is inserted a diffusion barrier is likely created since Arfs are probably unable move into the negative curvature surrounding the positive curvature of the budding membrane (tubule or vesicle). A high local concentration of Arf-GTP with lowered off rate is obtained that can recruit effector molecules to this precise location where budding is in progress. Subsequently, coat proteins are recruited that help to stabilize the budding vesicle.

DISCUSSION

Cellular processes such as morphogenesis and signaling are complex, and regulatory proteins are required to orchestrate and drive the membrane remodeling, cargo sorting and cytoskeletal rearrangements that must occur during such events. Small G-proteins of the Arf and Arl families function as such regulatory units, due to their abilities to define processes in a spatial and temporal manner and transmit this information to effector molecules.

In this study we show that Arf family proteins generate and sense membrane curvature through an amphipathic helix that can be inserted into the lipid bilayer in a GTP-driven manner. Given that amphipathic helix insertion leads to the induction of local positive membrane curvature, this can result in curvature sensitivity in the rate of Arf insertion. Further, once a helix has inserted this will help to drive the curvature preferred by the next Arf molecules. Through this mechanism, GTP-loaded Arfs are organized in high concentrations at local sites of positive membrane curvature. In this context, membrane curvature functions as a base for protein recruitment. This ability makes Arfs unique among small G-proteins and might explain why they are used in membrane budding. Previous studies on Arf1 and Sar1p has shown that mutations in the helix that renders it less hydrophobic results in an increased off rate from the membrane [START_REF] Lee | Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle[END_REF][START_REF] Antonny | N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange[END_REF]. This is consistent with our results and shows that mutations in the helix affect both membrane binding and curvature sensitivity. Also, the binding of Arf-GTP to a membrane feeds back to generate or stabilize even higher curvature which will drive the progression of membrane budding directly, and indirectly through increased recruitment of curvature effector proteins. Since insertion of the helix is dependent upon the GDP/GTP status of Arf, the procedure can be arrested or promoted by GAPs and GEFs, respectively. Interestingly several Arf-specific GAPs and GEFs are predicted to contain BAR domains that are known to sense, stabilize and generate membrane curvature [START_REF] Peter | BAR domains as sensors of membrane curvature: the amphiphysin BAR structure[END_REF]. This is in agreement with our model for the function and regulation of Arfs. Our data could also suggest that the need for GEFs in membrane budding might be to initially recruit Arfs and that once the curvature is generated Arfs can drive the process via their curvature sensitivity. It has been proposed that ArfGAP1, which is curvature sensitive due to helix insertion, promotes hydrolysis of Arf-GTP at the positive curvature on the vesicle but that Arf-GTP at the vesicular neck with negative curvature is unaffected [START_REF] Antonny | Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation[END_REF]. However, from our results it is difficult to envisage how Arf-GTP can be located at sites of negative curvature while ArfGAP cannot, since they both localize better to increasing positive membrane curvature.

Arf6 has been ascribed several roles which include the regulation of membrane trafficking, actin rearrangements and membrane-cytoskeleton interactions [START_REF] Souza-Schorey | ARF proteins: roles in membrane traffic and beyond[END_REF][START_REF] Doherty | Mediation, modulation, and consequences of membrane-cytoskeleton interactions[END_REF]. We found that the amphipathic helix of Arf6 is shorter than the helix in other Arf family members. In addition, Arf6 is thought to be continuously associated with membranes, perhaps since core residues in Arf6 bind more efficiently to membranes than those of others such as Arf1. This suggests that the role of Arf6 may have evolved to function more as a sensor than as a generator of membrane curvature. Arf6 has been shown to function in clathrin-independent endocytic and recycling pathways for MHC class I proteins and CD59 [26,27]. Morphologically, these pathways are characterized by tubular rather than vesicular structures. Our data suggest that Arf6 is adapted to localize and function at such membrane tubules. It is possible that the shorter helix in Arf6 is more suitable in this system. The role of Arf6 has been suggested to be largely dependent upon effects on phosphatidylinositol (4,5) bisphosphate levels [START_REF] Brown | Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic[END_REF]. Our in vitro data however show that Arf6 also has a direct effect on lipid packing resulting in, and depending upon, membrane shape modulation.

In conclusion, we propose that proteins in the Arf and Arl families of small G-proteins serve as GTP-dependent sensors and drivers of membrane shape. This ability will help to define local areas of activity that are fine-tuned by the characteristics of effector molecules. Likely, the unresolved functions of the less studied Arfs will also require the ability to sense membrane curvature. Whether this includes direct roles in vesicular budding, or indirect regulation of membrane remodeling, remains to be seen.

Biochemical

  Journal Immediate Publication. Published on 03 Jul 2008 as manuscript BJ20081237 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20081237 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Curvature sensitive activation of Arf family small G-proteins ! &

Biochemical

  Journal Immediate Publication. Published on 03 Jul 2008 as manuscript BJ20081237 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20081237 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 03 Jul 2008 as manuscript BJ20081237 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20081237 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Curvature sensitive activation of Arf family small G-proteins ! )

Figure 1 |

 1 Figure 1 | Arfs induce membrane curvature in a GTP-dependent manner. (A), Helical wheel representation of amino acids 2-13 in Arf1 (top wheel) and 2-11 in Arf6 (bottom wheel). Hydrophobic and charged amino acids are highlighted by dark grey circles or light grey circles, respectively. Myristoylation of the second residue (glycine) is depicted by (myristoyl). (B), Sequence alignment of the N-terminal amino acids of Arf/Arl family members. Hydrophobic amino acids are highlighted in dark grey. (C), Liposome co-sedimentation assays. Liposomes generated from brain-derived lipids (FOLCH fraction I) were incubated with the indicated proteins and GTP or GDP before centrifugation and analysis of supernatants (S) and pellets (P) by SDS-PAGE and Coomassie staining. A control experiment where protein was incubated in the absence of liposomes is shown. The proteins used were: myristoylated Arf1 purified from Sf9 insect cells (Myr-Arf1 (Sf9)), Arf1 purified from bacteria (Arf1 (bacteria)), myristoylated Arf1 purified from bacteria co-expressing N-myristoyltransferase (myr-Arf1 (bacteria)) and Nterminally truncated Arf1 purified from bacteria ( N-term Arf1 (bacteria)). (D), Electron micrographs of negatively-stained 800nm liposomes incubated in the presence of equal concentrations of proteins (10µM), and nucleotides (100µM) as indicated. Arrowheads indicate tubules that are approximately 45nm in diameter and asterisks indicate tubules that are 13-17nm wide. Scale bars represent 100nm.

Figure 2 | 1 Biochemical

 21 Figure 2 | Arf family proteins are activated by high positive membrane curvature. (A-C), Indicated proteins were incubated together with or without liposomes (-lip) of the indicated diameters and GTP. GTP-loading was analyzed in real time by measuring tryptophan fluorescence (see Experimental section). Fluorescence is presented as % of the value at 1000s for 50nm liposomes and Arf1 and Arf6, respectively. Insets in (A) and (B) shows liposome binding assays of Arf1 (A) and Arf6 (B) in the absence or presence of liposomes of different diameters.

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

Many thanks to Sven Carlsson for advice. Richard Lundmark was supported by the Swedish Research Council, the Medical Faculty of Umeå University and the Magn. Bergvalls foundation. Gary Doherty was supported by a Trinity College Cambridge Internal Graduate Studentship. The authors declare no competing interests.