Karolin V Dorn

Felix Willmund

Christian Schwarz

Christine Henselmann

Thomas Pohl

Barbara Hess

Daniel Veyel

Björn Usadel

Thorsten Friedrich

Jörg Nickelsen

Barbara Heß

Michael Schroda
email: michaelschroda@mpimp-golm.mpg.de

Chloroplast DnaJ-like proteins 3

Keywords: J-domain proteins, bacterial ferredoxin, chloroplast chaperones, redox regulation, RNA-binding proteins

come

INTRODUCTION

Molecular chaperones of the HSP70 class are highly conserved and found in all living organisms, except for some archaea. HSP70 chaperones are involved in a plethora of different cellular functions, like folding of newly synthesized proteins, refolding of denatured proteins after stress, protein quality control, transport of proteins across membranes, or assembly and disassembly of protein complexes [START_REF] Mayer | Hsp70 chaperones: cellular functions and molecular mechanism[END_REF]. HSP70s contain two functionally interconnected domains, an N-terminal ATPase and a C-terminal substrate-binding domain. Substrate proteins typically expose hydrophobic regions and substrate binding to HSP70 in the ATP state stimulates ATP hydrolysis [START_REF] Laufen | Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones[END_REF]. This results in tight binding of the substrate when HSP70 is in the ADP state. The specificity of HSP70 function is mediated by so-called J-domain proteins, which interact with selected substrates and deliver them to HSP70s in the ATP state [START_REF] Craig | The diverse roles of Jproteins, the obligate Hsp70 co-chaperone[END_REF][START_REF] Miernyk | The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones[END_REF]. ATP hydrolysis is further stimulated by the interaction of the J-domain with the ATPase domain of the HSP70 partner. Exchange of ADP by ATP in most prokaryotictype HSP70s is catalyzed by GrpE-type nucleotide exchange factors [START_REF] Harrison | GrpE, a nucleotide exchange factor for DnaK[END_REF].

Compared to the well-studied HSP70 systems in bacteria and most compartments of the eukaryotic cell (cytosol, ER, mitochondria) rather little is known about HSP70 systems in chloroplasts. This is surprising, as chloroplasts contain a compartment providing the basis for almost all life on earth -the thylakoid membranes -and knowledge about chloroplast proteostasis is mandatory to understand the biochemical mechanisms underlying the conversion of light energy into carbohydrates. To remedy this situation, we are studying the chloroplast HSP70 system in the unicellular green alga Chlamydomonas reinhardtii. Chlamydomonas possesses only one major chloroplast HSP70, termed HSP70B, and therefore is less complex than higher plants or moss containing two and three major chloroplast HSP70 homologs, respectively [START_REF] Rensing | The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants[END_REF][START_REF] Schroda | The Chlamydomonas genome reveals its secrets: chaperone genes and the potential roles of their gene products in the chloroplast[END_REF][START_REF] Schroda | Chaperones and Proteases[END_REF][START_REF] Su | Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds[END_REF].

HSP70B itself is assisted by its escort protein HEP2 to assume the functional state [10]. HSP70B cooperates with its GrpE-type nucleotide exchange factor, CGE1 [START_REF] Schroda | The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing[END_REF], and appears to be constitutively in complex with chloroplast HSP90C [START_REF] Willmund | The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90[END_REF]. HSP70B has been shown to protect PSII from photoinhibition and to play a role in the repair of photodamaged PSII [START_REF] Schroda | A chloroplasttargeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition[END_REF]. To date, two chloroplast-targeted J-domain protein co-chaperones of HSP70B have been characterized: CDJ1 in addition to the J-domain contains the Gly/Phe-rich and the Zn-finger substrate-binding domains characteristic for type I J-domain proteins [START_REF] Cheetham | Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function[END_REF] and therefore most likely is involved in supplying HSP70B (and HSP90C) with unfolded substrate proteins [START_REF] Willmund | The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90[END_REF]. CDJ2 lacks the Gly/Phe-rich and Zn-finger domains and therefore is a type III J-domain. CDJ2 binds to the vesicle inducing protein in plastids (VIPP1) and recruits the HSP70B-HSP90C chaperone complex [15,[START_REF] Liu | J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1[END_REF]. HSP70B, CDJ2 and CGE1 were shown to catalyze assembly and disassembly of VIPP1 oligomeric structures, and might therefore be involved in biogenesis/maintenance of thylakoid membranes [START_REF] Liu | The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas[END_REF].

Here, we report on three novel chloroplast-targeted type III J-domain proteins, termed CDJ3-5, and present a molecular and biochemical analysis of CDJ3 and CDJ4.

Biochemical Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091412 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091412 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

EXPERIMENTAL

Strains and Culture Conditions

Chlamydomonas reinhardtii strains were grown mixotrophically in TAP medium [START_REF] Harris | The Chlamydomonas sourcebook: A comprehensive guide to biology and laboratory use[END_REF] on a rotatory shaker at 25°C and an illumination of ~30 µE m -2 s -1 . For chloroplast isolation, cells were grown in TAP medium supplemented with 0.5% (w/v) peptone.

Cloning, Expression, and Purification of CDJ3 and CDJ4

A 623-bp DNA fragment encoding the N-terminal part of mature CDJ3 was amplified by PCR from cDNA clone AV628957 with primers 5'-GGACTAGTGCTCTTCTAACGCAGCTGATGAAGCAGCA-3' and 5'-GGATTCGCGACTGCCGCCGCTGCCAGGC-3'. The PCR product was digested with SpeI and cloned into SpeI-SmaI-digested pBluescript (Stratagene, La Jolla, CA), giving pMS330. pMS330 was digested with SpeI-NcoI and the resulting 501-bp SpeI-NcoI fragment was ligated into the SpeI-NcoI-digested cDNA clone AV628957, giving pMS332. Next, cDNA clone AV628957 was digested with NcoI-MfeI and the resulting 894-bp NcoI-MfeI fragment was ligated into NcoI-EcoRI-digested pMS330 to generate pMS333. At last, pMS333 was digested with SapI-XhoI and the resulting 1415-bp SapI-XhoI fragment was cloned into SapI-XhoI-digested pTYB11 (NEB, Frankfurt, Germany), giving pMS336. The coding sequence for CDJ4 was amplified by PCR from cDNA clone AV643891 with primers 5'-GGACTAGTGCTCTTCTAACGCAAGCAGTGATGTTGCTTC-3' and 5'-TACCACTCGAGAGCGGCGGAG-3'. The resulting 1006-bp PCR product was digested with SapI-XhoI and ligated into SapI-XhoI-digested pTYB11, generating pMS337. pMS336 and pMS337 were expressed in Escherichia coli ER2566 and purified by chitin affinity chromatography according to the manufacturer's instructions (NEB). Mature CDJ3/4 were cleaved from the intein/chitin binding domain by incubation with cleavage buffer (20 mM Tris/HCl, 0.5 M NaCl, 1.0 mM EDTA, 50 mM DTT, pH 9.0) at 25°C for 16 h and collected in five 1-ml fractions. Fractions 1-3 and 5 were pooled and subjected to three successive runs of concentration/dilution with KMH buffer (20 mM HEPES-KOH pH 7.2, 80 mM KCl, 2.5 mM MgCl 2 ) using Amicon Ultra-4 tubes (Millipore, Molsheim, France). Proteins were supplemented with 10% glycerol and 1 mM DTT, frozen in liquid nitrogen, and stored at -80°C. Yields were around 300 µg pure CDJ3/4 per liter E. coli culture. About 1 mg each of purified CDJ3 and CDJ4 were dissolved in 6 M urea, 20 mM Tris pH 8.0, 0.5 M NaCl and used for the generation of polyclonal rabbit antisera. Affinity purification of antibodies was done as described previously [19].

Heat Shock and Dark-to-Light Shift Kinetics, RNA and Protein Extractions, RNA Gels, and Hybridizations

Heat-shock and dark-light-shift kinetics, isolation of protein and RNA, and preparation of RNA blots were carried out as described in [START_REF] Liu | J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1[END_REF]. Membranes were hybridized with DNA probes prepared by the random priming technique using [α-32 P]dCTP (Hartmann Analytic GmbH, Braunschweig, Germany). Hybridization was done as described previously [START_REF] Schroda | A chloroplasttargeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition[END_REF]. Probes used were a 2-kb NheI-AatII fragment containing the HSP70B coding region, the entire CDJ3 and CDJ4 cDNAs, and a 1-kb cDNA of CBLP [START_REF] Von Kampen | Stress-dependent transcription of a gene encoding a Gb-like polypeptide from Chlamydomonas reinhardtii[END_REF]. Radioactive signals were detected with BAS-IP MS 2040 phosphorimager plates (raytest, Straubenhardt, Germany). β-mercaptoethanol, 0.005% bromphenol blue) was added to the samples and protein concentrations were determined by amido black [START_REF] Popov | Eine störungsfreie Mikromethode zur Bestimmung des Proteingehalts in Gewebshomogenaten[END_REF]. Proteins in gels were stained with colloidal Coomassie (Invitrogen) or transferred to nitrocellulose membranes (Hybond-ECL, GE-Healthcare) by semidry blotting using a discontinuous transfer system. Blocking and immunodecorations were performed in phosphate buffered saline (containing 3% nonfat dry milk, immunodetection was done by enhanced chemiluminescence (GE-Healthcare). Antisera were against HSP70B [START_REF] Schroda | A chloroplasttargeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition[END_REF], CGE1 [START_REF] Schroda | The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing[END_REF], mitochondrial carbonic anhydrase [START_REF] Eriksson | Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii[END_REF], and cytochrome f [START_REF] Pierre | Identification of two 4-kDa miniproteins in the cytochrome b 6 f complex from Chlamydomonas reinhardtii[END_REF]. ECL signals were detected with Hyperfilm-ECL (GE-Healthcare).

Cell Fractionations

Isolation of chloroplasts and fractionation into stroma, thylakoids and low density membranes was done as described previously [START_REF] Zerges | Low density membranes are associated with RNA-binding proteins and thylakoids in the chloroplast of Chlamydomonas reinhardtii[END_REF]. Mitochondria were isolated according to [26], but using a BioNebulizer™ (Glas-Col, Terre Haute, IN) for disrupting cells instead of vortexing with glass beads.

