Clare Jelinska

Biljana Petrovic-Stojanovska

W John Ingledew

Malcolm F White

Dimer:dimer stacking Interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

Keywords: stranded DNA, ssDNA, single stranded DNA, SDSL, site-directed spin-label, MTSSL, (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate, HSQC, heteronuclear single quantum coherence, DEER, double electron-electron resonance archaea, Alba, NMR, site-directed spin labelling, electron spin resonance Biochemical

Dimer stacking by the archaeal chromatin protein Alba Abbreviations used: dsDNA, double

INTRODUCTION

In contrast to the eukaryotic histone, there is no universal archaeal chromatin protein. Instead, archaea tend to utilise two or more unrelated proteins to package their DNA. The archaeal histone is conserved in the euryarchaeal branch of the archaeal lineage and like its eukaryotic counterpart it forms a nucleosome that wraps dsDNA (reviewed in [START_REF] Sandman | Molecular components of the archaeal nucleosome[END_REF]). Histone proteins are also present in the marine archaeon Cenarchaeum symbiosum [START_REF] Cubonova | Histones in crenarchaea[END_REF], but are absent from most crenarchaea and also from euryarchaeal thermoacidophiles such as Thermoplasma acidophilum [START_REF] White | Holding it together: chromatin in the archaea[END_REF]. The second widely distributed chromatin protein, Alba (also known as Sac10b or Sso10b) is present in one or two copies in the genomes of all archaea sequenced to date with the exception of the Methanosarcinas and Halophiles [START_REF] Sandman | Archaeal chromatin proteins: different structures but common function?[END_REF].

Alba is a dimeric, highly basic protein with a subunit size around 10 kDa. The Alba1 protein is highly abundant in the Sulfolobus species from which it was originally purified [START_REF] Lurz | Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius[END_REF], representing approximately 4% of the total soluble protein [START_REF] Xue | An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion[END_REF]. The crystal structure of Alba1 from Sulfolobus solfataricus revealed a compact structure similar to DNaseI, translation initiation factor IF3 and other bacterial proteins [START_REF] Wardleworth | Structure of Alba: an archaeal chromatin protein modulated by acetylation[END_REF]. The structural similarity to RNA binding proteins, coupled with the observation that Alba can bind to RNA in vivo and in vitro [START_REF] Guo | Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo[END_REF], has prompted the suggestion that Alba may also function as an RNA binding protein in vivo [START_REF] Aravind | The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism[END_REF]. A role in DNA binding in vivo has been confirmed by chromatin immunoprecipitation experiments, which show a wide distribution of S. solfataricus Alba1 at different gene loci [START_REF] Marsh | Sir2 and the acetyltransferase, Pat, regulate the archael chromatin protein, Alba[END_REF]. In S. solfataricus, a second Alba paralogue, Alba2, is expressed at 5-10% of the Alba1 protein level. Alba2 forms heterodimers with Alba1, resulting in a reduction in DNA binding affinity, and it has been suggested that this may provide a mechanism for the control of chromatin packaging in this organism [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF].

The Alba1 protein in S. solfataricus is reversibly acetylated on a single lysine residue, and this results in a reduction in the DNA binding affinity [START_REF] Bell | The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation[END_REF]. In vitro, acetylation of Alba1 reduces the repressive effects of the protein on transcription [START_REF] Bell | The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation[END_REF] and on strand separation by the replicative helicase MCM [START_REF] Marsh | Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM[END_REF]. However, the relevance of these findings in other archaeal species is not yet clear [START_REF] Sandman | Archaeal chromatin proteins: different structures but common function?[END_REF]. DNA binding by Alba is a highly cooperative process, with a final stoichiometry of one Alba dimer for every 6 bp of dsDNA bound, suggesting a high binding density [START_REF] Lurz | Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius[END_REF][START_REF] Xue | An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion[END_REF]. Electron microscopy studies of DNA binding by Alba from Sulfolobus acidocaldarius revealed the formation of fibre-like structures that were thought to indicate extended interwound helical protein fibres [START_REF] Lurz | Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius[END_REF]. These findings were later confirmed using the recombinant protein from S. solfataricus [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF], where it was further shown that the presence of the Alba2 protein caused subtle changes in the chromatin structures visualised. The crystal structure of Alba1 from S. solfataricus and Alba1 proteins from other species have highlighted a strongly conserved dimer:dimer interface centred on the Phe-60 residue, which stacks with its counterpart in the adjacent Alba1 dimer in the crystal lattice. It has been proposed that this interaction has biological relevance [START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF] and may relate to the fibre formation observed by electron microscopy [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. It has also been suggested that one role of the Alba2 subunit is to weaken or disrupt the dimer:dimer interface in protein fibres, as this interface is not conserved in Alba2 [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF].

In this paper we use NMR to map the amide backbone chemical shift changes of S. solfataricus Alba1 upon binding to dsDNA, ssDNA and RNA. This highlights differences between the three nucleic acid types, both in terms of the residues that are perturbed and the nature (size and shape) of the complexes formed. We show, using gel electrophoretic mobility shift experiments, that dsDNA is bound more tightly than either ssDNA or RNA. Site directed spin labelling (SDSL) is used as a sensitive probe of dimer:dimer formation on DNA binding, and the relevance of the Alba1 crystallographic dimer:dimer interface is confirmed using an F60A site directed mutant form of the Alba1 protein.

EXPERIMENTAL

Protein expression, purification and mutagenesis

Recombinant Alba1 proteins were expressed in E.coli BL21 Rosetta strain and purified as described previously [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF][START_REF] Wardleworth | Preliminary crystallographic studies of the double-stranded DNA binding protein Sso10b from Sulfolobus solfataricus[END_REF]. Protein concentrations were determined by measuring the optical absorbance at 280 nm. The F60A and R59C mutant versions of Alba1 were constructed using the Quikchange mutagenesis protocol (Stratagene), following the manufacturer's instructions. The oligos used for mutagenesis, F60Af; F60Ar; R59Cf; R59Cr, are available from the corresponding author on request. Mutated genes were sequenced fully to confirm that no spurious mutations had been introduced. The mutant proteins were purified as for the wild-type protein.

Oligonucleotides for binding studies

DNA and RNA oligonucleotides were purchased from MWG Biotech Ltd and Operon Biotechnologies, Cologne, Germany. The sequence of the 39mer ssDNA was 5'-CCTCGGTGCTAAGTTGATGCTGGTACTCGGAGTATCCCG-3' and that of the 16mer ssDNA and ssRNAs were 5'-CCCGGCGT(U)GCGGCCCG-3', where uracil (U) replaces thymine in the RNA oligomer. The 16 and 39mer ssDNA sequences correspond to one complimentary strand of the 16 and 39 bp DNA duplexes, respectively. 28mer polyadenine (28A) and 30mer polyuridine (30U) ssRNA oligonucleotides were also used.

DNA electrophoretic gel retardation assays

Binding assays were performed, and the data analysed as described previously [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. Apparent dissociation constants for the interaction between wild-type Alba1 and a 39 bp DNA duplex, 39 mer ssDNA, 30U, 28A and 16 mer ssRNAs, and for the interaction between F60A Alba1 and 16 and 39 bp DNA duplexes were determined. Data were fitted to a two state binding model where the fraction bound = 1/(1 + Kd/[Alba1]) m and the exponent, m, corrects for deviations in slope resulting from cooperativity. Data-fitting and graphical representations were generated using the program KALIEDOGRAPH (Synergy Software).

NMR Spectroscopy

The backbone assignment of Alba1 was obtained as described previously [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. A 420 M solution of 15 N labelled Alba1 in 20 mM Bis-Tris, 0.3 M NaCl, 1 mM NaN 3 , pH 6.5 containing 10 % D 2 O was titrated with a solution of 1.2 mM 16 bp DNA duplex to give a final molecular ratio (duplex:Alba dimer) of 0.2. Similar titrations were performed with the 16 mer ssDNA 16 mer ssRNA and 28A ssRNA oligomers to final molecular ratios of 0.21, 0.33 and 0.14, respectively. Sub-stoichiometric quantities were used in order to maximise any potential contributions from additional protein-protein interactions formed upon binding of Alba1 to each oligonucleotide. After each addition the samples were allowed to equilibrate at 55 C for ~15 min after which a 1 H-15 N HSQC spectrum was recorded. Spectra were acquired on a 500 MHz Brüker DRX spectrometer. All spectra were referenced in the direct dimension to the carrier frequency at 4.75 ppm and to the relative gyromagnetic ratios of 1 H and 15 N in the indirect dimension. NMR data were processed using FELIX 2000 (Accelrys, San Diego).

Plots of the average relative signal intensity as a function of mole fraction of oligonucleotide were generated using KALIEDOGRAPH (Synergy Software). The initial intensity of each amide resonance was normalized to 1. In order to reflect only the effect on correlation time, data relating to residues with an associated chemical shift change (where [( n/10) 2 + nh 2 ] > 0.015) were excluded.

Site-directed spin-labelling (SDSL)

Site directed mutagenesis was used to convert residue Arg-59 to a cysteine residue, as described. As there were no cysteines in the native S. solfataricus Alba1 sequence, the R59C mutant could be directly modified with the thiol-specific methanethiosulfonate spin label MTSSL (Toronto Research Chemicals Inc.). The protein was purified as for the wild-type protein, and dialysed against 10 mM TES buffer pH 7.4, 100 mM NaCl overnight at 4 °C. The spin labelling reaction was carried out using 20 µM R59C protein, 100 µM MTSSL in 10 mM TES buffer pH 7.4 and 100 mM NaCl in a volume of 2.5 ml [START_REF] Rabenstein | Determination of the distance between two spin labels attached to a macromolecule[END_REF]. After incubation at 4 °C for 1 hr, the unincorporated label was removed from the protein sample using a sephadex G-25 minicolumn (GE Healthcare) according to the manufacturer's instructions. The purified protein was concentrated to achieve a final concentration of 100-150 µM in 300 µl total volume, and the incorporation of the spin label was confirmed by mass spectrometry. For experiments in the presence of DNA, a 16bp duplex assembled from the oligo 5'-CCCGGCGTGCGGCCCG-3' and its complement, was added to the protein sample to a final concentration of 100 µM.

Electron Spin Resonance Spectroscopy

Samples were prepared for ESR by buffer-exchanging them into D2O/deuterated ethyleneglycol media (TES pH 7.4, 100 mM NaCl). The spin-labelled protein was exchanged into D 2 O (Aldrich) buffer and 20 % deuterated ethylene glycol-d 6 (Cambridge Isotope Laboratories) by sequential concentration and dilution with a centricon spin concetrator. The protein solution was then transferred into a clear fused quartz ESR tube, 3 mm internal diameter under an Argon atmosphere. The samples in ESR-tubes were quick-frozen by immersion into an isopentane/hexane freezing mixture (~ -70 °C) and then stored under liquid nitrogen until use. Continuous wave electron spin resonance spectra (cw-ESR) were measured at X-band using a Bruker ESP 300 ESR spectrometer as described previously [START_REF] Hastings | Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase-cytochrome bd from Escherichia coli[END_REF]. Spectra acquisition conditions are given in the figure legend. The pulsed DEER (Double Electron-Electron Resonance) experiments were carried out using a Brüker ELEXSYS E580 spectrometer operating at X-band with a dielectric ring resonator (ER 4118X-MD5-EN) and a Brüker 400U second microwave source unit. All measurements reported here were made at 50 K with an over-coupled resonator giving a Q factor of approximately 100. The measurements used the four pulse, dead-time free, sequence with the pump pulse frequency positioned at the centre of the nitroxide spectrum; the frequency of the observer pulses was increased by 70 MHz [START_REF] Rabenstein | Determination of the distance between two spin labels attached to a macromolecule[END_REF]. The observer sequence used a 32 ns -pulse; the pump -pulse was typically 30 ns. The resulting dipolar coupling evolution data was analyzed by Tikhonov regularization to a distance distribution using DeerAnalysis2006 developed and made freely available by Gunnar Jeschke (www.mpip-mainz.mpg.de/~jeschke/wpub.html) [START_REF] Jeschke | Data analysis procedures for pulse ELDOR measurements of broad distance distributions[END_REF].

RESULTS

Comparison of nucleic acid binding affinities of Alba1

To compare the binding of Alba1 to dsDNA, ssDNA and ssRNA, we determined the binding A c c e p t e d M a n u s c r i p t affinities by electrophoretic mobility shift assay using 39 bp dsDNA, 39mer ssDNA and 30U, 28A and 16mer ssRNA oligonucleotides (Fig. 1). The RNA oligonucleotides were designed to test for sequence-specific binding and to avoid the possibility of base pairing. The apparent dissociation constants were calculated from experiments carried out in triplicate. The data show that Alba1 binds about 5 fold less tightly to ssDNA (KD 270 nM) and about 20 fold less tightly to ssRNA (KD 960 nM) than it does to dsDNA (KD 50 nM) (Table 1). In contrast, Guo et al. [START_REF] Guo | Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo[END_REF] reported that KD's for all three species were "similar" (~ 100-300 nM) although a rigorous quantification of the dissociation constants was not included in their paper. This difference can be accounted for by the fact that our experiments were conducted at relatively high salt (0.3 M vs 0.01 mM), which weakens the interaction with RNA [START_REF] Guo | Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo[END_REF], but does not affect the dsDNA binding affinity (data not shown). Higher ionic strengths are likely to be closer to the physiological condition. Notably, we also found the RNA binding affinity of Alba1 to be independent of the length of oligonucleotide in contrast to dsDNA, where binding to longer duplexes is tighter [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. This suggests that the molecular nature of the interaction of Alba1 with ssRNA is also different from that with dsDNA. dsRNA, which adopts the A-form helical conformation, was not studied.

Residues involved in binding dsDNA, ssDNA and ssRNA

Initial 1 H 1D experiments, where Alba1 was titrated with a 28 bp duplex, resulted in the formation of a very large complex suggestive of a DNA-templated assembly process involving multiple protein-protein interactions (data not shown). A much smaller complex was formed upon binding to a shorter duplex (16bp) under the same conditions (0.3 M NaCl, pH 6.5, 55 C). Residues perturbed upon binding this duplex and the 16 mer ssDNA and ssRNA oligomers were then determined by measuring the changes in chemical shift of fast exchange peaks observed in the 1 H-15 N HSQC spectrum of Alba1 upon titration with the relevant oligonucleotide. It was found that amide resonances, corresponding to G4 and R86, were not present in the 1 H- 15 N HSQC spectrum at pH 6.5, presumably due to intermediate exchange with solvent. In addition, no peaks were observed for K16, R44 or Q80 regardless of pH. In Figure 2 the summed, weighted changes in 1 H-and 15 N chemical shifts are plotted against residue number. These changes cannot unequivocally be attributed to a direct interaction between protein and oligonucleotide, but their localised nature implies that large changes in protein conformation are not involved. Where the sum chemical shift changes ( 0.015) are plotted onto a surface representation of Alba1, the general binding regions can be clearly identified. It is clear that the basic surface predicted to interact with DNA following the initial structural studies [START_REF] Wardleworth | Structure of Alba: an archaeal chromatin protein modulated by acetylation[END_REF] does constitute the major portion of the binding interface. It is apparent that the residues involved in binding the DNA duplex and ssDNA are the same whilst only a subset of these are involved in binding to the equivalent ssRNA. Additionally, there is a distinct region (residues 10-15) that appears to be associated more specifically with RNA binding as well as a DNA specific region (residues 19-21). Data obtained from a titration of Alba1 with 28 poly-A ssRNA were very similar to those obtained with the 16mer ssRNA, suggesting that the changes observed were not strongly sequence dependent. The NMR acquisition parameters, processing and experimental conditions for the titrations of Alba1 with DNA and RNA were identical. The degree to which peaks are attenuated upon addition of the various oligonucleotides therefore provides further evidence that the interaction of Alba1 with DNA is distinct from that with RNA (Fig. 3). Provided that contributions from chemical and conformational exchange resulting from a direct binding interaction are removed, the degree of line-broadening reflects the tumbling rate (i.e the size and shape) of the complex. The greater degree of line-broadening indicates that a larger complex is formed upon binding of Alba1 to the 16mer ss-and duplex DNAs than to 16mer ssRNA. These data clearly show that the increase in correlation time upon binding to DNA is larger than that upon binding to RNA. This indicates that the DNA complex is larger and/or more elongated than the RNA complex and is consistent with the formation of additional protein-protein interactions.

Importance of the dimer:dimer interface for DNA binding.