Immunoprecipitations

Chlamydomonas CF185 cells [START_REF] Schroda | A chloroplasttargeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition[END_REF] were grown to a density of ~8 x 10 6 cells/mL, harvested in two equal fractions by centrifugation and either resuspended in 50 mL TAP medium prewarmed to 40°C for heat shock or in 50 mL TAP medium at 25°C for control. Heat shock and control treatments were performed for 1 h under shaking, then cells were harvested and resuspended in lysis buffer (20 mM HEPES, pH 7.2, 10 mM KCl,1 mM MgCl 2 , 154 mM NaCl, 0.25 x protease inhibitor cocktail (Roche, Mannheim, Germany)). Cells were lysed by sonication on ice. Lysates were loaded onto sucrose cushions (20 mM HEPES-KOH pH 7.2, 0.6 M sucrose) and centrifuged in a TI50 rotor for 30 min at 152,000 g and 4°C. The supernatants were supplied with Triton X-100 to a final concentration of 0.5% and incubated for 5 min. Protein A-Sepharose beads with coupled antibodies were equilibrated in lysis buffer and incubated with the cell lysates under agitation for one h at 10°C. Beads were washed four times with lysis buffer (containing 0.1% Triton X-100) and twice with 10 mM Tris-HCl, pH 7.5, and proteins were eluted by boiling 45 s in 2 x Laemmli sample buffer (Figure 5B), or by shaking 30 min at 25 °C with 2 x Laemmli sample buffer lacking βmercaptoethanol (Figure 5A).

UV-vis spectroscopy

200 µL of purified CDJ3 (30.5 µM) or CDJ4 (22.0 µM) in cleavage buffer were analyzed in a 1 cm pathlength UV micro cuvette (Brand, Wertheim) with a TIDAS-II diode array spectrophotometer (J&M, Aalen). The samples were reduced by the addition of 5 µL Na 2 S 2 O 4 (0.5 M) at 25°C and measured instantaneously. The molar extinction coefficient at 280 nm was calculated from the amino acid sequence according to [START_REF] Gill | Calculation of protein extinction coefficients from amino acid sequence data[END_REF] giving values of 35360 M - 1 cm -1 for CDJ3 and 49180 M -1 cm -1 for CDJ4, respectively.

Miscellaneous

Glutaraldehyde crosslinks were performed as described in [START_REF] Willmund | The NH 2terminal domain of the chloroplast GrpE homolog CGE1 is required for dimerization and cochaperone function in vivo[END_REF] and gel filtration analysis of purified CDJ3/4 as reported in [START_REF] Liu | The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas[END_REF]. Recombinant HSP70B containing a C-terminal hexahistidine tag was coexpressed with HEP2 in E. coli and purified by Ni-NTA affinity chromatography as reported previously [10]. Stromal chloroplast proteins for size exclusion chromatography were extracted as described in [START_REF] Zerges | Low density membranes are associated with RNA-binding proteins and thylakoids in the chloroplast of Chlamydomonas reinhardtii[END_REF]. For RNase treatment stromal proteins were incubated for 60 min at 4°C with 200 µg of RNase A. After application of proteins to a Superose 6 column, proteins were eluted with 23 cm/h (50 mM KCl, 5 mM ε-aminocaproic acid, 2.5 mM EDTA, 20 mM tricin/KOH pH 7.5) using an ÄKTApurifier system (GE Healthcare, Germany). Due to low abundance of CDJ3, samples were concentrated using AmiconUltra centrifugal filter devices (Millipore; USA) prior to western analysis. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

CDJ3-5 are conserved from green alga to higher plants

To obtain cDNAs that encode chloroplast-targeted J-domain proteins we have searched Chlamydomonas EST libraries [START_REF] Asamizu | Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii[END_REF][START_REF] Asamizu | A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags[END_REF] with the amino acid sequence of the J-domain of E. coli DnaJ. This has led to the identification of CDJ1 and CDJ2, two chloroplast-targeted Jdomain proteins that were shown to interact with chloroplast HSP70B [START_REF] Willmund | The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90[END_REF][START_REF] Liu | J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1[END_REF]. Further, we identified two ESTs that potentially encoded additional J-domain proteins with N-terminal extensions which by the ChloroP program [31] were predicted to be chloroplast transit peptides (Figure 1B). We termed them CDJ3 and CDJ4 (chloroplast DnaJ-like proteins 3 and 4) and determined the full sequences of their cDNAs. The complete cDNA sequences revealed that the two proteins lacked the Gly/Phe-rich and Zn-finger domains typical for type I J-domain proteins of the DnaJ/HSP40 prototype and therefore qualified CDJ3 and CDJ4 as type III J-domain proteins [START_REF] Cheetham | Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function[END_REF]. Strikingly, both proteins contain a bacterial ferredoxin domain that is followed by an extended C-terminal domain of unknown function (Figure 1B). Only version 3.0 of the Chlamydomonas genome sequence [32] revealed a further gene (CDJ5) with full EST coverage that encodes another J-domain protein with a bacterial ferredoxin domain. Analysis by ChloroP qualifies the N-terminal sequence of CDJ5 as potential chloroplast transit peptide. Unfortunately, CDJ5 was identified at a late stage of this study and therefore was not characterized in more detail. Information on molecular weights of CDJ3-5 precursors and mature proteins and similarities between their amino acid sequences is compiled in Table 1.

Database searches revealed that true homologs of CDJ3-5 existed only in green algae, moss and higher plants, each encoding at least two homologs of CDJ3-5 (note that one Ostreococcus sequence was incomplete and therefore excluded from our analysis). We wondered, whether the diversification of the CDJ3-5 family occurred early in the development of the green lineage, or rather late at the level of its different branches. Phylogenetic analysis of CDJ3-5 homologs from algae, moss, gymnosperms and angiosperms supported both hypotheses (Figure 1A): we could clearly distinguish two clades, one containing homologs of CDJ3 and CDJ4, the other containing homologs of CDJ5. The two clades must have diverged early, i.e. in the progenitor of the green lineage. But we also found diversification within the clades, e.g. in rice, which encodes at least three members of the CDJ3/4 clade.

CDJ3 is inducible by light but not by heat stress

J-domain proteins potentially support their HSP70 chaperone partner in refolding of stressdenatured proteins. Thus it was possible that the CDJ3/4 genes were heat shock-inducible. As shown in Figure 2A, the opposite was observed: both, CDJ3 and CDJ4 mRNA levels declined in response to heat shock and only started to recover when the expression of heat shock genes, e.g. HSP70B, attenuated. Interestingly, CDJ3 mRNA levels strongly increased in cells that were shifted from a 16-h dark period to low light (Figure 2B). Here, CDJ4 mRNA levels were below the detection limit. Consistently, the mRNA level of the Arabidopsis CDJ5 ortholog atDjC17 (AT5G23240) (extracted from publicly available microarray data) increased after illumination, as well (Supplemental Figure 1). Furthermore, the mRNA levels of atDjC17 showed strong circadian cycling with a ~30 fold increase in expression towards subjective dusk [START_REF] Edwards | FLOWERING LOCUS C mediates natural variation in the high-temperature response of the Arabidopsis circadian clock[END_REF]. This was even more marked for atDjC17 mRNA levels in plant rosettes grown under 12h/12h light dark diurnal cycles [START_REF] Usadel | Global transcript levels respond to small changes of the carbon status during progressive exhaustion of carbohydrates in Arabidopsis rosettes[END_REF]. In the case of the Arabidopsis CDJ3/4 ortholog atDjC18 (AT2G42750) weaker circadian/diurnal cycling could be observed, whereas the A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. expression peaked shortly after dawn. atDjC18 was upregulated by iron starvation and both Arabidopsis genes appeared to be downregulated by sugar.

CDJ3 and CDJ4 contain redox-active Fe-S clusters

To biochemically characterize CDJ3 and CDJ4 we expressed both proteins without their predicted N-terminal chloroplast transit peptide in E. coli as C-terminal fusions to the yeast VMA intein/chitin binding domain. As shown in Figure 3, the CDJ3/4 fusion proteins were well expressed in E. coli, but barely soluble. When the fusion proteins present in the soluble fraction were concentrated on the chitin columns, the columns became yellow-brownish which indicated that Fe-S clusters were already assembled into the fusion proteins. Following thiol-induced cleavage, mature CDJ3 and CDJ4 proteins with apparent molecular masses of ~38 and ~34 kDa were recovered, which corresponded well with those calculated (Table 1). Also the purified mature proteins had a yellow-brownish colour (Figure 3).

To assay whether CDJ3 and CDJ4 contained redox-active Fe-S clusters, we characterized the UV-vis spectroscopic properties of mature CDJ3 and CDJ4. The spectra revealed broad absorption maxima at 390 nm that bleached in the presence of sodium dithionite (Figure 4). Both, the oxidized and the reduced spectra are similar to those observed for P. aerogenes ferredoxin [START_REF] Stombaugh | Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen[END_REF]. The extinction coefficients at 390 nm of the oxidized spectra and the difference extinction coefficients at 425 nm are in the range reported for a single [4Fe-4S] cluster [START_REF] Gibney | Ferredoxin and ferredoxin-heme maquettes[END_REF]. It was not possible to obtain an EPR signal at various temperatures and microwave power settings, neither with the oxidized nor the reduced samples (data not shown). It is possible that the putative [4Fe-4S] clusters of CDJ3 and CDJ4 have a spin-ground state different of S=1/2. The concentration of the samples was too low to detect any contribution to the g = 4.5 region with the X-band spectrometer.

CDJ3 and CDJ4 interact with ATP-bound HSP70B in vitro

As CDJ3 and CDJ4 were predicted to be targeted to the chloroplast, they were likely to interact with HSP70B, which is the major (if not the only) HSP70 in the chloroplast of Chlamydomonas [START_REF] Schroda | The Chlamydomonas genome reveals its secrets: chaperone genes and the potential roles of their gene products in the chloroplast[END_REF][START_REF] Schroda | Chaperones and Proteases[END_REF]. To test this, we performed glutaraldehyde crosslinking experiments with the purified proteins. We used HSP70B that was coexpressed with its escort protein HEP2 and therefore functional [10]. As J-domain proteins interact with their HSP70 chaperone partners in the ATP-state (see e.g. [START_REF] Weitzmann | The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum -a quantitative approach[END_REF]), we performed crosslinks in the presence and absence of ATP. When crosslinked proteins were detected with an antiserum against HSP70B, we mainly detected HSP70B monomers (migrating slightly above the 72-kDa marker protein) and oligomers (migrating above the 170-kDa marker protein). However, we also detected crosslink products with apparent molecular masses of ~140 kDa in the lanes containing ATP, HSP70B, and CDJ3 or CDJ4 (Figure 5, left gel). These ~140-kDa crosslink products were also detected with antisera against CDJ3 (Figure 5, middle gel) and CDJ4 (Figure 5, right gel). Hence, CDJ3 and CDJ4 appear to interact with HSP70B, but only in the ATP state. As judged from the migration of these complexes at ~140 kDa they might consist of an HSP70B monomer and a CDJ3/4 dimer. However, it is also possible that these complexes consist of monomers of HSP70B and CDJ3/4 whose migration was retarded by crosslinks that preserved higher order structures. In addition to the ~140-kDa complex with HSP70B, only monomers of CDJ3 were detected (Figure 5, middle gel). In contrast, CDJ4 existed as monomers and two oligomeric forms, which migrated little below and little above the 72-kDa marker protein (Figure 5, right gel). Most likely, these represent CDJ4 dimers, where in the form with higher apparent molecular mass crosslinks might have preserved higher order structures. We could also observe oligomer formation of CDJ4 by gel filtration. As shown in Figure 6, CDJ3 and CDJ4 monomers migrated with slightly larger apparent than calculated molecular masses (~47 and ~42 kDa compared to ~38 and ~33 kDa, respectively). While CDJ3 was only found as monomers, a small fraction of CDJ4 appeared to form oligomers. When we take into account the larger apparent molecular mass of CDJ4, the peak corresponding to a 98 kDa oligomer rather points to a CDJ4 dimer (Figure 6).

CDJ3 and CDJ4 are very weakly expressed chloroplast proteins that respectively locate to stroma and membranes

To test, whether we could verify expression of both proteins by western analysis we used antisera that were raised against CDJ3 and CDJ4. As revealed in Figure 7, we could not, or at most barely, detect either of the two proteins in whole-cell extracts, even when we used the antisera at high concentrations. However, when we used the antisera against CDJ3 and CDJ4 to enrich the proteins by immunoprecipitation from whole-cell extracts, we could detect immunoreactive proteins with apparent molecular masses of ~38 and ~34 kDa. These masses corresponded to those calculated for mature CDJ3 and CDJ4, respectively (Figure 7A and Table 1). In the αCDJ3 and αCDJ4 immunoprecipitates we could also detect HSP70B. Hence, these data suggest that the CDJ3 and CDJ4 proteins are expressed at low levels in Chlamydomonas and that they may interact with HSP70B in vivo.

To verify the predicted chloroplast localization of CDJ3 and CDJ4 and to determine their suborganellar distribution, we isolated mitochondria and chloroplasts from Chlamydomonas cells. Chloroplasts were subsequently sub-fractioned into stroma, thylakoids and low-density membranes, which are considered to consist of inner envelopes and of transitory membranes between inner envelope and thylakoids [START_REF] Zerges | Low density membranes are associated with RNA-binding proteins and thylakoids in the chloroplast of Chlamydomonas reinhardtii[END_REF]. The purity of the fractions was tested with antisera against mitochondrial carbonic anhydrase, stromal CGE1 and the integral thylakoid membrane protein cytochrome f. As judged from the signals obtained with these antisera (Figure 7B), chloroplasts contained mitochondrial contaminations, stroma fractions were free from thylakoid and mitochondrial contaminations, thylakoids were free from stromal contaminations, but were strongly contaminated with mitochondria, and mitochondria were pure. CDJ3 was detected in the chloroplast and stromal fractions, whereas CDJ4 was weakly detected in chloroplast and strongly detected in thylakoid fractions (Figure 7B).

CDJ3 appears to be in complex with chloroplast transcripts

As outlined in more detail in the discussion, one possible function of CDJ3-5 might be the redox-regulation of transcription/translation initiation and/or transcript stability. In this case, we expected CDJ3-5 to be associated with chloroplast transcripts. To test this hypothesis, we subjected untreated stromal extracts and extracts that have been treated with RNase to gel filtration and monitored the fractions for the presence of CDJ3. We limited our analysis to CDJ3, as we could detect it in the stroma. We also monitored gel filtration fractions for the presence of RBP40 as control, since RBP40 specifically binds to the psbD mRNA [START_REF] Schwarz | Synthesis of the D2 protein of photosystem II in Chlamydomonas is controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the translational activator RBP40[END_REF]. As expected, in untreated stroma RBP40 was detected in fractions with complexes ranging from ~160 to ~2800 kDa, whereas in RNase-treated stroma it was shifted to fractions with complexes in the ~160 to ~550 kDa range (Figure 8). Interestingly, CDJ3 in untreated stroma was detected in fractions with complexes ranging from ~550 to ~2800 kDa. RNase treatment extended the fractions in which CDJ3 was detected down to the ~160 kDa region, at the expense of material in the high molecular weight range. This suggests that part of the complexes containing CDJ3 is associated with RNA. To test, whether CDJ3 is binding to psbD transcripts, e.g. in association with RBP40, we isolated stromal fractions from the PRB2A mutant. In this mutant, the 7-nt long so-called PRB2 sequence in the 5' UTR of the psbD mRNA is mutated, leading to destabilization specifically of the psbD transcript [START_REF] Nickelsen | Mutations at three different nuclear loci of Chlamydomonas suppress a defect in chloroplast psbD mRNA accumulation[END_REF]40]. As shown in Figure 8, RBP40 in the PRB2A mutant was detected only in stroma fractions containing complexes in the ~160 to ~660 kDa range, corroborating the specific interaction of RBP40 with the psbD message [START_REF] Schwarz | Synthesis of the D2 protein of photosystem II in Chlamydomonas is controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the translational activator RBP40[END_REF]. As the distribution of CDJ3 was not altered in stroma fractions from the PRB2A mutant, CDJ3 appears to be associated with transcripts other than psbD.

DISCUSSION

We report here on the molecular and biochemical analysis of CDJ3 and CDJ4, two novel Jdomain proteins encoded by the Chlamydomonas genome. We present the following evidence that CDJ3/4 are chloroplast-targeted co-chaperones of chloroplast HSP70B: First, CDJ3/4, like all their homologs in other algae, moss and higher plants, contain N-terminal extensions that by ChloroP/TargetP are predicted to be chloroplast transit peptides. The apparent molecular masses of mature CDJ3/4 observed in SDS-PAGE (Figure 7A) correspond to those calculated for the processed proteins (Table 1). Accordingly, fractionation experiments revealed that mature CDJ3 and CDJ4 are targeted to the chloroplast. Here they are localized to stroma and thylakoids, respectively, where also chloroplast HSP70B was localized (Figure 7B; refs. [START_REF] Schroda | The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing[END_REF][START_REF] Willmund | The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90[END_REF]15,[START_REF] Liu | J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1[END_REF]19]). Second, HSP70B coprecipitated with both CDJ3 and CDJ4 from Chlamydomonas cell extracts (Figure 7A) and recombinant HSP70B was found to interact with recombinant CDJ3 and CDJ4 in the ATPstate in vitro (Figure 5). Third, recombinant CDJ3/4 stimulated the ATPase activity of HSP70B, albeit at a weaker extent than the bona-fide chloroplast DnaJ homolog CDJ1 (data not shown).

On the evolution of ferredoxin-containing J-domain co-chaperones

A striking feature of CDJ3 and CDJ4 is that they contain redox-active Fe-S clusters (Figures 3 and4), which according to their spectral properties and amino acid sequences appear to be of the [4Fe-4S] type of bacterial ferredoxins (Figures 1, 3 and4; refs [START_REF] Stombaugh | Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen[END_REF][START_REF] Gibney | Ferredoxin and ferredoxin-heme maquettes[END_REF][START_REF] Fukuyama | Structure of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus refined at 2.3 A resolution. Structural comparisons of bacterial ferredoxins[END_REF]). The presence of a ferredoxin domain within a J-domain co-chaperone is highly unusual. Accordingly, we have found genes encoding homologs of CDJ3/4 only in algae, moss, and higher plants (Figure 1). In fact, Chlamydomonas harbours a third member of this gene family that we termed CDJ5 and whose deduced amino acid sequence is more distantly related to those of CDJ3 and CDJ4 (Table 1). Each plant species analyzed encodes at least two members of the CDJ3-5 family, of which at least one belongs to the CDJ3/4 or CDJ5 subfamilies (Figure 1).

Interestingly, proteins containing both a J-domain and a bacterial ferredoxin domain are also encoded by the genomes of several members of the mesophilic Crenarchaeota (now also referred to as Thaumarchaeota) [42][START_REF] Brochier-Armanet | Mesophilic Crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota[END_REF][START_REF] Lopez-Garcia | Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers[END_REF]. However, the domain architecture of the archaeal proteins differs from that of the CDJ3-5 homologs: whereas J-and ferredoxin domains are separated by a linker of 22-41 amino acids in CDJ3-5 homologs, this linker may contain up to 117 amino acids in the archaeal proteins (Figure 1B, Supplemental Figure 2). Instead of this extended linker, the CDJ3-5 homologs contain C-terminal domains of up to ~300 amino acids, which are absent in the archaeal proteins. Within their linkers, the archaeal proteins contain conserved patches of aromatic amino acids flanked by positively and negatively charged residues, which at a lower level of conservation are also found in the C-terminal domains of CDJ3-5. This might indicate that functions exerted by the archaeal linker are located to the C-terminal domains of CDJ3-5.

Genes encoding DnaK and its co-chaperones appear to be absent from the genomes of hyperthermophilic Crenarchaeota and most likely have been transferred horizontally from bacteria to mesophilic Euryarchaeota, which might have been a prerequisite for these to adapt to life at lower temperatures [START_REF] Lopez-Garcia | Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers[END_REF]. As to the evolution of the ferredoxin-containing Jdomain co-chaperones, three scenarios come to mind: i) the ancestor of CDJ3-5 appeared early during the evolution of the "green lineage" and was then acquired by a crenarchaeote by horizontal gene transfer. (ii) The J-domain/ferredoxin fusion occurred first in crenarchaeotes after they had acquired components of the bacterial DnaK system and was then transferred to an early ancestor of the "green lineage", giving rise to CDJ3-5. (iii) The Jdomain/ferredoxin fusion occurred independently in crenarchaeotes and an early ancestor of the "green lineage". Phylogenetic analyses suggest that the first scenario is the most likely one (D. Moreira and P. Lopez-Garcia, personal communication). In any case, development/maintenance of these specialized co-chaperones is likely to be the solution to a selective pressure similarly affecting mesophilic crenarchaeotes and members of the green lineage.