Upon binding of Alba1 to DNA, chemical shift changes were observed for residues associated with a conserved crystallographic dimer-dimer interface (Figure 2). These observations are consistent with the formation of additional protein-protein interactions in the Alba1-DNA complex that may indicate a mechanism for the assembly of nucleo-protein filaments. Crystal structures of the Alba1 protein from several species highlight the conserved dimer:dimer interface [START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF]. In contrast, the S. solfataricus Alba2 protein is quite divergent in this region, and this interface is not present in the crystal structure [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. Phe-60 is central to the dimer:dimer crystallographic interface of Alba1, where it forms astacking interaction with an adjacent dimer. Mutation of this position (F54R) in Archaeoglobus fulgidus Alba1 resulted in a qualitative decrease in the apparent binding affinity for plasmid DNA [START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF]. The F60A mutant version of Alba1 was made to test the significance of this residue with respect to DNA binding. The apparent binding affinities of the mutant with 16 and 39 bp duplexes were determined by electrophoretic mobility shift assay and compared with the wild-type protein (Fig. 4 and Table 1). The F60A mutant bound the 16 mer and 39 mer duplexes more weakly than the wild-type protein (3.5-fold and 9-fold, respectively), resulting in an equal affinity for both duplexes. This was consistent with the hypothesis that the dimer:dimer interface is involved in the assembly of Alba1-DNA nucleoprotein complexes and suggests that additional protein-protein interactions formed upon binding of the wild-type protein to longer duplexes are not present in the F60A complexes. These data therefore support the assertion that a protein-protein interaction surface, similar to the crystallographic dimer:dimer interface is involved in the assembly of Alba1 nucleoprotein filaments.

Site-directed spin-labelling (SDSL) as a probe for DNA-templated protein assembly.

In the absence of DNA, Alba1 exists as a dimer in solution, and Arg-59 in one subunit is separated from the same residue in the second subunit of the dimer by a distance of approx 40 Å (Figure 5A). The optimum form of ESR spectroscopy to measure interspin distances depends on the separation of the spin labels: below 20 Å, dipolar splittings observed by conventional continuous wave (cw) ESR can be used; above 20 Å more sophisticated ESR methods are required. We used pulsed DEER spectroscopy (pulsed Double Electron-Electron Resonance) to measure the distance between spin labels in the Arg-59C SDSL sample, estimating an average inter-spin distance of 41 Å with a half height distribution of +/-3.5 Å (Figure 4B) in good agreement with the crystal structure (Figure 5A).

When 16bp duplex DNA was added to the spin labelled Alba R59C to a final concentration of 100 µM a dramatic change in the spin-spin interaction between spin-labels on adjacent monomers occurred. A splitting of the cw-ESR spectrum due to dipolar interaction between close paramagnets was observed. In Fig. 4C the cw-ESR spectrum of the spin-labelled Alba R59C is shown in the absence (A-top spectrum) and the presence of 16 bp duplex DNA (B-middle spectrum) or 21mer ssDNA (C-bottom spectrum). Splitting of the spectrum due to dipolar interaction between closely adjacent paramagnets was induced by dsDNA binding (these are indicated in Figure 4 A c c e p t e d M a n u s c r i p t the Alba R59C present was expected to exhibit this interaction as the 16 bp duplex is only long enough to bind three dimers and there was also more DNA present than required for saturation of binding. Therefore the ESR spectrum shows a mixture of the interacting and non-interacting species because some of the spin-label will be at an exposed 'free-end' and not involved in a close dipolar interaction with an adjacent dimer. The dipolar splitting observed is in the order of 72 Gauss peak to peak (7.2 x10 -3 Tesla) which corresponds to an inter-spin distance of approx. 7.5 Å [START_REF] Rabenstein | Determination of the distance between two spin labels attached to a macromolecule[END_REF]. This confirms the close assembly of two spin labels, as would be expected if consecutive Alba dimers stack end-to-end on DNA binding as predicted from the crystal structure. The addition of ssDNA (21 bases) appeared to have a grossly similar but less well resolved effect to the addition of dsDNA. There are splittings due to interactions between close spin-labels, but these are broader and less well resolved, indicating the structure is more heterogeneous, perhaps due to a greater flexibility in the Alba-ssDNA complexes.

DEER spectra could not be readily obtained in the presence of dsDNA or ssDNA because the close dipolar interactions enhance the transverse relaxation times, which shortens the available experimental timing window to an extent that the technique can't be readily used. Direct measurement of the transverse relaxation (not shown) confirmed a considerable enhancement of the relaxation due to the close interactions between spin labels when bound to DNA.

DISCUSSION

The majority of sequenced archaeal genomes contain at least one gene encoding the Alba protein [START_REF] White | Holding it together: chromatin in the archaea[END_REF], making it perhaps the most conserved nucleic acid binding protein in the archaea. Although Alba clearly binds dsDNA in vitro, there has been some debate about its true role in vivo. On the one hand, UV crosslinking data has suggested that an interaction with RNA might be more physiologically relevant [START_REF] Guo | Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo[END_REF]. On the other, chromatin immunoprecipitation experiments have demonstrated that Alba1 is associated with dsDNA [START_REF] Marsh | Sir2 and the acetyltransferase, Pat, regulate the archael chromatin protein, Alba[END_REF]. Alba1 from S. solfataricus has been shown to stabilize dsDNA against DNA melting by the cognate single stranded DNA binding protein, consistent with a role in the stabilization of dsDNA in vivo [START_REF] Cubeddu | DNA damage detection by an archaeal singlestranded DNA-binding protein[END_REF]. Our data demonstrates that Alba1 binds dsDNA more tightly than either ssDNA or RNA in vitro, and suggests that differences exist in the interface with DNA and RNA species that are still not fully understood. The stabilization of RNA as well as DNA is likely to be particularly important in thermophiles, where chemical and physical damage to nucleic acid is accelerated by elevated growth temperatures [START_REF] Grogan | Stability and repair of DNA in hyperthermophilic Archaea[END_REF]. A recent study of nucleic acid binding proteins in the crenarchaeote Thermoproteus tenax, which lacks any clear ssDNA binding protein encoding gene, revealed that the two main detectable ssDNA binding proteins in this organism were the Alba protein and a novel protein named CC1 [START_REF] Luo | CC1, a novel crenarchaeal DNA binding protein[END_REF]. Both proteins bound ssDNA and dsDNA. Thus, it is possible that archaea utilize a number of highly-expressed general nucleic acid binding proteins, such as Alba, to stabilize all classes of nucleic acid in vivo.

The DNA interaction region as determined by NMR is only partially consistent with previous binding models [START_REF] Wardleworth | Structure of Alba: an archaeal chromatin protein modulated by acetylation[END_REF][START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF] and differs significantly from that determined by Cui et al [START_REF] Cui | Two conformations of archaeal Ssh10b. The origin of its temperature-dependent interaction with DNA[END_REF]. Our experiment is different in that sub-stoichiometric quantities of duplex were used in order to maximize the contribution from protein-protein interactions, which were not evident at the 3:1 excess of duplex used previously [START_REF] Cui | Two conformations of archaeal Ssh10b. The origin of its temperature-dependent interaction with DNA[END_REF]. We also found widespread changes in chemical shift upon the addition of salt, which we measured prior to commencing our titrations. We found that whilst the basic, positively charged surface is highlighted, the extended -loop region (residues 78-84), previously thought to bind in the minor groove of the double helix does not appear to be involved. The evidence for this is two-fold; first the A c c e p t e d M a n u s c r i p t lack of changes in chemical shifts in this region and second, that the loop remains flexible upon binding. This flexibility is exemplified by R83, at the tip of the loop region which gives rise to a distinctive sharp resonance throughout the titrations (not shown). The NMR data also provided evidence that higher order complexes of Alba1 are a feature of DNA binding. Oligomerization of Alba1, through a dimer-dimer interface, into linear rod-like structures has been observed in all but one of the 5 crystal structures published to date [START_REF] Wardleworth | Structure of Alba: an archaeal chromatin protein modulated by acetylation[END_REF][START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF][START_REF] Wang | Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii[END_REF]. This has been argued to be of functional importance as the residues involved at the oligomerisation interface are highly conserved within the archaea [START_REF] Zhao | Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding[END_REF]. The NMR data suggest that this protein-protein interface might indeed be important for the assembly of the Alba:DNA nucleoprotein complex. This was confirmed by site-directed mutagenesis of the central residue of the interface, Phe-60, which resulted in weaker DNA binding.

Additionally, we have shown using SDSL that Alba1 dimers assemble on DNA with consecutive dimers binding end-to-end, consistent with the crystal structures. This presumably relates to the extended helical protein fibres observed by electron microscopy when Alba1 is complexed with plasmid DNA [START_REF] Lurz | Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius[END_REF][START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. An attractive hypothesis is that the dimer:dimer interface may provide a mechanism by which DNA could be rapidly sequestered into high order structures. The next challenge is to formulate a molecular description of the chromatin structure formed by DNA-bound Alba1. This represents, to the best of our knowledge the first example of the use of spin labeling to study nucleoprotein formation, and demonstrates the utility of SDSL to study this type of molecular interaction. One advantage of SDSL over FRET is that only a single type of reporter group is required in SDSL, whereas donor and acceptor dyes are required for FRET studies, which can pose a technical challenge. This parameter was used to improve the fit and reflects the steepness of the curve. b These values are reproduced from [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. to either a 39 bp or 16 bp duplex respectively. The sequence of the 39mer ssDNA was 5'-CCTCGGTGCTAAGTTGATGCTGGTACTCGGAGTATCCCG, which was annealed with a complementary strand to produce the 39 bp duplex. Gel-shift analysis was carried out in triplicate and analysed as described previously [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. Standard error bars are shown for each data point. The apparent dissociation constants measured for the wild-type and F60A mutant proteins to the 39 bp duplex were 56 ± 2 µM and 520 ± 13 µM, respectively. The equivalent values for the 16 bp duplex were 100 ± 4 µM and 710 ± 20 µM. A significant increase in apparent binding affinity (Kd) for the 39 bp duplex when compared to the 16 bp duplex is observed for the wild-type but not for the F60A mutant (Table 1).