In fact, an Hsp70 homolog (HscA), a J-domain protein (HscB) and a Ferredoxin (Fdx) are encoded in close proximity on the bacterial isc operon and they themselves and their mitochondrial homologs Ssq1p, Jac1p, and Yah1p, respectively, are essential for Fe-S cluster biogenesis [45][START_REF] Kessler | Iron-sulfur cluster biosynthesis in photosynthetic organisms[END_REF][START_REF] Lange | A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins[END_REF]. The HscB/Jac1p J-domain proteins facilitate the interaction of HscA/Ssq1p with scaffold proteins that bind Fe-S cluster intermediates. The chaperones may assist cluster formation by maintaining the scaffold proteins in a conformation suitable for cluster assembly; alternatively, they may facilitate the transfer of the cluster from the scaffold to an acceptor apoprotein [45]. The yeast mitochondrial ferredoxin homolog Yah1p was suggested to provide the reducing power for an essential step in cluster biogenesis, e.g. for cysteine reduction, iron reduction, or reduction of cluster intermediates prior to release from the scaffold proteins [START_REF] Lange | A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins[END_REF]. If the chaperones were indeed involved in the release of cluster intermediates and ferredoxin was essential for prior reduction, the integration of J-domain cochaperone and ferredoxin into the same polypeptide might have made these processes more efficient.

On possible functions of CDJ3-5

A role of the stromal HSP70 chaperone system in Fe-S cluster biogenesis might be supported by the finding that stromal HSP70s are essential [START_REF] Su | Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds[END_REF], which is a typical feature shared by all proteins with important roles in Fe-S cluster biogenesis [START_REF] Lill | Iron-sulfur-protein biogenesis in eukaryotes[END_REF]. However, there are arguments in disfavour: first, the Fdx/Yah1p proteins, for which homologs in Arabidopsis and Chlamydomonas mitochondria exist [START_REF] Godman | Genome analysis of Chlamydomonas reinhardtii reveals the existence of multiple, compartmentalized iron-sulfur protein assembly machineries of different evolutionary origins[END_REF], contain [2Fe-2S] clusters with a polypeptide fold distinct from that of [4Fe-4S] type clusters [50]. Second, if CDJ3-5 were involved in such a fundamental and conserved process like Fe-S cluster biogenesis, it would be surprising to find so many family members only in the green lineage and not in cyanobacteria or nongreen alga. Third, a possible role of CDJ3 in Fe-S cluster biogenesis is difficult to explain in light of its observed association with RNA-binding protein complexes (Figure 8).

Which functions of CDJ3-5 other than in Fe-S cluster biogenesis can we envision? We found CDJ3 to be strongly induced by light and the Arabidopsis ortholog of CDJ5 (atDjC17) to be light-inducible and to be under strong circadian control (Figure 2 and Supplementary Figure 1). The Fe-S clusters in CDJ3/4 are redox-active (Figure 4), both proteins interact with HSP70B (Figures 5 and7A), and CDJ3 appears to be part of an RNAbinding protein complex (Figure 8). J-domain co-chaperones interact via specialized domains with specific substrate proteins and deliver them to their HSP70 partner for processing [START_REF] Craig | The diverse roles of Jproteins, the obligate Hsp70 co-chaperone[END_REF][START_REF] Miernyk | The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones[END_REF]. Hence, it is tempting to speculate that CDJ3-5 interact with specific substrates via their Cterminal domains and that substrate binding or delivery to HSP70B occurs only at a redox state defined by their ferredoxin domains, e.g. via an internal conformational change or a reduction step. The light inducibility of CDJ3 might suggest that redox signals inflicted by light, like the oxidation state of the thioredoxin system or the accumulation of reactive oxygen species (ROS), determine the oxidation state of CDJ3-5 and, therefore, whether substrate processing by HSP70B occurs. The association of CDJ3 with RNA again might point to a chaperone-mediated remodelling of RNA-binding protein complexes that, for example, are involved in translation initiation/elongation or mRNA stability. These are found in the stroma A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. and associated with thylakoids [START_REF] Marín-Navarro | Chloroplast translation regulation[END_REF], where also CDJ3/4 and HSP70B are located (Figure 7B).

Well-studied examples of chaperone-mediated remodelling of replication initiation are known from E. coli, where DnaK and DnaJ monomerize RepA dimers and dissociate DnaB helicase-Lambda P complexes to trigger replication of plasmid P1 and lambda phage, respectively [START_REF] Alfano | Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication[END_REF][START_REF] Wickner | Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin[END_REF]. By means of their Fe-S clusters, the transcription and translation regulators SoxR, FNR, Aconitase, and IscR sense signals like ROS, NO, cellular oxygen or iron concentrations and relay them to transcriptional or translational activities [START_REF] Kiley | The role of Fe-S proteins in sensing and regulation in bacteria[END_REF]. In fact, gene expression in the chloroplast is mainly regulated by nuclear-encoded factors at the level of translation initiation and/or transcript stability and, therefore, is distinct from cyanobacteria [START_REF] Marín-Navarro | Chloroplast translation regulation[END_REF]. As this post-transcriptional regulation of the expression of many chloroplast genes, including psbA, psbD and rbcL, is strongly regulated by light [START_REF] Marín-Navarro | Chloroplast translation regulation[END_REF], we propose that CDJ3-5 might represent such nuclear-encoded factors that act as redox switches by recruiting HSP70B for the reorganization of regulatory protein complexes. aromatic and charged residues. Alignments were done by CLUSTALW and the GeneDoc program was used for presentation.

Figure 2 CDJ3 and CDJ4 mRNA accumulation after heat shock and dark-to-light shift mRNA from Chlamydomonas wild type cells that were exposed to a heat shock at 40°C or that were grown for 16 h in the dark and then shifted to low light (30 µE m -2 s -1 ) was separated on agarose gels (10 µg per lane) and transferred to nylon membranes. These were hybridized with probes generated from the coding regions of HSP70B (positive control), CDJ3, CDJ4, and CBLP (loading control).

Figure 3 Heterologous expression of CDJ3 and CDJ4

CDJ3 and CDJ4 were expressed as C-terminal fusions to the Sce VMA intein containing a chitin binding domain, purified by chitin affinity chromatography, and eluted after thiolinduced cleavage of the intein. Aliquots obtained during the purification steps were separated on 10% SDS-polyacryamide gels and stained with Coomassie Brilliant blue. Lane 1: lysates of E. coli host cells after induction with IPTG. Lane 2: supernatant of cell lysates after a 20min centrifugation at 20.000 g. Lane 3: flow-through. Lane 4: 0.5% of eluate (fraction 4) after thiol-induced cleavage. Lane 5: 0.3% of fractions 1-3 & 5 after concentration. Lane 6: proteins remaining on the chitin column after elution. The positions of the fusion proteins (FP), cleaved CDJ3/4, and of the Saccharomyces cerevisiae VMA intein/chitin binding domain (CBD) are indicated. Pictures of eluted proteins at concentrations of 3 µg/µl (CDJ3) and 1 µg/µl (CDJ4).

Figure 4 UV-vis spectra of purified proteins (A)

The spectra of oxidized (-) and dithionite reduced CDJ3 (⋅⋅⋅⋅) are shown. The dominant absorbance below 400 nm in the spectra of the reduced samples derives from dithionite. The inset represents the reduced-minus-oxidized difference spectrum. (B) Spectra of CDJ4 recorded as described in (A) for CDJ3.

Figure 5

Glutaraldehyde-crosslinking of HSP70B and CDJ3/4 0.5 µM of purified HSP70B, CDJ3 and CDJ4 were incubated alone or in the combinations indicated for 30 min at 30°C in the presence of 0.5 U apyrase (-ATP) or 200 µM ATP (+ATP). Proteins were crosslinked for 15 min with 0.05% glutaraldehyde (GA), separated on a 4-18% SDS-polyacrylamide gel and analyzed by immunoblotting.

Figure 6 Gel filtration of CDJ3 and CDJ4

50 µg of purified CDJ3 (solid gray line) and CDJ4 (dotted black line) were loaded onto a Superdex 200 gel filtration column and developed at a flow rate of 0.5 ml/min. The calibration curve for thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) is shown in the inset. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. and subfractions were separated on a 7.5-15% SDS-polyacrylamide gel and analyzed by immunoblotting with antisera against HSP70B, CDJ3, CDJ4, thylakoidal cytochrome f (Cytf), stromal CGE1, and mitochondrial carbonic anhydrase (CA). Antibodies against CDJ3 and CDJ4 were affinity-purified.

Figure 8 Gel filtration analysis of CDJ3 complexes

Stromal proteins were separated according to their native size by size exclusion chromatography before being subjected to SDS-PAGE and western analysis using indicated antisera. Analysed protein extracts included wild-type stromal proteins without (WT) and with RNase treatment (WT+RNase) and those from the PRB2A mutant lacking the psbD mRNA (PRB2A). Elution profiles of marker proteins (in kDa) are given at the top together with respective fraction numbers.

Supplemental Figure 1 Circadian regulation of Arabidopsis CDJ3-5 homologs

The log2 scaled mRNA expression levels of AtDjC17 and AtDjC18 in various microarrays pertaining to sugar, light, and cycles are shown. The figure shows from left to right, the response of seedlings to light from the Atgenexpress data set; plants left in the dark compared to the expression after plants were shifted from the dark to the light at compensation point CO 2 ; plants shifted from the dark to the light at compensation point or ambient CO 2 ; the expression of AtDjC17 and AtDjC18 in a seedling culture grown under full nutrition (FN), carbon starvation (-C), and 30 minutes or 3 h of sucrose re-addition; the expression in seedlings in constant light after entraining a 12h/12h light dark cycle where subjective night phases are indicated by dashed gray lines; the expression throughout the diurnal cycle, where the light and dark phases are indicated by yellow and gray backgrounds, respectively.

Supplemental Figure 2 Alignment of J-domain/bacterial ferredoxin domain proteins from mesophilic crenarchaeota (Thaumarchaeota) Sequences used were from the following organisms: uncultured crenarchaeote DeepAnt-EC39 (Ucr1, accession AAR24498), uncultured marine crenarchaeote AD1000-207-H3 (Ucr2, accession ACF09820), uncultured marine crenarchaeote AD1000-56-E4 (Ucr3, accession ACF09658), uncultured crenarchaeote 74A4 (Ucr4, accession AAK96090), Nitrosopumilus maritimus SCM1 (Nm1, accession YP_001582358), and Cenarchaeum symbiosum A, (CsA, accession YP_875357). Amino acids highlighted in black are conserved in all 6 proteins; those highlighted in gray are conserved in at least 5 of them. Asterisks indicate cysteines involved in [4Fe-4S] cluster binding and boxed regions represent patches enriched in aromatic and charged residues. ------------------------------ -------------------------------251 CrCDJ4 RARVISQTSDSVEDVQIAIECCPVDCIHYVTLPQLSLLEAALGTMSRIEVSTMQRFGRSGG--NVFEVAYKAWERRMAAIE------------------------------- ------------------------------ ------------------------------------------------------NQARSRMMKRKNTDETP--------SYWDNLWGKQN--------------------- ------------------------------------------------------RRVAEATSSAAYSAPRQPAAKKAGWGFWNNAVEFGAAAGSQFYNDANSARAKKQAQQQ 309 CrCDJ4 -------------------------------------------------------QRAAAAAAAAGGGKAR------VDWSFWTNGASL--------------------QYDE 318 AtDjC17 RSS---------------PYTKPLSPESNMSLTFTKRKKAVDPDIRKLQDVVAAMKQADQSGRTKE-----KGSAYLLGEDYWSPS--------------- --------------------------------------------------ARARANKYYSPQQEAARRKAAEELARQHLGFFAQFTSAFETAFAG-----------------323 Syn ------------------------------------------------------------------------------------------------------------------⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ C-terminal domain ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ AtDjC18 ----EYQKSEEE---VQERAQRAAAAARRWREYSRRGVDKRPTFKLPDSASRGDN---------------------------------------------------------344 Ps1 ----RSTNPSSENYSARERASRAAAAARKWREYSRKGVDRRPTCSLPGKKSSVEEQKEVQTV--------------------------------------------------367 Pp1 ----GDEDFRPDDMGARKRAAKTAAASRRWREYSRQNAGRRSVFSLPRQSGTEDETDSKKETAVK-----------------------------------------------364 CrCDJ3 EQQQTTGNGDRESRAVAGLAARAARAARTWRRYQEVAAASRREKLVITSMSSASSMDSIDSASGSMDEADRMVAGVVKGGIALR----------------------------393 C CDJ4 QAEERAAVMDPEQRRIANLAASAARAARMWRLYSRSTGG RSLLSESSSGSSMGSGSSMASMSEL 382 CrCDJ4 QAEERAAVMDPEQRRIANLAASAARAARMWRLYSRSTGG----RSLLSESSSGSSMGSGSSMASMSEL--------------------------------------------382 AtDjC17 SKASSNPQVTRKTFPSEEKPTSRRENRRQFRIKKFPIGTAIVAVFLVQYQASYRAASELNDHIGGSLALSIVNSPWQQILLAGVTWYFIGAMLLQLVEAVQHKLEDKET---465 Ps2 ERQERSIGEEAKEVNMKQRESKKDEDLKKRILSGIPLATSVGAAIIVGFGSGGGEAGGLEKHLAGSLAVQLINSFSLQVFLAATVWYIIGTLVASLIAVFVFSRERL-----523 Pp2 GKPGNSYEPKWAGISDLQMGDEVLSGWLKSGLSAIPVTASLIGAVSASMTNGLG-AANSSDISSGPLPAEITSGIFMQVFLAAAVWYMIGAVVGGLATLLVVSAITIKRNKD 521 CrCDJ5 --VGNSVSTGNETEELRQVGRRKRAAGKRWDWLEQQRARGGWMVPPERALVPISVYAESLRN--------------------------------------------------383 Syn ----------------------------------------------------------------------------------------------------------------- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

AtDjC18 MAQILSPVCTDLLKFQNSALSSRSGASPRFSAKTTGASSSWYLPRYAGKRRTDSIGRLRVATEDASSLSTGDVADDYYAVLGLLPDATQEEIKKAYYN 98 Ps1 --MAVTQPQVCTNSLKFQPRDSNTRFRTRDWKPRSLDTSKRMLSQGSGKDLLSERLFMVKLARRGRVRVVGVKAKARDEEVNLYGDSAADDYYSVLGVLPDATPAEIKHAYYS 111 Pp1 -------MAAYQATLPVAPTSRVPAQVYKDHKSSKRYCKPDACPSWLRPRDLLSMRGAPACTKSPMGRVETRAKFGGDGDVDLNGDVIGEDFYSVLGLTPDATQEEIKKAYYS 106 CrCDJ3 ---------------------------------MLRLKATFQRGSNFMAQRRASLSSFAQATRRPYKRPNRLRTAAVASQSAAADEAAEEDFYSLLGVSPLADGKEIKAAYYR 80 CrCDJ4 MHMCRYKNLSTMFSQQESARHGLSQHRPPGRPTPPRSCRQQLHDTNIRAARYSPVDGFVRGLTLTVQRARGGTIPRASSDVASVGLFGGEDFYTILGVTPSAEPRDIKRAYHS 113 AtDjC17 -----------------------------------------MTPAIFSPTTLPPSTATWPCSTSQKLITVRSPLKFKCRATSSSSSITDFDLYDLLGIDRSSDKSQIKSAYRA 72 Ps2 -------------------MLTMAAATIPYGLAVKIPKSSSAFNYSLPLGRRWSSKSQRQRKQPWRRSSYRRRCRCSALPGGVDFENLGYDLYDLLGVESKAGQDQIRRAYRW 94 Pp2 ---------------MATAQLHGRWNLTPSRALPDRKSRFENLRLCGLPLLTEQADLKGLRIAKKSSRRRNNGLVTAAHTARGSAIDHGYDLYELLGVETDAPFPEIKQAYRW 98 CrCDJ5 ------------------------MAFTALQRSATPACTSGRATLTPVPRTAFGRGSAYATRSICGVSSRRIKCDVIAAGNTYLLSNGMIDYYEVLGVDDDAPYDEIKKAYRA 89 Syn ----------------------------------------------------------------------------------------------------------------- - ⎯⎯⎯⎯⎯⎯⎯⎯⎯
-268 Ps1 RARACCQSGN-PDLVQQAIDSCPVDCIHWTSAAQLSLLEDEMRRVERVNVGLMLANMGSAV-PDVFSQASVRWEKRQRRAL--------------------------------281 Pp1 RARVHCQVGD-VRAAQEAIDTCPVDCIHWVTAAQLTLLEDEMRRVERVNVGMMLSGMGYQS-PDVFAQASWRWEKRQAKAM--------------------------------276 CrCDJ3 RARVMLQDADSEAKLQEAIDTCPVSCIHWVTAPQLNLLEGAMARMERIAVWSMMGGSGMGANKDVFVEASYAWQRRQSEIR-
-278 Syn RSRAFSQNGDDEAVIQEAIDTCPVDCIHWVPYDELKHLEEKRKHQQIRPLGYPQINPHL------------------------------------------------------133 * ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ C-terminal domain ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ AtDjC18 -
-296 Ps1 -------------------------------------------------------ERAVIRMRKQKKGGKAQ--------SYWEGAWGVDN----------------------309 Pp1 -------------------------------------------------------EQARIRMMKEKGKDAFG--------AWWQG-WSNPG----------------------303 CrCDJ3 -

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091412 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091412

  Biochemical Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091412 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091412 A c c e p t e d M a n u s c r i p t

Biochemical

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Biochemical

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Figure 7

 7 Figure 7 Enrichment and subcellular localization of CDJ3/4(A) Enrichment of CDJ3/4 from whole-cell extracts. Chlamydomonas whole cells were lysed by sonication in the presence of 2% Triton X-100 and solubilized material was separated from non-soluble matter by centrifugation through a 0.6 M sucrose cushion. The supernatant was incubated with Sepharose beads coupled to preimmune serum (Pre) or to anti-CDJ3 and anti-CDJ4 antisera. Whole-cell proteins (WC) and precipitated proteins were separated on 7.5-15% SDS-polyacrylamide gels and analyzed by immunoblotting. (B) Subcellular localization of CDJ3/4. Chlamydomonas chloroplasts (Cp) were separated into soluble stroma (St), low density membranes (LM), and thylakoid membrane fractions (Th). Mitochondria (Mt) were isolated from the same strain. 7 µg proteins of whole cells (WC)

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⎪⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯

  Figure 1

  J-domain ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎪⎯⎯⎯⎯⎯⎯⎯⎯⎯ Linker ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎪⎯⎯⎯ Ferredoxin domain ⎯⎯⎯ AtDjC18 CMKSCHPDLSGND--PETTNFCMFINDIYEILSDPVQRMVYDEIHGYTVTAINPFLD-------------------DSTPRDHVFVDEFACIGCKNCANVAPDIFQIEED-FG 189 Ps1 CMKSCHPDLSGND--ADATNFCIFVNEVYEVLSDPVQRMVYDEIHGYSLRTVNPFLD-------------------DSRPRDHVFVDEFSCIGCKNCANVASEIFAIEED-FG 202 Pp1 CMKACHPDLSGNN--SDSTDFCMFVNEIYEVLSDPEQRMVYDEINGYALTSANPFLF-------------------PKQERDHAFVDEFTCIGCKNCANVASDTFEIEEE-YG 197 CrCDJ3 MVRTCHPDRSGD---DEATDFCAMLNEVYETLSDPTKRALYDELAGFSAESVNPFLD-------------------DRYPADRVFVDEFSCIGCRNCNAVCPKTFGMEED-YG 170 CrCDJ4 LMREFHPDRAPEGLRDGMADLCVLLNEIYATLSDEEKRCVYDSIAGFSSSAINPFLD-------------------GSFARDQVFVDEISCIGCGKCVRACPMTFEIEDSKYG 207 AtDjC17 LQKRCHPDIAGDPG----HDMAIILNEAYQLLSDPISRQAYDKEQAKLEELRGYTGKPIYSVWCG-----------PETEQRAAFVDEVKCVGCLKCALCAEKTFAIETA-YG 169 Ps2 LQKKCHPDIAGPDG HHMSILLNHAYSVLSDPNARFAYDQTRVETLDLEGYSGQPLYSKWFG SASEERALFVDEVKCVGCLKCALVAPKTFAIETV YG 191 Ps2 LQKKCHPDIAGPDG----HHMSILLNHAYSVLSDPNARFAYDQTRVETLDLEGYSGQPLYSKWFG-----------SASEERALFVDEVKCVGCLKCALVAPKTFAIETV-YG 191 Pp2 LQKRCHPDIAGPIG----HDMSILLNDAYATLQDPMQRAAYDVKRVERAAFDGYTGKPLYSKWLG-----------PEEEDRAIFVDESQCVGCLKCALIASNTFAIENR-YG 195 CrCDJ5 LAKSCHPDYLGEKG----HEICIMLNEAYQILGDADARANYNNKLEQALLDEDDNYTGLPLSKWMPTVKPNMAKNEDPAERRGVFVDEFTCIGCKQCVWCASATFRIEPE-HG 197 Syn --------------------------------------MVIADLNFPPDHRSGLEPELGGDWRNFDDRSGLEPELGGELRERGVYVDEVTCIGCKNCAHVAPNTFTIEQE-HG 74 * * * ⎯⎯⎯ Ferredoxin domain ⎯⎯⎯⎪⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ C-terminal domain ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ AtDjC18 RARACNQRGN-PDLVQQAVETCPVDCIHQTSAAQLSLLEDEMRRVERVNVALMLSGMGSGA-VDVFRMARSRWEKRQAKVL-

  --EELGRLFEAARRRRQGN----GEEGNVLAEDDYWTPVKHVSR----SVPSEEKRATGPRRESKP 410 CrCDJ5 -

Biochemical

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Figure 2 0

 2 Figure 2

  Figure 3

  Figure 6

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© 2010 The Authors Journal compilation © 2010 Portland Press Limited

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ACKNOWLEDGMENTS

We would like to thank the Kazusa DNA Research Institute for providing cDNA clones AV628957 and AV643891, encoding CDJ3 and CDJ4, respectively. We would also like to thank Francis-André Wollman for the antibody against cytochrome f and Mats Eriksson for the antibody against mitochondrial carbonic anhydrase. We are grateful to P. López-García for sharing unpublished insights.

FUNDING This work was supported by the Deutsche Forschungsgemeinschaft [Schr 617/2-4].

TABLES AND FIGURES

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. Here we show that PrP Sc bound to and replicated within both GT1 neuronal cells and murine cortical neurones.

). We report that PrP Sc digested with PI-PLC (PrP Sc -inositolphosphoglycan (PrP Sc -IPG)) did not bind readily to GT1 cells or cortical neurones and did not lead to the formation of PrP Sc in recipient cells over the time course of these experiments. Surprisingly, although PrP Sc that had been digested with PLA 2 (PrP Sc -G-lyso-PI) bound readily to GT1 cells and cortical neurones, it had little capacity to convert endogenous PrP C into PrP Sc .

Experimental.

Cell lines. GT1 cells were grown in medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 200 nM retinoic acid and 2 % foetal calf serum (PAA, Yeovil, UK). GT1 cells were plated in 6 well plates (10 5 cells/well for PrP Sc replication studies and 10 6 cells/well for binding studies) and allowed to adhere overnight.

Primary cortical neurones.

Cortical neuronal cultures were prepared from the brains of mouse embryos (day 15.5) derived from wildtype (PrP +/+ ) or PrP knockout mice (PrP 0/0 ) after mechanical dissociation and cell sieving. Neuronal precursors were plated at (10 6 cells/well) in 6 well plates that had been pre-coated with 5 µg/ml poly-L-lysine, in Ham s F12 containing 5% foetal calf serum for 2 hours. Cultures were shaken (600 r.p.m for 5 minutes) and non-adherent cells removed by 2 washes in phosphate buffered saline (PBS). Neurones were subsequently grown in neurobasal medium containing B27 components (PAA, Yeovil, UK) for 7 days before use. Immunostaining showed that the cells were greater than 97% neurofilament positive. Less than 3% stained for GFAP (astrocytes) or for F4/80 (microglial cells).

Test PrP Sc preparations. Two distinct PrP Sc preparations were used in these assays. The first consisted of the supernatants collected from ScGT1 cells which were centrifuged (500 x g for 5 minutes) to remove cell debris. Supernatants were digested with 0.2 units/ml PI-PLC (Bacillus cereus) or 10 units/ml bee venom PLA 2 (Sigma) with intermittent shaking at 37 O C for 24 hours. The enzymatic activity was destroyed by heating at 70 O C for 5 minutes. This method eschewed proteinase K digestion in order to preserve protease sensitive forms of PrP Sc . The amount of PrP Sc in these supernatants was determined by enzyme linked immunoassay (ELISA) and diluted to 1 ng/ml for further studies. We also used partially purified PrP Sc preparations isolated from ScGT1 cells homogenised in distilled water. Cell debris and nuclei were removed by centrifugation at 500 x g for 5 minutes. Cell membranes collected from the supernatant by centrifugation (10 minutes at 14,000 x g) and solubilised in buffer containing 10 mM Tris-HCL, 10 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40 and 0.5% sodium deoxycholate. PrP C and PrP Sc were immunoprecipitated by incubation with 0.5 µg/ml ICSM35 which recognises native PrP Sc (D-Gen, www.d-gen.co.uk) and µMACS protein G microbeads (10 µl/ml) (Miltenyi Biotech, Bisley, UK). Protein G bound antibody complexes were isolated using a µMACS magnetic system (Miltenyi) and digested with 0.2 units/ml PI-PLC or 10 units/ml bee venom PLA 2 with intermittent shaking at 37 O C for 24 hours. Precipitates were further digested with 5 µg/ml proteinase K for 1 hour at 37 O C to remove PrP C .

Trifluoroacetic acid was added to achieve a 0.1% final concentration, samples were shaken in a cell disruptor (10 minutes) and applied to C18 columns (Waters, Elstree, UK). PrP Sc was eluted under a gradient of acetonitrile in water and 0.1% trifluoroacetic acid and fractions were tested for PrP by ELISA. PrP positive fractions were pooled and run on second C18 column. Samples containing PrP were pooled, washed twice with PBS and the amount of PrP Sc determined by ELISA. Samples were frozen at -20 o C for storage. On the day of use, PrP Sc preparations were diluted to 1 ng/ml and solubilised in culture medium.

Cell treatments. For PrP Sc replication experiments, cells were pulsed with PrP Sc preparations for 24 hours, washed 5 times in warm PBS to remove unbound PrP Sc and incubated in fresh medium for a further 6 days (GT1 cells) or 9 days (cortical neurones). For binding studies, cells were incubated with PrP Sc preparations for 1 or 24 hours. Cells were washed 5 times in warm PBS and cell membrane extracts collected.

Cell membrane extracts. Treated cells were homogenised in an extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and 0.2% SDS) at 10 6 cells/ml. Membranes were homogenised and nuclei and large fragments were removed by centrifugation (300 x g for 5 minutes). To determine the amount of PrP Sc in cells these extracts were digested with 5 µg/ml proteinase K for 1 hour at 37°C; digestion was stopped with mixed protease inhibitors. The soluble material was heated to 95 o C for 5 minutes to denature PrP Sc and tested in a PrP ELISA. Some samples were mixed with an equal volume of Laemmli buffer (Bio-Rad) and subjected to electrophoresis on a 15 % polyacrylamide gel. Proteins were transferred onto a Hybond-P PVDF or nitrocellulose membranes (Amersham Biotech, UK) by semi-dry blotting. Membranes were blocked using 10 % milk powder in Triz-buffered saline, pH 7.2 containing 0.2 % Tween 20. PrP was detected by incubation with mAb ICSM35 (D-gen), followed by a secondary anti-mouse IgG conjugated to peroxidase. Bound antibody was visualised using an enhanced chemiluminescence kit (Amersham Biotech, UK).

Isolation of GPI anchors. PrP Sc samples were further digested with 100 µg/ml proteinase K at 37 O C for 24 hours to release GPI anchors. The released GPIs were extracted with water-saturated butan-1-ol, washed 3 times with ice cold water, lyopholised and dissolved in ethanol. Extracted GPIs were applied to silica gel 60 high performance thin layer chromatography (HPTLC) plates (Whatman) and developed using a mixture of choloroform/methanol/water (4/4/1 -v/v/v). GPI anchors were detected by immunoblotting as described [START_REF] Bate | Role of glycosylphosphatidylinositols in the activation of phospholipase A 2 and the neurotoxicity of prions[END_REF]. Briefly, plates were soaked in 0.1 % poly(isobutyl methacrylate) in hexane, dried, and blocked with PBS containing 5 % milk powder. They were probed with 1 µg /ml of a mouse mAb that binds to phosphatidylinositol, washed with PBS-Tween, and incubated with goat antimouse IgG conjugated to peroxidase (Sigma) for 1 hr. The bound antibody was washed and visualised using an enhanced chemiluminescence kit (Amersham Biotech, UK).

Isolation of detergent resistant membranes (lipid rafts).

Treated cells were lysed in an ice-cold buffer containing 1% Triton X-100, 10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA at 10 6 cells/ml. Membranes were prepared by homogenisation followed by mechanical agitation on a cell disruptor (10 minutes full power), and nuclei and large fragments were removed by centrifugation (300 x g for 5 minutes). The supernatant was incubated on ice for 60 minutes prior to further centrifugation (16,000 x g for 30 minutes at 4 o C). The soluble material was reserved as the normal cell membrane. Insoluble proteins were solubilised in extraction buffer (10 mM Tris-HCL, 10 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and 0.2% SDS and mixed protease inhibitors, centrifuged (10 minutes at 16,000 x g), and the supernatant retained as the lipid raft fraction. Both fractions were digested with proteinase K (5 µg/ml for 1 hour at 37 0 C) to remove PrP C , heated to 95 o C for 5 minutes to denature PrP Sc and tested in a PrP ELISA.