*Data for the wild-type protein are reproduced from reference [START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF], but were measured at the same time as those for the F60A mutant. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 18 Jan 2010 as manuscript BJ20091841 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091841 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

  Biochemical Journal Immediate Publication. Published on 18 Jan 2010 as manuscript BJ20091841 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091841

Biochemical

  Journal Immediate Publication. Published on 18 Jan 2010 as manuscript BJ20091841 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091841 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

  and also shown at x10 amplification). Not all Biochemical Journal Immediate Publication. Published on 18 Jan 2010 as manuscript BJ20091841 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091841

Biochemical

  Journal Immediate Publication. Published on 18 Jan 2010 as manuscript BJ20091841 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091841

  a

Figure legends Figure 1 .Figure 2 .

 12 Figure legends Figure 1. DNA and RNA binding by Alba1. Binding Curves were obtained by gel electrophoretic retardation analysis. Data relating to binding of Alba to 39 bp dsDNA*, 39 mer ssDNA and 30U ssRNA, 28A ssRNA and 16 mer ssRNA are denoted by open circles, open squares, closed triangles, closed squares and closed circles, respectively. Standard error bars are shown for each data point and curves obtained from fitting the data to the binding equation described in the text are represented as solid black lines.*Data reproduced from[START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]

Figure 3 .

 3 Figure 3. Global NMR line broadening effects during titration of Alba1 with nucleic acid.Alba1 was titrated with a 16 bp duplex (circles), a 16 mer ssDNA (squares) and the corresponding 16 mer ssRNA (triangles). For each point in the titration, the average relative signal intensity for peaks in the15 N-1 H HSQC spectrum of Alba1 was plotted with standard error bars. Residues with an associated chemical shift change were omitted from the calculation to remove contributions from chemical and conformational exchange resulting from direct binding interactions. Plots of the average relative signal intensity as a function of mole fraction of oligonucleotide were generated using KALIEDOGRAPH (v 3.6 Synergy

Figure 4 .

 4 Figure 4. The F60A Alba1 mutant binds DNA more weakly than the wild-type protein.Alba-DNA Binding Curves Obtained by Gel Electrophoretic Retardation Analysis. Open and closed symbols are used to denote binding of Wild-type* (circles) and F60A (squares) Alba1 to either a 39 bp or 16 bp duplex respectively. The sequence of the 39mer ssDNA was 5'-CCTCGGTGCTAAGTTGATGCTGGTACTCGGAGTATCCCG, which was annealed with a complementary strand to produce the 39 bp duplex. Gel-shift analysis was carried out in triplicate and analysed as described previously[START_REF] Jelinska | Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging[END_REF]. Standard error bars are shown for each data point. The apparent dissociation constants measured for the wild-type and F60A mutant proteins to the 39 bp duplex were 56 ± 2 µM and 520 ± 13 µM, respectively. The equivalent values for the 16 bp duplex were 100 ± 4 µM and 710 ± 20 µM. A significant increase in apparent binding affinity (Kd) for the 39 bp duplex when compared to the 16 bp duplex is observed for the wild-type but not for the F60A mutant (Table1).

Figure 5 .

 5 Figure 5. SDSL confirms the DNA-dependent assembly of consecutive Alba dimers A. Overlaid cartoon and surface representations of three Alba1 dimers in the end-to-end arrangement found in various crystal structures are shown in grey. The side chains of R59 and F60 for each molecule, coloured blue and orange respectively, are shown as sticks. The distance in Angstroms between two R59 guanidinium N atoms belonging to the same dimer unit is highlighted in black. This image was generated using MacPymol (© 2008 Delano Scientific LLC). B. The interspin distance profile is obtained from transformation of the background corrected DEER spectrum, using Tikhonov regularisation of the four pulse DEER spectrum of spin labelled Alba R59C (no DNA). The minor peak is not significant. The normalized background corrected DEER spectrum is also shown (insert), with the best fit by Tikhonov regularisation shown in red. The experimental conditions and spectral analysis procedure are outlined in the Methods section. A four pulse DEER sequence was used at X-band frequencies (approx. 9.5 GHz) and a sample temperature of 50K. C. First derivative cw-ESR spectra of spin labelled Alba R59C are shown in the presence and absence of 16 bp DNA and 21 mer ssDNA. The black trace shows the cw-ESR spectrum in the absence of DNA, the green trace the spectrum in the presence of 16 bp DNA, and the red trace in the presence of 21 mer ssDNA. The inserts show an amplification (x10) of salient features of the spectrum. ESR conditions: temperature, 140 K; modulation frequency, 100 KHz; modulation amplitude, 0.4 mT, microwave frequency 9.5 GHz and microwave power 0.2 mW.

Table 1

 1 Comparison of the binding affinity of Alba1 for DNA and RNA

	Oligonucleotide Apparent Kd (nM)	m a
	16 bp (dsDNA) 200 4 b (710 20)	1.3 0.1 (0.9 0.1)
	39 bp (dsDNA) 56 2 b (520 13)	1.9 0.1 (1.4 0.1)
	39mer (ssDNA) 270 ± 13	2.0 ± 0.2
	30U (ssRNA)	900 38	1.6 0.1
	28A (ssRNA)	960 ± 30	2.1 ± 0.1
	16mer (ssRNA) 920 69	2.2 0.4
	Values are mean SEM for triplicate measurements
	Values obtained for the F60A mutant are shown in parentheses.

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

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

ACKNOWLEDGEMENTS

Thanks to Dr Catherine Botting and Dr Sally Shirran for mass spectrometry services, which are funded by the Wellcome Trust. Thanks to Prof. Jonathan P. Waltho for critical discussion of the manuscript and Dr. M. J. Cliff and and Dr Hassane El-Mkami for assistance with data analysis. We are grateful to Dr Graham Smith of the School of Physics, University of St. Andrews for the use of the Bruker ElexSys 580 Spectrometer and for advice and support.

FUNDING

This work was funded by the UK Biotechnology and Biological Sciences Research Council through BBSRC grant BB/F004583/1 and by the Engineering and Physical Sciences Research Council EPSRC by grant EP/F039034/1.


Richard S Marshall

Lorenzo Frigerio

Lynne M Roberts
email: lynne.roberts@warwick.ac.uk

Disulfide formation in plant storage vacuoles

Keywords: Disulfide, vacuole, endosperm, endoplasmic reticulum, ricin, tobacco ER (endoplasmic reticulum), PDI (protein disulfide isomerise), PSV (protein storage vacuole), RCA (Ricinus communis agglutinin

come

Introduction:

During endosperm development in the castor oil seed of Ricinus communis, lipids and proteins accumulate in oil bodies and protein storage vacuoles (PSV), respectively. The most abundant of the storage proteins, which provide an early source of amino acids to fuel post-germinative growth, are the 11S (legumin-type) crystalloid proteins and 2S albumins. The remaining PSV proteins are the 7S globulin (vicilin-like) lectins, ricin (Ricinus communis agglutinin II; RCA II) and its tetrameric relative Ricinus communis agglutinin I (RCA I, henceforth referred to as RCA). It is known that the glycosylated 7S lectins and non-glycosylated 2S albumins are co-translationally imported into the ER and transported as precursors via the Golgi to the PSV [START_REF] Lord | Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean[END_REF][START_REF] Jolliffe | Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors[END_REF]. One of the two 7S lectins, the cytotoxin ricin, is a disulfide-linked dimer comprising a galactose-binding B chain with 4 intrachain disulfides (apparent molecular mass of 34 kDa), and a ribosome inactivating A chain (apparent molecular mass of 32 kDa). Ricin is initially folded and transported as a ~67 kDa precursor known as proricin, in which the A and B chains are coupled by a 12 residue linker peptide containing a vacuolar targeting signal [START_REF] Frigerio | The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting[END_REF][START_REF] Jolliffe | The position of the proricin vacuolar targeting signal is functionally important[END_REF]. Upon delivery into the PSV the linker is processed, resulting in the release of mature ~66 kDa ricin. RCA is also made as a precursor, with an identical signal peptide and linker propeptide (Figure 1). The RCA A chain (~32 kDa) is 93% identical to ricin A chain, while the B chains of the two proteins are 84% identical, with RCA B chain (~36 kDa) possessing an additional glycan [START_REF] Lord | Ricinus communis agglutinin B chain contains a fucosylated oligosaccharide side chain not present on ricin B chain[END_REF]. Two of the 18 differences between the respective A chains of RCA and ricin involve the substitution of cysteine residues into RCA, one of which (Cys 156) is reported to form a disulfide link with an adjacent RCA molecule to generate a mature ~136 kDa lectin with the subunit arrangement B-A-A-B [START_REF] Cawley | Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin[END_REF][START_REF] Sweeney | Preliminary crystallographic characterization of ricin agglutinin[END_REF][START_REF] Gabdoulkhakov | Structure function investigation of the complex of the agglutinin from Ricinus communis with galactoaza[END_REF]. It has been assumed that all 11 disulfide bonds in mature RCA are formed in the ER to link the two proRCA precursors together, as depicted in Figure 1. During seed maturation, the two lectins accumulate to ~10% particulate protein.

Here, using a biochemical approach, we have examined the assembly of RCA in tobacco cells and in developing castor oil seed endosperm tissue. Surprisingly, we observed that neither covalent nor non-covalent association of two proRCA molecules occurred within the ER lumen. From analysis of lectin biosynthesis in maturing castor oil seeds, it was shown that the final disulfide bond between the RCA A subunits occurred after deposition and processing in the vacuole, such that a mixed population of ~136 kDa and ~68 kDa RCA accumulated during the later stages of endosperm maturation, before the onset of seed dessication. These observations show that disulfide bond formation can occur at sites other than the ER in the endomembrane system of plant cells.

Experimental:

Materials

Polyclonal rabbit anti-ricin antiserum was supplied by Vector Laboratories, while an RCA-specific peptide antibody was prepared by CovalAb, both of Peterborough, Cambridgeshire, UK. Complete™ protease inhibitor cocktail tablets were provided by Roche Applied Sciences, Mannheim, Germany, and the QuickChange™ in vitro mutagenesis system by Stratagene, La Jolla, California, USA. [ 35 S]-methionine), Amplify™ and Percoll™ were from GE Healthcare, Amersham, Buckinghamshire, UK, while all other reagents were from Sigma Aldrich, Poole, Dorset, UK.

4

Recombinant DNA All DNA constructs were generated in the CaMV 35S-promoter-driven expression vector pDHA [START_REF] Tabe | A biotechnological approach to improving the nutritive value of alfalfa[END_REF]. Expression constructs encoding preproricin (ppRT) and preproricin with a mutated vacuolar sorting signal (ppRT I271G ) have been described previously [START_REF] Frigerio | The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting[END_REF][START_REF] Frigerio | Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts[END_REF]. A pDHA construct containing the Ricinus communis agglutinin precursor cDNA, preproRCA (ppRCA), was utilised [START_REF] Roberts | The primary sequence of Ricinus communis agglutinin: comparison with ricin[END_REF]. The equivalent mutation within the vacuolar sorting signal of RCA (ppRCA I270G ) was generated using the QuickChange™ in vitro mutagenesis system, using the mutagenic primer 5'-CGTCACAGTTTTCTTTGCTTGGAAGGCCAGTGGTGCC-3' (and its reverse complement, not shown). The site of codon mutation is underlined.

Transient transfection and in vivo radiolabelling of tobacco leaf protoplasts Protoplasts were prepared from axenic leaves (4 to7 cm long) of Nicotiana tabacum cv. Petit Havana SR1 [START_REF] Maliga | Streptomycin-resistant plants from callus culture of haploid tobacco[END_REF], and subjected to polyethylene glycol-mediated transfection with plasmids as previously described [13]. Cells were radiolabelled with [ 35 S]-Promix and chased for the times indicated in the figures, as previously described [START_REF] Frigerio | Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts[END_REF]. In some experiments, before radioactive labelling, protoplasts were incubated for 1 hour at 25°C in K3 medium (3.78 g/l Gamborgs B5 basal medium with minimal organics, 750 mg/l CaCl 2 .2H 2 O, 250 mg/l NH 4 NO 3 , 136.2 g/l sucrose, 250 mg/l xylose, 1 mg/l 6benzylaminopurine, 1 mg/l α-naphthalenacetic acid) supplemented with 36 µM brefeldin A (7mM stock in 100% ethanol). At the desired time points, 3 volumes of cold W5 medium (9 g/l NaCl, 0.37 g/l KCl, 18.37 g/l CaCl 2 .2H 2 O, 0.9 g/l glucose) were added and protoplasts were pelleted by centrifugation at 60 × g for 10 minutes at 4°C. Separated cell and media samples were frozen on dry ice and stored at -80°C, unless further manipulations were to be performed.

Immunoprecipitation from tobacco protoplast protein extracts

Frozen samples were homogenised by adding 2 volumes of cold protoplast homogenisation buffer (150 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1.5% (w/v) Triton X-100) supplemented immediately before use with Complete™ protease inhibitor cocktail. Homogenates were used for immunoprecipitation with polyclonal rabbit anti-ricin antiserum, which recognises both proricin and proRCA. Protein A-Sepharose beads were washed three times with NET-gel buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% (w/v) Nonidet P-40, 0.25% (w/v) gelatin, 0.02% (w/v) NaN 3 ), and immunoselected polypeptides were analysed by 15% reducing or non-reducing SDS-PAGE. Gels were fixed, treated with Amplify™, and radioactive polypeptides revealed by fluorography.

Transient transfection, in vivo radiolabelling and immunoprecipitation from Arabidopsis suspension culture protoplasts A suspension culture of Arabidopsis thaliana cells, ecotype Columbia T87 [START_REF] Axelos | A protocol for transient expression in Arabidopsis thaliana protoplasts isolated from cell suspension culture[END_REF], was obtained from the Max-Planck Institut für Molekulare Pflanzenphysiologie, Potsdam, Germany. This was maintained axenically at 25°C in JPL Arabidopsis cell culture medium [START_REF] Jouanneau | Growth and synthesis of proteins in cell suspensions of a kinetin dependent tobacco[END_REF] 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. mediated transfection with plasmids was also performed as previously described [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]. Cells were radiolabelled, homogenised and proteins immunoprecipitated with anti-ricin antiserum as described above. Immunoselected polypeptides were analysed by 15% non-reducing SDS-PAGE and visualised by fluorography.

Velocity gradient fractionation 600 µl homogenised cell samples containing approximately 2 million protoplasts were loaded on top of 11 ml continuous 5% to 25% (w/w) linear sucrose gradients made in 20 mM Tris-HCl pH 8.0. Gradients were formed in Beckman 12 ml ultracentrifuge tubes using a Searle Densiflow IIC filler device connected to the pumps of an FPLC system (GE Healthcare, Amersham, Buckinghamshire, UK). These were centrifuged at 100,000 × g for 20 hours at 20°C in a Beckman SW40 rotor (Beckman Coulter, High Wycombe, Buckinghamshire, UK), before unloading manually from the top in fractions of 600 µl. Radiolabelled proteins were immunoselected from gradient fractions and resolved by non-reducing SDS-PAGE and fluorography, as above. Size markers resolved from a parallel gradient were bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), catalase (232 kDa), ferritin (440 kDa) and thyroglobulin (660 kDa). This were visualised following reducing SDS-PAGE by staining with Coomassie Brilliant Blue.

In vivo radiolabelling of Ricinus communis endosperm tissue Ricinus communis plants were grown from dry seeds in an equal mix of multi-purpose compost and vermiculite under greenhouse conditions at 15°C with a 16 hour light / 8 hour dark cycle. Prior to planting, seeds were imbibed in running water overnight. The development of Ricinus communis seeds is divided into seven stages (A to G) based on size, testa formation and state of hydration [START_REF] Roberts | Protein biosynthetic capacity in the endosperm tissue of ripening castor bean seeds[END_REF]. Endosperm tissue was excised from the ripening seeds during testa formation at various developmental stages, as indicated in the figures (typically 7 to 9 weeks after flowering, when the lectins and storage proteins are being rapidly synthesised [START_REF] Lord | Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean[END_REF]). Intact endosperm halves were treated with 25 µCi of [ 35 S]-Promix, which was added directly to the abaxial surface. After a pulse of 1 hour at room temperature, the [ 35 S]-Promix was washed off with sterile water, and 10 µl of 2.5 mM unlabelled cysteine and methionine was added to each half. At each time point, six endosperm halves were frozen and ground to a fine powder in liquid N 2 . The frozen powder was mixed with 1.0 ml of PC buffer (1% (w/v) Nonidet P-40, 0.2 M galactose), supplemented immediately before use with Complete™ protease inhibitor cocktail. Samples were then stored at -80°C.

Immunoprecipitation from Ricinus communis endosperm tissue Samples were defrosted, and membranes were removed from the suspension by centrifugation at 100,000 × g for 30 minutes. Duplicate 25 µl aliquots of the supernatant were added to 1.0 ml of cold 10% (w/v) trichloroacetic acid. The precipitated protein was collected by filtration onto a Whatman GF/A filter disc, washed with 10% trichloroacetic acid, and its radioactivity content determined by scintillation counting after immersing the disc in 5.0 ml of scintillant. A volume of supernatant containing 10 6 counts per minute was taken at each stage for immunoprecipitation. The crude endosperm homogenates were dispersed in an equal volume of IP buffer (1% (w/v) Nonidet P-40, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.4 M galactose) supplemented immediately before use with Complete™ protease inhibitor cocktail. Immunoprecipitation was carried out with polyclonal rabbit anti-ricin antiserum as above, although Sepharose beads were this time washed three times with 0.2% 6 Nonidet P-40, twice with 0.2% Nonidet P-40, 0.5 M NaCl, and once with 10 mM Tris-HCl pH 7.5. Immunoselected polypeptides were analysed by 15% reducing or nonreducing SDS-PAGE and visualised by fluorography.

Isopycnic organelle gradient fractionation

Ricinus communis endosperm tissue isolated from maturing castor oil seeds at developmental stage F/G was homogenised in a Petri dish on ice by continuous chopping for 15 minutes using a hand-held razor blade, after addition of ice-cold grinding buffer (150 mM Tris-HCl pH 7.5, 10 mM KCl, 1 mM MgCl 2 , 1 mM EDTA, 100 mM lactose, 12% (w/w) sucrose) supplemented immediately before use with Complete™ protease inhibitor cocktail, using 6 ml of buffer per gram of endosperm tissue .The homogenate was centrifuged at 1,000 × g for 10 minutes at 4°C. 600 µl of supernatant was loaded on top of a 9 ml 30% to 60% (w/w) linear sucrose gradient, formed as above, overlaid with 2 ml of 20% (w/w) sucrose. These were centrifuged at 150,000 × g for 2 hours at 4°C in a Beckman SW40 rotor (Beckman Coulter, High Wycombe, Buckinghamshire, UK), before unloading manually from the top in fractions of 600 µl. Non-reducing Western blot analysis of 60 µl aliquots of these fractions was performed as previously described, using either an anti-RCA peptide antibody (described below) or anti-BiP antiserum [13].

Purification of ricin and Ricinus communis agglutinin Ricin and RCA were purified as previously described [START_REF] Nicolson | The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces[END_REF][START_REF] Nicolson | Characterization of two plant lectins from Ricinus communis and their quantitative interaction with a murine lymphoma[END_REF], with slight modifications. 10 g of endosperm tissue at different developmental stages was frozen in liquid N 2 and ground to a fine powder. This was further ground for 5 minutes in 100 ml PBS containing 0.2 M NaCl. After 3 hours at 4°C the extract was filtered and centrifuged twice at 10,000 × g for 30 minutes. The resulting supernatant was adjusted to 60% saturation with ammonium sulphate. This was left stirring overnight at 4°C, before centrifugation as above. The resulting pellet was dissolved in ~10ml PBS containing 0.2 M NaCl, and the ammonium sulphate removed by dialysis. The freshly prepared soluble protein was then immediately loaded onto a 50 ml column of propionic acid-treated Sepharose 6LB, pre-equilibrated with PBS, and unbound proteins were removed by washing with 300 ml of PBS, with 10 ml fractions being collected. Bound ricin was then eluted with 300 ml PBS containing 5 mM N-acetylgalactosamine, before PBS containing 100 mM galactose was used to elute bound RCA. Fractions were assayed for protein content by spectrophotometric measurement at 280 nm, and aliquots of suitable fractions were analysed by non-reducing SDS-PAGE and Coomassie Brilliant Blue staining or silver staining, and Western blotting with an anti-RCA peptide antibody. The peptide antibody was raised in a rabbit against a surface-exposed 11 amino acid sequence of Ricinus communis agglutinin B chain (single letter code ISKSSPRQQVV), of which 8 amino acids are different to the equivalent position in ricin B chain.

Results:

Mature Ricinus communis agglutinin 1 (RCA) is reported to be a B-A-A-B tetramer containing an interchain disulfide bond between each subunit (Figure 1 and [START_REF] Sweeney | Preliminary crystallographic characterization of ricin agglutinin[END_REF]). The primary sequence of RCA shows it to contain a signal peptide for targeting to the endoplasmic reticulum, followed by A chain and B chain sequences that are separated by a 12 amino acid linker (Figure 1 and [START_REF] Roberts | The primary sequence of Ricinus communis agglutinin: comparison with ricin[END_REF]). An identical linker peptide was originally observed in the primary translation product of the ricin precursor, and this was shown A c c e p t e d M a n u s c r i p t 7 to contain a vacuolar targeting signal [START_REF] Frigerio | The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting[END_REF][START_REF] Jolliffe | The position of the proricin vacuolar targeting signal is functionally important[END_REF]. Previous studies with ricin have also revealed that events during its synthesis and trafficking can be mimicked when it is ectopically expressed in tobacco leaf protoplasts [START_REF] Frigerio | Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts[END_REF]. Using this system to investigate the assembly of RCA, we observed from pulse-labelling that preproRCA is made as a ~69 kDa precursor which disappears as it is converted to mature A and B subunits during a chase with cold amino acids (Figure 2A). Such biochemical processing of the related proricin molecule has been recognised as a hallmark of vacuolar deposition, since this is the location of the relevant proteases (Figure 2A and [START_REF] Lord | Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean[END_REF][START_REF] Butterworth | Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor[END_REF]22]). However, when identical samples were examined on non-reducing gels, it was evident that disulfide-bonded proRCA dimers of an expected molecular mass of ~136 kDa did not form (Figure 2B, asterisk). Very similar results were obtained using protoplasts from Arabidopsis cell suspension cultures (Figure 2C). The multiple bands observed when these proteins are resolved on non-reducing gels are typical of proteins constrained by a variable number of disulfide bonds. The slight decrease in overall sizes of the precursor bands during the early chase points most likely reflects glycan modifications (Figure 2B, 2C and [23, 24]) as the proteins traffic through the Golgi in a brefeldin A-sensitive manner (Figure 3A), as has been previously reported for proricin [START_REF] Frigerio | Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts[END_REF]. The marked size reduction observed after 3 hours of chase is indicative of proteolytic removal of the linker upon arrival at the vacuole [START_REF] Frigerio | Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts[END_REF]. If the RCA linker is mutated within its putative vacuolar targeting sequence (I270G) then, by default, it becomes secreted with time and avoids vacuolar processing (Figure 3B).

Since there is no evidence for the formation of covalent disulfide-linked dimers in tobacco protoplasts, we checked whether proRCA assembled non-covalently into a ~136 kDa multimer. This was achieved by separating the newly made proteins on nondenaturing velocity gradients following a 1 hour pulse and 0 or 3 hour chase. Fractions were then immunoprecipitated and samples resolved by non-reducing SDS-PAGE (Figure 4). The position of RCA was compared to that of protein markers run on a parallel gradient (Figure 4, top panel). It is evident that proRCA and processed RCA colocalise around the peak fractions of bovine serum albumin (67 kDa; labelled I) and not around lactate dehydrogenase (140 kDa; labelled II).

Since we could observe no formation of tetrameric RCA in tobacco or Arabidopsis cells, we next analysed its endogenous synthesis during the maturation of Ricinus communis seed endosperm ( [START_REF] Roberts | Protein biosynthetic capacity in the endosperm tissue of ripening castor bean seeds[END_REF]; stages depicted in Figure 5A). Since ricin and RCA are co-synthesised in Ricinus communis seeds, and since they are such closely related polypeptides, polyclonal antibodies raised against either protein interact with both antigens. We therefore prepared an RCA-specific peptide (Figure 5B) in which 8 out of 11 residues of RCA B chain differed from the equivalent peptide in RTB. Antibodies raised to this peptide were able to differentiate between the two non-reduced lectins when denatured, as in a Western blot (Figures 5C and5D), but were found to not to interact with either protein upon immunoprecipitation (data not shown). We then purified and separated the two Ricinus lectins from young and mature seed tissue. This was achieved by eluting the lectins from propionic acid-washed Sepharose 6LB, using N-acetylgalactosamine to elute ricin followed by galactose to elute RCA. This is possible due to the different sugar binding specificities of the B chains of ricin and RCA [START_REF] Nicolson | The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces[END_REF][START_REF] Debray | Specificity of twelve lectins towards oligosaccharides and glycopeptides related to Nglycosylproteins[END_REF][START_REF] Green | Lectin affinity highperformance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II[END_REF]. An elution profile is shown for stage F/G tissue, where two clear peaks are evident (Figure 5C). The first, eluting with N-acetylgalactosamine, was shown by Coomassie blue staining of a non-reducing gel, and by the absence of any interaction with the RCA-specific peptide antibody, to be ricin (Figure 5C, left-hand 8 gels). The second peak, eluting from the column with galactose, was shown to contain two forms of RCA, a ~68 kDa form and a ~136 kDa form (Figure 5C, right-hand gels). In younger endosperm tissue (stage B/C), overall lectin yields were much lower, and so silver staining was used instead of Coomassie to visualise the purified protein, due to its additional sensitivity. It is clear from both silver stained gels and from Western blots that very little tetrameric RCA is evident in this younger tissue (Figure 5D, right-hand gels). To further explore the formation of the fully assembled RCA, tissue extracts from a wider range of tissue stages were analysed by Western blotting with the RCA-specific antibody. It is clear that predominantly dimeric RCA (~68 kDa) accumulates early in seed development, while the 136 kDa tetrameric form is observed only at later stages of development (Figure 5E, top panel). However, some RCA remains as A-B dimers throughout. That the tetrameric form is truly a disulfide-bonded species is shown in the reducing gel of these samples (Figure 5E, lower panel).

To examine exactly where in the cell tetramer assembly occurs, we fractionated late stage developing endosperm, separating the organelles by sucrose density gradient ultracentrifugation (as described in, for example, [START_REF] Lord | Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm[END_REF][START_REF] Lord | Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm[END_REF]). In such gradients, ERderived membranes are recovered just below the interface between 20% and 30% sucrose, as revealed here by the location of the ER resident chaperone BiP (Figure 6A, lower panel), while disrupted PSVs release their contents to be recovered at the top of the gradient [START_REF] Butterworth | Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor[END_REF]. In the RCA blot, the RCA-specific antibody that does not cross react with ricin was used. It is clear that the material in the first few fractions contains both ~68 and ~136 kDa forms of RCA. However, the ~136 kDa form of RCA is not detectable in the fractions commensurate with ER (ascertained from the localisation of BiP and from the known sucrose density of endosperm-derived ER microsomes (Figure 6A and [START_REF] Lord | Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm[END_REF])). That a disulfide-bonded ~136 kDa RCA did not form within the ER was confirmed by pulse-chase and immunoprecipitation using late stage developing endosperm tissue (stage F/G). Only during the chase can it be seen that larger B-A-A-B forms (~136 kDa), which are the hallmark of fully assembled RCA, become evident (Figure 6B, lanes 2 and 3). These forms disappear when resolved on a reducing gel (Figure 6B, lanes 5 and 6), confirming that upon delivery into PSV, the RCA precursors are processed and assembled in a disulfide-bonded manner. The precise fraction that is converted from ~68 kDa to the larger disulfide-bonded forms is not known, since the antibody used for immunoprecipitation here is, necessarily, the polyclonal Ricinus lectin antibody that also detects ~66 kDa ricin.

Discussion:

We reasoned that the assembly of RCA1 would require the formation of disulfide bonds (11 in total) within and between two RCA precursors during their oxidative folding in the ER lumen, in a manner analogous to the assembly of other multimeric, disulfidelinked proteins. However on analysing newly synthesised RCA expressed ectopically in tobacco and Arabidopsis protoplasts or endogenously in developing castor oil seed endosperm, we found no evidence for disulfide-associated precursors in the ER. Indeed, in immature endosperm there was very little, if any, disulfide-linked association of the RCA heterodimers. Here we show that not only is the final stage in assembly of tetrameric RCA (the disulfide-linked association of two RCA A chains) observed exclusively in the later stages of seed development, but that it also occurs outside the environment of the endoplasmic reticulum, most likely within the PSV. Disulfide bonds between the thiol groups of cysteine residues help stabilise the folded structure of proteins, and their formation is usually catalysed by oxidised members of the protein disulfide isomerase (PDI) family in the oxidising environment of the ER. PDIs can act as chaperones, but are primarily thioredoxin-like oxidoreductases that form and reshuffle disulfide bonds. Continued PDI action relies on an electron transfer cascade to recycle it from its reduced to its oxidised form. This is accomplished by Ero1 (endoplasmic reticulum oxidoreductin 1), which in turn transfers electrons to molecular oxygen with the generation of H 2 O 2 [START_REF] Frand | Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum[END_REF][START_REF] Gross | Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p[END_REF]. The latter is potentially very damaging, but through feedback regulation to reduce the oxidase activity of Ero1 [START_REF] Sevier | Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1[END_REF][START_REF] Appenzeller-Herzog | A novel disulfide switch mechanism in Ero1α balances ER oxidation in human cells[END_REF][START_REF] Baker | Low reduction potential of Ero1α regulatory disulfides ensures tight control of substrate oxidation[END_REF], and possibly through detoxification by an ER resident peroxidoredoxin [34], oxidative damage caused by H 2 O 2 can be prevented. In plant systems, Ero1 homologues have been identified in Arabidopsis [START_REF] Dixon | Cloning and initial characterization of the Arabidopsis thaliana endoplasmic reticulum oxidoreductins[END_REF], while a role for Ero1 in disulfide formation in the endosperm of rice has recently been demonstrated [START_REF] Onda | ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm[END_REF].

PDIs have been localised in subcellular compartments other than the ER [START_REF] Turano | Proteins of the PDI family: unpredicted non-ER locations and functions[END_REF], including chloroplasts [START_REF] Kim | Protein disulfide isomerase as a regulator of chloroplast translational activation[END_REF][START_REF] Trebitsh | The protein disulfide isomerase-like RB60 is partitioned between stroma and thylakoids in Chlamydomonas reinhardtii chloroplasts[END_REF][START_REF] Shimada | Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase[END_REF]. Their functions in the more reducing conditions that prevail outside the ER are often unclear, but they are likely to involve the reduction of disulfides rather than their formation [41]. For example in animal cells, the reducing activity of cell surface PDI has been shown to play a role in nitric oxide signalling pathways [START_REF] Zai | Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide[END_REF][START_REF] Ramachandran | Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cellsurface protein disulfide isomerase[END_REF], reduction and release of surface receptors [START_REF] Couët | Cell surface protein disulfide-isomerase is involved in the shedding of human thyrotropin receptor ectodomain[END_REF] and cell adhesion [START_REF] Pariser | The cell adhesion molecule retina cognin is a cell surface protein disulfide isomerase that uses disulfide exchange activity to modulate cell adhesion[END_REF]. Various hypotheses have been put forward to assign a function to PDI members reported in the nucleus and cytosol, but often these require further confirmation [START_REF] Turano | Proteins of the PDI family: unpredicted non-ER locations and functions[END_REF]. An alternative oxidising compartment where disulfide bond formation is known to occur concomitant with protein folding is within the intermembrane space of mitochondria, where a disulfide relay system has recently been identified and characterised (reviewed in [46]).

In Arabidopsis, 22 PDI-like proteins have been identified [START_REF] Houston | Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins[END_REF], although only 12 of these possess a classical PDI structure [START_REF] Lu | Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana[END_REF]. However, while it is clear that ER residents such as BiP and PDI can be transported to lytic vacuoles for degradation [49,[START_REF] Tamura | 14 Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation[END_REF] and that oxidised proteins are sent to the vacuole for degradation by autophagy [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF], there is no evidence for PDI activity in vacuoles. Recently, the Arabidopsis PDI5 isoform was found to localise to both the ER and vacuoles in developing seed tissues. Through disulfide exchange, this protein interacts with and inhibits cysteine proteases on their journey to vacuoles [START_REF] Ondzighi | Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds[END_REF]. Such chaperoning precedes the initiation of programmed cell death. There is therefore no available evidence for the presence of a disulfide relay, or supporting enzymatic activity of PDI, in plant vacuoles, leaving it unclear as to how the final disulfide that connects each RCA heterodimer, to complete the assembly of tetrameric RCA in castor oil seed PSV, is formed. Whether this event involves simply air oxidation, and whether it is promoted by protein accumulation, remains to be established. It should be noted that the final disulfide in this lectin occurs after processing of the RCA precursor by the vacuolar processing enzyme (VPE) [22,[START_REF] Hara-Nishimura | Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni[END_REF]. This type of proteolysis is common in the maturation of seed storage proteins [54][START_REF] Hiraiwa | An aspartic endopeptidase is involved in the breakdown of propeptides of storage proteins in protein storage vacuoles of plants[END_REF][START_REF] Shimada | Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana[END_REF][START_REF] Wang | The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice[END_REF]. Proteolytic removal of the linker (Figure 1) cannot occur outside the PSV in castor oil seeds, since newly synthesised VPE is transported in latent form and is activated only upon vacuolar delivery [START_REF] Hara-Nishimura | Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni[END_REF]. A preliminary crystallographic structure of RCA reveals a disulfide bridge between Cys 156 of two adjacent A chains [START_REF] Sweeney | Preliminary crystallographic characterization of ricin agglutinin[END_REF]. We speculate that processing of the proRCA linker may subtly alter the conformation of the resulting A-B heterodimer such that neighbouring dimers, accumulating and concentrating over time, 10 have their respective Cys 156 residues optimally juxtaposed to favour spontaneous disulfide formation.

If RCA1 has two quaternary structural forms (dimeric and tetrameric), why does the literature refer to this protein as a tetrameric lectin? Commercially purified RCA is supplied as a homogenous disulfide bonded B-A-A-B protein. However, as we demonstrate here, RCA in the endosperm of mature castor oil seeds is actually present as a mixture of ~68 kDa A-B and ~136 kDa B-A-A-B forms. Indeed, in early developing endosperm, only the ~68 kDa A-B dimer is present. One likely explanation for the description of RCA as a tetrameric lectin is that in dry seeds (from which the lectin is typically purified), the larger ~136 kDa species predominates. When purifying RCA, castor oil seed extracts are subjected to affinity chromatography and gel filtration. The less abundant smaller species, being the size of mature ricin and being indistinguishable from it with polyclonal antisera, could readily be mistaken for ricin contamination and therefore discarded.

In summary, we show that the ER of plant cells is not the exclusive location for disulfide bond formation. In the assembly of tetrameric RCA1, the final disulfide between two RCA A subunits occurs within the PSV. While the mechanism remains to be elucidated, it is clear that conditions within the seed PSV are conducive for such a post-translational modification. Top panel: The marker proteins bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), catalase (232 kDa), ferritin (440 kDa) and thyroglobulin (660 kDa) were subjected to velocity gradient centrifugation on a 5 to 25% (w/w) linear sucrose gradient. Fractions were resolved on reducing SDS-PAGE and revealed by Coomassie staining. The range of each protein across the gradient is indicated by a horizontal line and the numbers I, II, III, IV and V, respectively. Centre and lower panels: Tobacco protoplasts transfected with plasmid encoding ER-targeted preproRCA were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for 0 or 3 hours, as indicated. Total cell homogenates were subjected to velocity gradient centrifugation on a 5 to 25% (w/w) linear sucrose gradient. Fractions were immunoprecipitated with anti-ricin antiserum, analysed by non-reducing SDS-PAGE, and visualised by fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons. A: Endosperm halves from castor oil seeds at developmental stage F/G were homogenised and subjected to isopycnic centrifugation on a 9 ml linear 30% to 60% sucrose gradient overlaid with 2 ml of 20% sucrose. Fractions were resolved on non-reducing SDS-PAGE and immunoblotted with either anti-RCA peptide antibodies or anti-BiP antiserum. B: Endosperm halves from castor oil seeds at developmental stage F/G were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for the indicated periods of time, before immunoprecipitation from cell homogenates with anti-ricin antiserum and analysis by reducing or non-reducing SDS-PAGE and fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons.

S S S S S S S S S S

A chain linker SP B chain

♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆

SH

RCA1 primary translation product (~69kDa without SP)

S S S S S S S S S S S S S S S S S S

A chain linker

♆ ♆ ♆ ♆ B chain ♆ ♆ ♆ ♆ S S ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ S A chain linker B chain ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆

S

putative disulphide bonded proRCA1 precursors (ER, ~138kDa)

S S S S S S S S S S S S S S S S S S S S

A chain B chain Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ A chain B chain ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ ♆ mature RCA1 (

  Biochemical Journal Immediate Publication. Published on 24 Feb 2010 as manuscript BJ20091878 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091878

Figure legends : Figure 1 .

 :1 Figure legends:

Figure 2 .

 2 Figure 2. ProRCA reaches the vacuole as an A-B heterodimer in tobacco protoplasts, but does not form disulfide-bonded tetramers. Tobacco mesophyll (A and B) or Arabidopsis suspension culture (C) protoplasts transfected with pDHA (vector) alone or plasmids encoding ER-targeted preproricin or preproRCA were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for the indicated times, before immunoprecipitation from cell homogenates with anti-ricin antiserum, and analysis by either reducing (A) or non-reducing (B and C) SDS-PAGE and visualisation by fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons.

Figure 3 .

 3 Figure 3. ProRCA is transported to the vacuole in a sequence-specific manner via the Golgi complex. A: Tobacco protoplasts transfected with the indicated constructs were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for the indicated times, before immunoprecipitation from cell homogenates with anti-ricin antiserum, and analysis by reducing SDS-PAGE and fluorography. Where indicated, protoplasts were pre-incubated for 1 hour with 36µM BFA before radiolabelling. B: Tobacco protoplasts were transfected and treated as in A. Radiolabelled polypeptides were immunoprecipitated from separated cell (top panel) and medium (lower panel) homogenates and analysed by non-reducing SDS-PAGE and fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons.

Figure 4 .

 4 Figure 4. ProRCA does not form non-covalent tertramers in tobacco protoplasts.Top panel: The marker proteins bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), catalase (232 kDa), ferritin (440 kDa) and thyroglobulin (660 kDa) were subjected to velocity gradient centrifugation on a 5 to 25% (w/w) linear sucrose gradient. Fractions were resolved on reducing SDS-PAGE and revealed by Coomassie staining. The range of each protein across the gradient is indicated by a horizontal line and the numbers I, II, III, IV and V, respectively. Centre and lower panels: Tobacco protoplasts transfected with plasmid encoding ER-targeted preproRCA were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for 0 or 3 hours, as indicated. Total cell homogenates were subjected to velocity gradient centrifugation on a 5 to 25% (w/w) linear sucrose gradient. Fractions were immunoprecipitated with anti-ricin antiserum, analysed by non-reducing SDS-PAGE, and visualised by fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons.

Figure 5 .

 5 Figure 5. RCA can form teramers in maturing Ricinus communis seed endosperm.A: The different developmental stages of a maturing castor oil seed[START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]. B: The threedimensional crystal structure of ricin holotoxin, with the region of maximum sequence divergence (an exposed surface loop incorporating amino acids 66 to 76 of the B chain) circled (left). The three letter amino acid codes of these residues (and two flanking residues either side) are shown (top right), along with the equivalent residues in RCA

Figure 6 .

 6 Figure6. Tetrameric RCA is found only in the vacuoles of mature castor bean endosperm. A: Endosperm halves from castor oil seeds at developmental stage F/G were homogenised and subjected to isopycnic centrifugation on a 9 ml linear 30% to 60% sucrose gradient overlaid with 2 ml of 20% sucrose. Fractions were resolved on non-reducing SDS-PAGE and immunoblotted with either anti-RCA peptide antibodies or anti-BiP antiserum. B: Endosperm halves from castor oil seeds at developmental stage F/G were radiolabelled with [ 35 S]-amino acids for 1 hour and chased in the presence of unlabelled amino acids for the indicated periods of time, before immunoprecipitation from cell homogenates with anti-ricin antiserum and analysis by reducing or non-reducing SDS-PAGE and fluorography. In all cases, numbers on the left indicate molecular mass markers in kilodaltons.

Figure 1 .

 1 Figure 1. Marshall et al.

S

  vacuoles, ~136 kDa) S Biochemical Journal Immediate Publication. Published on 24 Feb 2010 as manuscript BJ20091878 THIS IS NOT THE VERSION OF RECORD -see doi:10.

Figure 2 .

 2 Figure 2. Marshall et al.

Figure 3 .

 3 Figure 3. Marshall et al.

30 -

 30 Biochemical Journal Immediate Publication. Published on 24 Feb 2010 as manuscript BJ20091878 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091878 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 4 .Figure 5 .

 45 Figure 4. Marshall et al.

Figure 6 .

 6 Figure 6. Marshall et al.

  35

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

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

Acknowledgements:

We are grateful to Marie Maitrejean for her assistance in the preparation of Arabidopsis protoplasts and to Aldo Ceriotti (Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milano), Chris Snowden and Robert Freedman (University of Warwick) for their helpful suggestions and critical reading of the manuscript.

Funding:

RSM was funded through a UK Biotechnology and Biological Sciences Research Council studentship.