PrP ELISA. The amount of PrP in cell extracts/supernatants was determined by ELISA as described [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]. Maxisorb Immunoplates (Nunc, Rosklide, Denmark) were coated with 0.5 µg/ml a capture mAb A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited (ICSM18 (D-Gen)) which recognises amino acids 146 to 159 of murine PrP. Samples were applied and detected with biotinylated mAb ICSM35 (D-Gen) (which recognises an epitope between amino acids 91 and 110). Biotinylated mAb was detected using extravidin-alkaline phosphatase and 1 mg/ml 4nitrophenyl phosphate (Sigma). Absorbance was measured at 450 nm and the amount of PrP in cell extracts was calculated by reference to a standard curve of recombinant murine PrP (Prionics, Zurich, Switzerland).

Cholesterol Assay. The amount of cholesterol was measured using the Amplex Red cholesterol assay kit (Invitrogen, Paisley, UK). Cholesterol is oxidised by cholesterol oxidase to yield hydrogen peroxide that reacts with 10-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red reagent) to produce highly fluorescent resorufin; measured by excitation at 550 nm and emission detection at 590 nm. The amount of cholesterol was calculated by reference to cholesterol standards.

Statistical Analysis. Comparison of treatment effects was carried out using one and two way analysis of variance techniques as appropriate. For all statistical tests significance was set at the 1 % level.

Results

Partial purification of phospholipase digested PrP Sc on C18 columns. Digestion with PI-PLC results in the removal of diacylglycerol from GPI anchors. As shown in Figure 1 the digestion of PrP Sc with PI-PLC resulted in PrP Sc -IPG. Whereas mock-treated PrP Sc (PrP Sc -GPI) bound to C18 columns and was eluted in fractions containing between 65 and 85% acetonitrile, PrP Sc -IPG did not bind to C18 columns and was eluted in the void volume (Figure 2A). PrP Sc digested with PLA 2 (PrP Sc -G-lyso-PI) bound to C18 columns and was eluted in fractions containing between 35 and 50% acetonitrile. Positive fractions were pooled, the amount of PrP Sc measured by ELISA and the pool diluted to 1 ng/ml for future assays. The amount of PrP Sc in cell preparations was not significantly altered following by digestion with either PLA 2 (4.8 ng/ml ± 0.4 compared with 4.6 ± 0.5, n=9, P=0.6) or PI-PLC (4.5 ng/ml ± 0.3 compared with 4.6 ± 0.5, n=9, P=0.8). Western blot analysis did not detect an observable difference in the apparent molecular weight of these preparations (Figure 2B). However, HPTLC analysis of GPI anchors isolated from phospholipase digested PrP Sc preparations showed that the GPI isolated from PrP Sc digested with PLA 2 or PI-PLC had different migration patterns to those isolated from mock treated PrP Sc (Figure 2C).

Phospholipase digestion of PrP Sc reduced infectivity for GT1 cells.

In the first set of experiments GT1 cells were pulsed with different amounts of mock-treated, or phospholipase digested ScGT1 supernatants (range 1 to 0.016 ng) for 24 hours. We report that mock treated supernatants infected GT1 cells and that the PrP Sc content of recipient GT1 cells was dependent upon the dose of the original innoculum (Figure 3A). In contrast, recipient GT1 cells pulsed with 1 ng of PI-PLC digested ScGT1 supernatants did not contain detectable levels of PrP Sc (less than 0.05 ng). GT1 cells pulsed with PLA 2 digested ScGT1 supernatants contained less PrP Sc than GT1 cells pulsed with mock treated supernatants. GT1 cells were also pulsed with partially purified PrP Sc preparations. The addition of PrP Sc -GPI resulted in a dose-dependent increase in the amount of PrP Sc in recipient GT1 cells (Figure 3B). The addition of 1 ng PrP Sc -GPI did not significantly affect cell survival, as measured by thiazyl blue tetrazolium. We did not detect PrP Sc in GT1 cells pulsed with 1 ng PrP Sc -IPG (less than 0.05 ng). GT1 cells pulsed with 1 ng PrP Sc -G-lyso-PI contained significantly less PrP Sc than GT1 cells pulsed with 1 ng PrP Sc -GPI (0.28 ng PrP Sc ± 0.25 compared with 6.1 ng PrP Sc ± 1.12, n=27, P<0.01). Western blots confirmed that after 7 days GT1 cells pulsed with PrP Sc -GPI contained greater amounts of PrP Sc than did cells pulsed with PrP Sc -Glyso-PI or PrP Sc -IPG (Figure 3C). We report that mock treated ScGT1 supernatants infected cortical neurones; there was a dose-dependent increase in the PrP Sc content of recipient cortical neurones (Figure 4A). Cortical neurones pulsed with PLA 2 digested ScGT1 supernatants also contained PrP Sc . However, digestion with PLA 2 significantly reduced the infectivity of ScGT1 supernatants. We did not detect PrP Sc in cortical neurones pulsed with ScGT1 supernatants containing 1 ng PrP Sc that had been digested with PI-PLC (less than 0.05 ng PrP Sc ).

Phospholipase digestion reduced PrP

The addition of partially purified PrP Sc -GPI resulted in a dose-dependent increase in the amount of PrP Sc in cortical neurones (Figure 4B). Thus, the addition of 1 ng PrP Sc -GPI was amplified to 44 ng PrP Sc ± 6.7, n=22 after 10 days in cortical neurones. We were unable to detect PrP Sc in cortical neurones pulsed with 1 ng PrP Sc -IPG. Cortical neurones pulsed with 1 ng PrP Sc -G-lyso-PI contained 3.63 ng PrP Sc ± 2, n=22. The PrP Sc in these cells bound to C18 columns and was eluted in fractions containing greater than 70% acetonitrile indicating that the PrP Sc contained a GPI anchor with 2 acyl chains. Thus, this PrP Sc had been synthesised by the recipient cortical neurones and was not a remnant of the original inoculum. Primary cortical neurones derived from PrP knockout mice were also pulsed with ScGT1 supernatants containing 1 ng PrP Sc or 1 ng partially purified PrP Sc -GPI for 24 hours and incubated for a further 9 days. We were unable to detect PrP Sc in these cells using either ELISA (less than 0.05 ng) or immmunoblot (Figure 4C).

The binding of PrP Sc to cells is reduced by phospholipase digestion.

To determine if GPI anchors affected the binding of PrP Sc to recipient cells, GT1 cells were incubated with 1 ng partially purified PrP Sc preparations for 1 or 24 hours, washed and the amount of cell-associated PrP Sc was measured. After 1 hour there was no significant difference in the amount of PrP Sc -GPI and PrP Sc -G-lyso-PI that had bound (0.95 ng ± 0.1 compared with 0.93 ± 0.12, n=12, P=0.7) (Figure 5A). After 1 hour incubation we were unable to detect any PrP Sc -IPG that had bound to cells (less than 0.05 ng) indicating that acylation of the GPI anchor facilitates the efficient binding of PrP Sc to neurones. However, increasing the incubation period to 24 hours resulted in a small amount of cell associated PrP Sc -IPG (128 pg ± 42, n=12).

Phospholipase A 2 digested PrP Sc does not target lipid rafts. Next we sought to determine why it was that although PrP Sc -G-lyso-PI bound readily to cells, only low amounts of PrP Sc were produced in recipient cells. PrP Sc production is thought to be dependent on the formation or function of cholesterolsensitive lipid rafts. While the addition of 1 ng PrP Sc -GPI significantly increased the amount of cholesterol in GT1 cell membranes (573 ng/10 6 cells ± 57 compared to 395 ± 38, n=8, P<0.01), the addition of 1 ng PrP Sc -G-lyso-PI had no significant affect (402 ng/10 6 cells ± 47 compared to 395 ± 38, n=8, P=0.78). PrP Sc is found within lipid rafts that are defined by their resistance to solubilisation by nonionic detergents [START_REF] Vey | Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains[END_REF]. We report that greater than 90% of PrP Sc -GPI was found in the detergent resistant (lipid raft) fraction of GT1 cell membranes. In contrast, less than 10% of PrP Sc -G-lyso-PI was found in the lipid raft fraction (Figure 5B). Similar results were obtained when these preparations were added to cortical neurones; PrP Sc -GPI was mainly found within lipid rafts and PrP Sc -G-lyso-PI within the normal cell membrane.

Discussion

The molecular mechanisms by which prions spread throughout an infected brain remain poorly understood. In this study we demonstrate the role of acyl chains of the GPI anchor on the ability of PrP Sc to bind neuronal cells and to replicate. PrP Sc with a conventional GPI anchor (PrP Sc -GPI) was able to bind and replicate within both GT1 neuronal cells and primary cortical neurones. As expected, PrP Sc did not replicate within cortical neurones derived from PrP knockout mice. Modification of the GPI anchor reduced the infectivity of PrP Sc ; PrP Sc -IPG did not bind to cells and although PrP Sc -G-lyso-PI did bind to cells it did not readily convert PrP C to PrP Sc . The partial purification of phospholipase digested PrP Sc molecules was facilitated by using PrP Sc isolated from ScGT1 cells rather than from infected brains. PrP Sc exists as aggregates of different sizes [START_REF] Silveira | The most infectious prion protein particles[END_REF] and large aggregates of PrP Sc are thought to cause steric hindrance of PI-PLC. In this study vigorous shaking increased the susceptibility of PrP Sc to phospholipases, presumably because it reduced the size of PrP Sc aggregates. Digested products were isolated by differential binding and elution from C18 columns in a gradient of acetonitrile and water. Western blot analysis of PI-PLC or PLA 2 digested preparations showed no apparent difference in molecular weight. This result was not surprising considering digestion with PLA 2 would reduce the molecular weight by only 0.2 kDa and PI-PLC digestion by 0.4 kDa. In contrast the removal of the whole GPI anchor would be expected to reduce the molecular weight by approximately 5 kDa. However, HPTLC analysis showed that digestion with either PI-PLC or PLA 2 altered the GPI anchors of these preparations.

Whereas PrP Sc -GPI bound rapidly to neurones, PrP Sc -IPG did not indicating that the acylation of the GPI anchor affected PrP Sc binding to cells. The binding of PrP Sc to cells is complex, and although there are many reports of specific PrP Sc -protein interactions [START_REF] Lee | Towards cellular receptors for prions[END_REF], the nature and presence of specific protein receptors for PrP Sc is yet to meet an agreed consensus. Our observation that the removal of both acyl chains greatly reduced the binding of PrP Sc to cells shows that they contribute strongly to the binding of PrP Sc to cells. Such results do not necessarily exclude prion-protein or prion-glycan interactions which may be facilitated after PrP Sc has inserted into the membrane. We conclude that in these studies PrP Sc -IPG in the supernatant had limited interactions with cell-associated PrP C .

At first glance our results appear to contradict studies from transgenic mice producing PrP C without a GPI anchor which produced high amounts of PrP Sc and infectivity [START_REF] Chesebro | Anchorless Prion Protein Results in Infectious Amyloid Disease Without Clinical Scrapie[END_REF]. However, in these transgenic mice anchorless PrP C was rapidly secreted from neurones. Moreover, a recent study showed that cell lines producing anchorless PrP C were resistant to scrapie infection [START_REF] Mcnally | Cells Expressing Anchorless Prion Protein are Resistant to Scrapie infection[END_REF]. A direct comparison with these results is invalid considering that the removal of the entire GPI anchor in these mice affected the glycosylation of PrP C and its expression at the surface of neurones. As the glycosylation status of PrP C can affect PrP Sc formation [START_REF] Tuzi | Host PrP Glycosylation: A Major Factor Determining the Outcome of Prion Infection[END_REF] it is not clear whether the effects of removing the entire GPI anchor was a direct effect of the loss of the GPI anchor, or an indirect effect resulting from of altered glycosylation. Another study in which recombinant PrP that did not contain a GPI anchor induced prion disease in transgenic mice [START_REF] Legname | Synthetic mammalian prions[END_REF] suggests that the GPI anchor is a not essential for infectivity. Our studies suggest that acylation of the GPI anchor facilitates PrP Sc binding but is probably not the sole factor involved in infectivity. Our results also differ from a study which reported that removal of the GPI anchor from PrP Sc by cathepsin D did not affect prion infectivity [START_REF] Lewis | Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity[END_REF]. However, digestion with cathepsin D removes the whole GPI anchor as well as a fragment of the C terminus, whereas phospholipases simply modify the GPI anchor by the removal of 1 or both acyl chains as shown in Figure 1. Several technical differences are also apparent in the 2 studies, those authors used PrP Sc derived from infected brain homogenates which was extracted using strong detergents and incubated uninfected cells with PrP Sc for 3 days. We used two PrP Sc preparations, the first (ScGT1 supernatants) contained PrP Sc that had been released from ScGT1 cells without the use of detergents and which had not been protease treated. Since protease sensitive forms of PrP Sc have been identified [START_REF] Tzaban | Protease-Sensitive Scrapie Prion Protein in Aggregates of Heterogeneous Sizes[END_REF] our preparations overcame the problem of protease digestion reducing the infectivity of PrP Sc . Although the second PrP Sc preparation was extracted using a mild detergent, most of the detergent was removed by the subsequent passage through C18 columns prior to the addition of PrP Sc to cells. The lack of detergent in our PrP Sc preparations is a possible explanation of the differences between our studies and previous reports.

The amount of PrP Sc -G-lyso-PI and PrP Sc -GPI that had bound to recipient cells after 1 hour was not significantly different indicating that a single acyl chain is sufficient to facilitate the binding of PrP Sc to neuronal cells. Crucially, although PrP Sc -G-lyso-PI bound to cells, it did not readily convert endogenous PrP C . While these preparations contained mostly PrP Sc -G-lyso-PI we cannot exclude the possibility that they also contained a small amount of undigested PrP Sc -GPI which was responsible for PrP Sc A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited recipient cells. Most GPI-anchored proteins are targeted to cholesterol dense lipid rafts and the conversion of PrP C to PrP Sc is cholesterol sensitive [START_REF] Bate | Squalestatin cures prion-infected neurones and protects against prion neurotoxicity[END_REF][START_REF] Taraboulos | Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform[END_REF]. GPI-anchors contain predominantly unsaturated acyl chains which encourage the solubilisation of cholesterol [START_REF] Schroeder | Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPIanchored proteins in liposomes and cells show similar behavior[END_REF]. In this study the addition of PrP Sc -GPI significantly increased the cholesterol content of cells whereas the addition of PrP Sc -G-lyso-PI had no effect. The amount of cholesterol in cell membranes affects lipid raft formation and greater than 90% of PrP Sc -GPI was targeted to lipid rafts. In contrast; less than 10% of PrP Sc -G-lyso-PI was found within lipid rafts indicating that these molecules were mostly in the normal cell membrane. The localisation of PrP Sc -G-lyso-PI to non-raft membranes may restrict its interactions with endogenous PrP C which is localised to lipid rafts. Since lipid rafts can regulate cell signalling [START_REF] Simons | Lipid rafts and signal transduction[END_REF] the localisation of PrP Sc -GPI to lipid rafts may enhance cell signalling pathways involved in PrP C conversion. In contrast, the localisation of PrP Sc -Glyso-PI to non-raft membranes may reduce cell signalling involved in PrP C conversion.

In summary we showed that PrP Sc was able to bind and replicate in both GT1 neuronal cells and primary cortical neurones. A combination of phospholipase digestion and shaking allowed the role of the GPI anchor on PrP Sc binding and replication to be determined. PI-PLC digestion of PrP Sc reduced its ability to bind to cells. Although PLA 2 digested PrP Sc bound to neuronal cells, it was not found within lipid rafts and infected cells contained only low amounts of PrP Sc . Such studies show that the GPI anchor attached to PrP Sc affects the targeting of PrP Sc to specific membranes and its ability to either interact with, or convert, endogenous PrP C . We speculate that compounds that alter GPI anchors may provide a novel therapeutic approach to combat prion disease.

Acknowledgements.

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  Journal Immediate Publication. Published on 03 Mar 2010 as manuscript BJ20091469 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091469

  Sc infectivity for cortical neurones. To confirm the GT1 cell data in non-dividing cells primary cortical neurones were pulsed with ScGT1 supernatants at different Biochemical Journal Immediate Publication. Published on 03 Mar 2010 as manuscript BJ20091469 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091469 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited concentrations as shown.

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  formation in Biochemical Journal Immediate Publication. Published on 03 Mar 2010 as manuscript BJ20091469 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091469

Figure 1 .

 1 Figure 1. Phospholipase digestion of PrP Sc GPI anchors. Cartoon showing the cleavage sites of PLA 2 and PI-PLC on the putative GPI anchor attached to PrP Sc . Glycan residues shown include mannose (Man), sialic acid (SA), galactose (Gal), N-acetyl galactosamine (GalNAc) and glucosamine (GlcN) as well as phosphate (P). Also shown are the predicted digested products (PrP Sc -IPG and PrP Sc -G-lyso-PI).

Figure 2 .

 2 Figure 2. Phospholipase digestion of PrP Sc alters C18 binding. (A) The amount of PrP in fractions eluted by reverse phase chromatography under a gradient of acetonitrile in water from C18 columns loaded with mock digested PrP Sc (PrP Sc -PrP Sc digested with PI-PLC (PrP Sc -Sc digested with PLA 2 (PrP Sc -G-lyso-Sc (ng/ml) ± SD, n=9. (B) Immunoblots showing PrP Sc -GPI (1), PrP Sc -G-lyso-PI (2) or PrP Sc -IPG (3). (C) HPTLC analysis of GPI anchors isolated from PrP Sc -GPI (1), PrP Sc -G-lyso-PI (2) and PrP Sc -IPG (3).

Figure 3 .

 3 Figure 3. PrP Sc replicates within GT1 cells. (A) The amount of PrP Sc in GT1 cells pulsed for 24 hours with different concentrations (inoculum) of mock 2 digested as shown and incubated for a further 6 days. Values show are the mean average amount of PrP Sc (ng/10 6 cells) ± SD, n=9. (B) The amount of PrP Sc in GT1 cells pulsed for 24 hours with different amounts of partially purified PrP Sc -Sc -G-lyso-shown and incubated for a further 6 days. Values show are the mean average amount of PrP Sc (ng/10 6 cells) ± SD, n= 27. (C) Immunoblots showing the amount of PrP Sc in GT1 cells had been pulsed or 24 hours with 1 ng PrP Sc -GPI (1), PrP Sc -G-lyso-PI (2) or PrP Sc -IPG (3) and grown for a further 6 days.

Figure 4 .

 4 Figure 4. Phospholipase digestion reduced PrP Sc replication in cortical neurones. (A) The amount of PrP Sc in cortical neurones pulsed for 24 hours with different concentrations (innoculum) of mock-treated ScGT1 cell 2 digested ScGT1 cell d incubated for a further 9 days. Values show are the mean average amount of PrP Sc (ng/10 6 cells) ± SD, n=9. (B) The total amount of PrP Sc in primary cortical neurones pulsed with different amounts of partially purified PrP Sc -PrP Sc -G-lyso-for 24 hours and incubated for a further 9 days. Values show are the mean average amount of PrP Sc (ng/10 6 cells) ± SD, n=22. (C) Immunoblots showing the PrP Sc content of primary cortical neurones derived from PrP wildtype (+/+) or PrP knockout (0/0) mice that had been pulsed for 24 hours with 1 ng PrP Sc -GPI and incubated for a further 9 days.

Figure 5 .

 5 Figure 5. Phospholipase A 2 digestion of the PrP Sc -GPI anchors affects detergent solubility. (A) The amount of PrP Sc -GPI, PrP Sc -G-lyso-PI or PrP Sc -IPG found in the in whole cell extracts from GT1 cells after 1 hour. Values shown are the mean average amount of PrP Sc (ng) ± SD, n=12. Inset -Immunoblot showing the amount of PrP Sc in GT1 cells had been pulsed for 1 hour with 1 ng PrP Sc -GPI (1), PrP Sc -Glyso-PI (2) or PrP Sc -IPG (3). (B) The amount of PrP Sc -GPI or PrP Sc -G-lyso-PI found in the in detergent-Values shown are the mean average amount of PrP Sc (ng) ± SD, n=12.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited