Véronique Santoni
email: santoniv@ensam.inra.fr

Lionel Verdoucq

Nicolas Sommerer

Joelle Vinh

Delphine Pflieger

Christophe Maurel

Joëlle Vinh ¶

Methylation of aquaporins in plant plasma membrane

Keywords: Aquaporin, methylation, N-terminal maturation, plasma membrane protein, post-translational modification, root dimethyl Lys3 ESI: electrospray ionization FW: fresh weight MALDI-TOF: matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry PIP: plasma membrane intrinsic protein PM: plasma membrane

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Aquaporins define a large family of ubiquitous integral membrane proteins [START_REF] Zardoya | A phylogenetic framework for the aquaporin family in eukaryotes[END_REF] which mediate the transport across membranes of water, small neutral solutes, and occasionally ions. In plants, aquaporins critically contribute to plant water homeostasis during development and in response to challenging environmental conditions [START_REF] Luu | Aquaporins in a challenging environment: molecular gears for adjusting plant water status[END_REF]. Most of aquaporins consist of small (25-34 kDa) hydrophobic proteins assembling in tetramers. All aquaporin monomers share a typical organization with six transmembrane -helices, the N-and C-terminus being located in the cytoplasm. The extra-membrane parts of the proteins, which include membrane connecting loops and the N-and C-termini, are the most divergent regions among aquaporin homologues. A large body of data shows that these regions play a critical role in aquaporin expression and function and that these regulations are mediated in part by various posttranslational modifications. For instance, the phosphorylation of Ser residues at multiple sites was shown to modulate the gating of plant aquaporins [START_REF] Tornroth-Horsefield | Structural mechanism of plant aquaporin gating[END_REF][START_REF] Johansson | Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation[END_REF][START_REF] Guenther | Phosphorylation of soybean nodulin 26 on serine 262 enhances water permeability and is regulated developmentally and by osmotic signals[END_REF] and the subcellular trafficking of animal aquaporins [START_REF] Kamsteeg | The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and non phosphorylated monomers[END_REF][START_REF] Van Balkom | The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel[END_REF]. N-linked glycosylation of mammalian aquaporin-2 (AQP2) in extracellular loop C was recently shown to regulate cell surface expression of the protein [START_REF] Hendricks | Glycosylation is important for cell surface expression of the water channel aquaporin-2 but is not essential for tetramerization in the endoplasmic reticulum[END_REF].

Intracellular redistribution of the plant vacuolar membrane aquaporin McTIP1;1 in response to osmotic stress also requires glycosylation [START_REF] Vera-Estrella | Novel regulation of aquaporins during osmotic stress[END_REF]. Mammalian AQP1 undergoes ubiquitination in fibroblasts, a process which controls the differential stability of the protein between normal and osmotic stress conditions [START_REF] Leitch | Altered ubiquitination and stability of aquaporin-1 in hypertonic stress[END_REF]. Finally, controlled proteolysis of the N-and C-terminal tails of AQP0 occurs in lens but the functional significance of this processing remains as yet unknown [START_REF] Ball | Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens[END_REF][12][START_REF] Schey | Modifications to rat lens major intrinsic protein in selenite-induced cataract[END_REF].

Proteins are made of 20 main amino acids but over 150 post-translationally modified amino acid forms have thus far been reported. Thus, a tremendous repertoire of modified proteins remain to be uncovered in both prokaryotic and eukaryotic organisms. For instance, protein methylation can occur as either N-methylation of residues such as Lys, Arg, His, Ala, Pro, Gln, Phe, Asn and Met or carboxymethylation i.e. O-methylesterification of Glu and Asp residues. S-adenosyl methionine is used as a universal methyl donor and the extent of methylation varies from partial to complete. Whereas methylation (at Glu residues) of bacterial chemotaxis receptors has been characterized as typically reversible [START_REF] Kim | Determinants of chemotactic signal amplification in Escherichia coli[END_REF], Nmethylation of eukaryotic proteins has long been considered as irreversible. Yet, histone lysine demethylases have recently been identified [START_REF] Tsukada | Histone demethylation by a family of JmjC domaincontaining proteins[END_REF][START_REF] Shi | Histone demethylation mediated by the nuclear amine oxydase homolog LSD1[END_REF]. Protein methylation affects various cell processes, including protein-protein and protein-nucleic acid interactions, chromatin remodeling, and cellular signalling [START_REF] Mcbride | State of the arg: protein methylation at arginine comes of age[END_REF]18]. These processes mainly involve the methylation of soluble proteins. There have been only a few reports on methylation of eukaryotic transmembrane proteins and its role has remained elusive [19,[START_REF] Kluck | Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation[END_REF]. Recently, co-expression studies in Xenopus oocytes and mouse cortical collecting duct cells have shown that a novel protein O-methyltransferase can enhance the activity of an epithelial sodium channel but the methylation site(s) was (were) not identified on the protein [START_REF] Edinger | Regulation of Epithelial Na + Channels (ENaC) by methylation: a novel methyltransferase stimulates ENaC activity[END_REF].

With the objective of revealing novel post-translational regulations of plant aquaporins, we have recently engaged a thorough analysis of the modification status of aquaporins expressed in root plasma membrane (PM) using mass spectrometry (MS). In a previous study we have shown that the Arabidopsis root PM contains various Plasma membrane Intrinsic Proteins (PIP) aquaporin isoforms including members of both the PIP1 and PIP2 subclasses [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF]. Here we show that the N-terminal tail of PIP aquaporins can exhibit multiple modifications and is differentially maturated between the two PIP subclasses. More interestingly, we observed that several residues can be methylated in PIP2 aquaporins.

Arabidopsis suspension cells were used to overexpress and characterize methylated wild-type (WT) PIP2;1 and mutants with altered methylation sites.

Protein extraction

Total proteins were extracted from suspension cells (5-10 g FW) as described [26], with the following modifications. The vacuum-dried pellet was resuspended in 0.1 ml/mg FW of a lysis buffer (9 M urea, 4% CHAPS, 0.5% Triton X-100, 65 mM DTT) for 30 min under agitation. The extract was then centrifuged for 10 min at 10,000 g and the supernatant containing total proteins was recovered. Protein concentration was measured using a modified Bradford procedure [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF].

Plasma membrane purification

A microsomal fraction was obtained from roots [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF] and suspension cells [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. In the latter case, cells were first homogenized with a Waring Blender for 10 sec and then fully disrupted in a cell disrupter (Constant System Ltd, Warwick, U. K.) at a pressure of 0.54 kBar. PM vesicles were purified by aqueous two-phase partitioning of a microsomal fraction, in a mixture of polyethylene glycol 3350/Dextran T-500, 6.4% (w/w) each in the presence of 5 mM KCl, as described [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF]. For both roots and suspension cells, the mean yield of PM extraction was 25 µg protein/g FW. Extrinsic membrane proteins were stripped with an urea and NaOH treatment according to a previously described procedure [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF].

Immunodetection

For western-blots, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11% acrylamide gels [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF] and probed with a primary antibody raised against a 17-amino acid C-terminal peptide of AtPIP2;1, as described [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF].

For ELISA assays, serial two-fold dilutions in a carbonate buffer (30 mM Na 2 CO 3 , 60 mM NaHCO 3 , pH 9.5) of 2 µg of total protein extracts or 0.1 µg of PM proteins were loaded in duplicate on Maxisorp immunoplates (Nunc, Denmark) overnight at 4°C. The immunodetection was performed according to the manufacturer's instructions with 0.1% Tween 20 and 1% BSA when required. A 1:2000 dilution and a 1:2500 dilution of primary anti-PIP2;1 antibody and of secondary peroxidase-coupled anti-rabbit antibody, respectively, were successively applied for 2 h at 37°C. A linear regression between the absorbance signal due to oxidized 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid diammonium salt, as read with a multiplate-reader (Victor, Perkin Elmer, USA), and the amount of total proteins was obtained for each ample and used for relative comparison between samples.

MS methods

Protein digestion and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS analysis were performed as described [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF]. Post-translationally modified peptides were searched using a FindMod software (http://us.expasy.org/tools/findmod/) [START_REF] Wilkins | High-throughput mass spectrometric discovery of protein post-translational modifications[END_REF] which systematically compares experimental peptide masses that did not match with native protein sequences, with those obtained by addition of a putative modification. Mono-, di-and trimethylation, and acetylation typically correspond to an increment in monoisotopic mass of 14.01 Da, 28.03 Da, 42.04 Da, and 42.01 Da, respectively.

Post-source decay (PSD) was performed on a BiFlex III mass spectrometer. Peptide parent ions were isolated with a resolution M/ M ~ 100. A FAST TM procedure (Bruker, Bremen, Germany) was used to step scan the reflector tension and to reconstruct the daughter ion spectra. Two hundred spectra were summed for each reflector tension. A MS-BioTools TM software (Bruker) was used to confirm peptide sequence and the final interpretation of spectra was done manually. Electrospray ionisation-(ESI)-MS/MS analyses were performed as described [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF].

Stopped-flow light scattering

Water transport measurements were performed at 20°C essentially as previously described [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. Briefly, membranes were diluted 100-fold into an equilibration solution which contained 20 mM KCl, 30 mM Tris-HCl, pH 8.3. The shrinking of vesicles was initiated by mixing, in a SFM300 stopped-flow spectrophotometer (Biologic, Claix, France), the vesicles with an equal volume of the same buffer containing 500 mM mannitol. The subsequent kinetics of PM vesicles volume adjustment were followed by 90° light scattering at ex = 510 nm. The exponential rate constant, k exp , fitted from the recorded data was used to estimate the osmotic water permeability of the vesicles [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]. It was assumed that because they were prepared in parallel, membrane vesicles from independent cell lines had similar size repartition.

RESULTS

The N-terminus of root PM aquaporins shows isoform-specific maturation A PM fraction was purified from Arabidopsis roots by aqueous two-phase partitioning and enriched in hydrophobic proteins with an urea and NaOH treatment. In previous studies, the proteomic analysis of a major band at 28 kDa identified the presence of at least six PIP isoforms in this fraction (PIP1;1 and/or PIP1;2, PIP1;5, PIP2;1, PIP2;2, PIP2;4, PIP2;7) [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF][START_REF] Javot | Role for a single aquaporin isoform in root water uptake[END_REF]. These studies relied on the tandem MS sequencing of twelve tryptic peptides. Here, a more thorough analysis allowed to identify additional tryptic peptides that cover new sequence regions of the previously identified PIP1 (Table 2) and PIP2 (Table 3). We also performed a systematic investigation of covalent modifications carried by the N-terminal tail of PIPs. For this, a FindMod software was used, which predicts from MALDI-TOF MS fingerprints, the presence of modifications based on the resulting mass shifts [START_REF] Wilkins | High-throughput mass spectrometric discovery of protein post-translational modifications[END_REF]. This study pointed to eleven new peptides that possibly map to the N-terminal region of PIPs and carry putative modifications, that were for most of them confirmed by tandem MS (Tables 2 and3).

In a more general context, FindMod predictions and subsequent sequence analyses allowed us to cover a substantial part of the N-terminus of all PIPs. This corresponded to the entire Nterminal tail of PIP2;1 (Ala2-Arg39) and of PIP1;1 or PIP1;2 (Met1-Arg52). The N-termini of PIP2;2 and PIP2;4 were covered at 80 % whereas that of PIP2;7 was covered at 42 % and those of PIP1;3 (or PIP1;4) and PIP1;5 were covered at 30 % (Tables 2 and3).

The peptide with a mass [M+H] + of 1134.52 Da (Table 2) corresponded to the perfectly conserved N-terminus of the PIP1;1, PIP1;2, PIP1;3 and PIP1;4 isoforms, with the initiating Met being acetylated (Table 2; Figure 1). The corresponding peptide in PIP1;5 whether acetylated or not, was too small to fall into the detection range of the MALDI instrument.

Tandem MS further established that, by contrast to PIP1s, excision of the initiating Met occurred in PIP2;1 and PIP2;2 (Table 3, Figure 2). PIP2;4 was also found in the PM fraction [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF] and the MALDI detection of a peptide with a mass [M+H] + of 1539.78 Da strongly suggested that the initiating Met was also cleaved (Table 3). One peptide specific for PIP2;7 was also detected in the PM fraction (Table 3) but the N-terminal sequence next to the initiating Met was not covered.

In summary, the initiating Met was co-translationally processed in all PIP isoforms studied. Whereas it was N -acetylated in members of the PIP1 subclass, Met was cleaved in PIP2 isoforms.

Modification of aquaporins by methylation

Fingerprint analysis of the tryptic digest also revealed putative PIP2;1 and PIP2;2 peptides with mass increments of ~ 14, 28, and 42 Da (Tables 3 and4). These can be attributed to methylation, dimethylation, and trimethylation or acetylation, respectively. The fragmentation by ESI-MS/MS of a PIP2;1 peptide with a mass [M+H] + of 1631.80 Da induced N-terminal (b-type), C-terminal (y-type) and internal fragments (Figure 2A). Analysis of these fragments revealed that Lys3 was dimethylated ( 2me Lys3) (Tables 3 and4, Figure 2A). Note that this modification can be unambiguously identified, due to the high resolution of MS. The fragmentation of a PIP2;1 peptide with a mass [M+H] + of 1645.80 Da revealed that in addition to being dimethylated at Lys3, the peptide was monomethylated at Glu6 ( 1me Glu6) (Tables 3 and4; Figure 2B). Sequence analysis of an homologous N-terminal peptide from PIP2;2 with [M+H] + = 1475.70 Da showed that in this isoform also, Lys3 was dimethylated (data not shown). An additional monomethylation site was detected but could not be unambiguously positioned (Table 3). Because of the presence of a trypsin miscleavage after Lys3, the N-terminal peptides of PIP2;1 and PIP2;2 with the highest signal were identified as Ala2-Arg16 and Ala2-Arg14 peptides, respectively. However, trypsin cleavage occurred occasionally and the Asp4-Arg16 and the Asp4-Arg14 peptides of PIP2;1 and PIP2;2, respectively, could also be detected (Table 3). Overall, the N-terminal extremity (Ala2-Arg16) of PIP2;1 occurred in the PM fraction under four distinct molecular forms (Tables 3 and4): unmodified, dimethylated on Lys3 ( 2me Lys3), monomethylated on Glu6 ( 1me Glu6), and trimethylated ( 2me Lys3 1me Glu6).

Ectopic expression of PIP2;1 in suspension cells

PIP2;1 is one of the most abundant PIPs in the Arabidopsis root PM [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF] and supposedly plays a central role in water uptake by roots. By contrast to root cells, Arabidopsis suspension cells show a low endogenous expression level of PIP aquaporins ( [START_REF] Gerbeau | The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH[END_REF]; Santoni et al.

unpublished results

). With the prospect of studying N-terminal methylation of PIP2;1 in a simplified system, we evaluated the ability of these cells to ectopically express this aquaporin.

Biolistic transformation using a CaMV70S::PIP2;1 expression cassette yielded seven independent transformed cell lines. Western blot analysis of total protein extracts using an anti-PIP2;1 peptide antibody revealed a significant overexpression of PIP2 aquaporins in these cells, as compared to untransformed (Untr.) cells or cells transformed with an empty vector (PG) (Figure 3A). The 28 kDa band of a PM extract from cells overexpressing PIP2;1 was further analysed by MS. Of the ~ 20 most abundant peptides of the spectrum, eleven were shown to derive from PIP2;1 (Figure 3B). Further tandem MS analysis of the PIP2;1 peptides revealed a modification status similar to that of endogenous PIP2;1 expressed in the root PM (Table 4): the initiating Met was cleaved, Lys3 and Glu6 were either native or present in dimethylated and monomethylated forms, respectively.

A semi-quantitative MS characterization of the methylation status of PIP2;1 was performed, based on the peak intensity of the detected peptides assuming equal ionisation efficiencies for peptides with identical primary sequences (Table 4). In both roots and suspension cells, the fully unmodified form was the most abundant. The proportion of peptides containing 1me Glu6 was slightly more abundant in suspension cells (42 %) than in roots (29 %) as indicated by the addition of their respective peak intensities in the MALDI spectrum (Table 4). By contrast, the proportion of peptides carrying 2me Lys3 was similar in suspension cells (34 %) and root cells (33 %) (Table 4). Thus, suspension and root cells appear to have comparable PIP2;1 methylation patterns and the former cells can be used as an expression system to further investigate N-terminal PIP2;1 methylation.

Modification profile of PIP2;1 mutants with altered methylation sites

With the prospect of dissecting the respective roles of PIP2;1 methylation at positions 3 and 6, we introduced punctual changes at these positions, the corresponding residues being in particular substituted to Ala (E6A-PIP2;1 and K3A-PIP2;1 mutants). The resulting mutants were then overexpressed in Arabidopsis suspension cells and their modification profile in purified PM was characterized as described above for WT-PIP2;1 (Table 4).

PM of cells expressing the E6A-PIP2;1 mutant revealed a unique N-terminal peptide.

As in the WT form, the initiating Met was cleaved (Table 4). In addition, the peptide carried no decoration. Thus, substitution of Glu6 by an Ala residue resulted in a complete lack of methylation at position 6 but also at position 3 (Table 4).

All N-terminal peptides of K3A-PIP2;1 typically showed Met excision but with a 42 N -terminal acetylation [29] (Table 4). Tandem MS also showed a residual mono-methylation on 4 % of the Ala2-Arg16 K3A-PIP2;1 peptide. Curiously, methylation occurred on Ala3 and not on the non-mutated methylation site at position 6 (Table 4). Thus, the K3A mutation was unable to fully prevent methylation at this position whereas it strongly interfered with methylation of the adjacent site. The complex alteration of the PIP2;1 N-terminus modification pattern induced by the K3A mutation prompted us to investigate the effects of a more conservative substitution of Lys3 by an Arg residue. Similar to WT-PIP2;1, the PIP2;1-K3R mutant showed a typical Met excision (Table 4). We also observed mono-methylation and acetylation, but on very minor fractions (2-4 %) of the N-terminal peptides (Table 4).

Thus, the K3R mutation resulted in an almost complete lack of methylations at position 3 and 6. Overall, these and the data obtained with the E6A-PIP2;1 and K3A-PIP2;1 mutants show mutual interactions of methylation at the two sites.

Intrinsic water transport activity of PIP2;1 mutants with altered methylation sites

Methylation of PIP2;1 N-terminus may modulate the intrinsic water transport activity of the aquaporin. To explore this possibility, we investigated the water transport activity of the WT and mutant PIP2;1 forms. PM vesicles were purified from at least two representative cell lines of each of the PG, WT-PIP2;1, E6A-PIP2;1, K3A-PIP2;1 and K3R-PIP2;1 genotypes, and water transport in these cells was characterized by means of stopped-flow spectrophotometry.

Figure 4A shows typical kinetic changes in light scattering as induced by a sudden hyperosmotic challenge on purified vesicles from PG and WT-PIP2;1 genotypes. The exponential rate constant (k exp ) of these changes is indicative of the membrane osmotic water permeability. Membranes purified from WT-PIP2;1 and all other transformed cell lines investigated showed a water transport activity that was about two-fold higher than in membranes from PG cells (Fig. 4A, data not shown). These results suggest that both WT and mutant PIP2;1 can be expressed as active forms in the PM of suspension cells. The relationship between the abundance of PIP2;1 forms in purified PM (as assayed by ELISA) and the water permeability of these membranes was used to evaluate the intrinsic transport activity of each form. Figure 4B shows that PIP2;1-E6A and PIP2;1-K3R mutants had an intrinsic water transport activity similar to that of PIP2;1-WT. By contrast, the PIP2;1-K3A mutant showed a tendency towards a smaller activity (Figure 4B). Altogether, the data show that mutation of PIP2;1 at Lys3 or Glu6 and/or reduced methylation had no major effect on PIP2;1 water transport activity.

DISCUSSION

Because of their relatively high abundance, aquaporins provide a unique model to address general aspects of membrane protein expression and regulation. In the present work, we have made use of the power and resolution of MS techniques to reveal, in the natural context of plant root membranes, novel co-and post-translational modifications of PM aquaporins. In roots, distinct maturation profiles of the initiating Met, through acetylation or excision were exhibited by native members of the PIP1 and PIP2 subclasses, respectively. In suspension cells, WT-PIP2;1 and its K3A, K3R and E6A derivatives also showed N-terminal Met excision. Excision of the N-terminal Met has been extensively studied in soluble proteins [START_REF] Moerschell | The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo[END_REF][START_REF] Giglione | Protein N-terminal methionine excision[END_REF] and to a much lesser extent in membrane proteins. This process was shown to depend on the nature of penultimate residues, and occurs in the presence of residues with side chains of reduced steric hindrance [START_REF] Moerschell | The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo[END_REF][START_REF] Giglione | Protein N-terminal methionine excision[END_REF]. In agreement with this rule, the three Arabidopsis PIP2s studied (PIP2;1, PIP2;2, PIP2;4), which exhibit an Ala residue at position 2, all had their Met excised whereas PIP1s, which have a perfectly conserved Glu residue, did not. N-terminal acetylation, which has been thoroughly studied in soluble mammalian and yeast proteins depends on N-terminal sequences [29]. For instance, of 64 eukaryotic proteins examined, which similar to PIP1s exhibited a Met-Glu (or a Met-Asp) N-terminal end, all were acetylated, as were PIP1s [29]. Similar analyses suggested that, although the presence of a Ser or Ala residue at the N-terminus as in PIP2s may permit acetylation, the presence of an adjacent basic residue usually prevents this process. Accordingly, WT-PIP2;1 and the K3R-PIP2;1 mutant were not or almost not acetylated (Table 4). By contrast, the substitution of Lys3 by an Ala yielded a K3A-PIP2;1 mutant with very significant N-terminal acetylation.

Curiously, we found that a minor proportion (~13 %) of the mutant was devoid of acetylation (Table 4). This form could reflect an incomplete modification or be indicative of an as yet unknown deacetylase activity.

In the present work, we also observed that PIP2;1 and PIP2;2 isoforms carried methylated residues, a yet discrete but less commonly described decoration. Lys3 and Glu6 of PIP2;1 although they existed in their native form, occasionally carried di-methyl (Lys3) or mono-methyl (Glu6) moieties (Tables 3 and4). The methylation of Lys residues has been observed in soluble proteins and recently described in a few mammalian membrane intrinsic [START_REF] Kluck | Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation[END_REF]. Also, a proteomic analysis of rat brain membranes uncovered 15 proteins which possibly carried mono-, di-or tri-methylated Lys [START_REF] Wu | A method for the comprehensive proteomic analysis of membrane proteins[END_REF], suggesting that Lys modification might be common in membrane proteins.

Glu methylesterification is by far less common than Lys methylation and its observation in PIP2s represents a novel modification for eukaryotic membrane proteins. In bacteria, chemotaxis receptors undergo reversible methylation at glutamyl residues, a process which modulates their signalling output [START_REF] Springer | Identification of a protein methyltransferase as the cheR gene product in the bacterial sensing system[END_REF][START_REF] Simms | The kinetic mechanism of S-adenosyl-Lmethionine: glutamylmethyltransferase from Salmonella typhimurium[END_REF][START_REF] Djordjevic | Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain[END_REF]. We were aware that artefactual carboxymethylation of proteins can take place, when these ones are simultaneously treated by trichloroacetic acid and alcohols (methanol), as during gel Coomassie blue staining [36].

These treatments were, however, carefully avoided in the present work.

Additional analysis indicated that alanine can also be methylated in the K3A-PIP2;1 mutant (Table 4). In that case, we assume that methylation occurs at the peptide bond. Such N-methylation has been described in several biologically active natural peptides and was shown to improve their lipophilicity and proteolytic stability [START_REF] Haviv | Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists[END_REF]. Its role in membrane proteins is to our knowledge as yet unknown.

The significance of observations made on PIP2;1 N-terminal tail surely goes beyond the context of the present study since putative methylated residues could be identified in other PIPs such as PIP1;1 (Table 2) and other PM proteins (Santoni et al. unpublished results).

These are, to our knowledge, the first observed methylations in aquaporins and even plant membrane proteins. Methylation of PIP2;1 was examined in details since this aquaporin is one of the most abundantly expressed aquaporins in plants. Although MALDI-TOF analyses do not allow an absolute quantification of the modified vs. unmodified forms of a peptide, their relative peak intensities can be indicative of their relative abundance. Such analyses revealed an interplay between adjacent methylations of PIP2;1 at Lys3 and Glu6 (Table 4). In WT-PIP2;1, the apparent degree of methylation at one position was enhanced by methylation at the other site.

For instance, 1me Glu6 was found in 26 % and 73 % of peptides carrying unmodified Lys3 or 2me Lys3, respectively. Conversely, 2me Lys3 was found in 15 % and 59 % of peptides carrying unmodified Glu6 or 1me Glu6, respectively. We also observed that a PIP2;1 mutant with a punctual E6A substitution did not display any methylation on Lys3 (Table 4). Conversely, the punctual K3A or K3R substitutions and possibly the altered N-terminal maturation of the former mutant protein prevented any methylation of Glu6 (Table 4). Such interactions between modifications at adjacent sites are reminiscent of observations made in histones. In histone H3 for instance, methylation of Lys4 prevents methylation of Lys9 (for review, see [START_REF] Turner | Cellular memory and the histone code[END_REF]). Our findings also provide primary information on the specificity of protein methyl transferases involved in modifying the PIP2;1 N-terminal end. They conform to the idea that these enzymes have local recognition sequences that encompass a few residues [START_REF] Chuikov | Regulation of p53 activity through lysine methylation[END_REF]. Enzymes involved in Lys and Glu methylation of PIP2;1 are very likely distinct in nature, and both remain to be identified.

In recent years, the significance of histone methylation in epigenetic control of gene expression has been extensively studied [40] and the effects of methylation on the activity or the subcellular trafficking of a few other soluble proteins have been elucidated [START_REF] Chuikov | Regulation of p53 activity through lysine methylation[END_REF][START_REF] Green | Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p[END_REF][START_REF] Liang | The N-terminal SH4 region of the Src family kinase fyn is modified by methylation and heterogeneous fatty acylation[END_REF][START_REF] Smith | Arginine methylation of RNA helicase A determines its subcellular localization[END_REF].

Although recent data points to a role of methylation in controlling the activity of an epithelial sodium channel [START_REF] Edinger | Regulation of Epithelial Na + Channels (ENaC) by methylation: a novel methyltransferase stimulates ENaC activity[END_REF], the functional significance of specific methylation events on eukaryotic membrane proteins remains to be elucidated. PIP aquaporins can be gated by phosphorylation of their cytosolic loop B or C-terminal tail [START_REF] Johansson | Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation[END_REF] or protonation of their cytosolic loop D [START_REF] Tournaire-Roux | Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression[END_REF].

To investigate whether methylation of PIP2;1 N-terminal tail plays a similar role, we coupled water transport measurements to aquaporin immunoquantification in PM vesicles expressing WT or mutant PIP2;1 forms. The E6A-PIP2;1 and K3R-PIP2;1 mutants which both are not methylated at Lys3 and Glu6 (Table 4) exhibited a water transport activity similar to that of WT-PIP2;1. These results suggest that methylation at Lys3 and Glu6 does not interfere with intrinsic water permeability. By contrast, the K3A-PIP2;1 showed a tendency to a lower water transport activity. We suppose that the non conservative Lys3 to Ala substitution and /or the resulting alteration of the protein maturation profile, including an abnormal Nacetylation, may result in a partially inactive protein.

As an alternative approach for investigating the functional significance of aquaporin methylation, we also searched for physiological conditions that would induce changes in the modification profile of root PM aquaporins. We observed that salinity that down regulates root water transport by inhibiting aquaporins [START_REF] Boursiac | Gating of aquaporins by cytosolic pH regulates root water transport during anoxic stress[END_REF] does not alter their methylation profile (Prak S. et al., unpublished). Since methylation of PIP2;1 seems not to control the gating of this aquaporin, we now investigate the role of this modification in the protein stability and sub-cellular localization.

In conclusion, the present work identifies methylation as a new post-translational modification of aquaporins, and plant membrane proteins in general, and pinpoints to interactions between adjacent methylated sites. Although the functional role of this modification remains as yet unclear, our work may represent a critical advance in identifying new regulatory mechanisms of membrane water transport. The table shows peptides that were identified from the 28 kDa band of a root PM fraction enriched in hydrophobic proteins [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF] and that corresponded to putative PIP1 tryptic digests.

TABLES Table 1 Oligonucleotide primer sequences

The masses [M+H] + of peptides are indicated in the first column. Masses in italics correspond to peptides that were described previously [START_REF] Santoni | A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots[END_REF].

The corresponding sequence predicted by mass fingerprinting, the sequence determined by MS/MS analysis, the type of modification, the isoform name and position inside the sequence are indicated in the following columns. Peptides with modified residues are indicated in bold characters.

Conventions for post-translational modifications are as follows: The table shows putative PIP2 peptides that were identified from the 28 kDa band of a root PM fraction as described in Table 2. Peptides with modified residues are indicated in bold characters.

Conventions for post-translational modifications are as follows:

1me: monomethyl; 2me: dimethyl; 1me E6: monomethyl Glu6; 2me K3: dimethyl Lys3.

*: peptide the ESI-MS/MS spectrum of which is shown in Figure 2.

**: the peptide sequence was deduced from PSD fragmentation. Lys3 was shown to be dimethylated ( 2me K3). The additionnal 14 Da mass increment indicates the presence of a methyl that must be positionned within the following sequence DVEGPEGFQTR. The first column refers to the form of PIP2;1, either WT or mutated. The second column refers to the measured peptide mass. The indicated peptides were identified after trypsin digestion of the proteins. They correspond to all identified forms of the 2-16 N-terminal peptide. Note that, because of an inefficient trypsin cleavage after Lys3, the Asp4-Arg16 peptide represents, with respect to Ala2-Arg16, a minor digestion product almost undetectable in the MALDI-TOF fingerprint. The predicted sequence is indicated in the third column. The last column refers to the relative abundance (± SE) in PM fractions from either roots or suspension cells. n refers to the number of MS spectra from at least two independent transformed cell lines or plant cultures.

*: sequence determined by tandem MS. £ : the position of the methyl group was not determined ac A: acetyl Ala; 1me E: monomethyl Glu; 2me K: dimethyl Lys. 2. The whole sequence can be deduced from the spectrum.

Ac M: acetylated Met. 3. The whole sequence can be deduced from the spectrum. The initiating Met is cleaved. Lys3 is dimethylated ( 2me K) whereas Glu6 is monomethylated ( 1me E). Z = K + 28 Da; J = E + 14 Da.

Figure 3 Ectopic expression of PIP2;1 in suspension cells

(A) Western blot analysis, using a anti-PIP2;1 peptide antibody, of total protein extracts from independent cell lines, either untransformed (Untr.) or transformed with an empty vector (PG)

or with a CaMV70S::PIP2;1 construct (lines 1-5). Equal amounts of proteins (5 µg) were loaded onto each lane.

(B) MALDI-TOF analysis of the trypsin-cleaved 28 kDa band from PM of suspension cells overexpressing PIP2;1. Peptides derived from PIP2;1 are described by their monoisotopic mass, sequence and position inside the protein sequence.

Three peptides were assigned to endogenous PIP aquaporins:

  used for the mutation and cloning of WT and mutated forms of PIP2;1 in a pGreenII 0179 vector, as described in the experimental section. All primers are given in the 5' to 3' orientation. Cloning sites are indicated in bold characters, mutated/inserted residues are indicated in italics and the start/stop codons are underlined.

  ac M1: acetyl Met1; 1me: monomethyl; *: peptide the ESI-MS/MS spectrum of which is shown in Figure1

Figure 1

 1 Figure 1 Representative sequence analysis of a N-terminal PIP1 tryptic peptide from root PM. A Met1-Arg9 peptide of PIP1 was analyzed by ESI-MS/MS and both N-terminal (b-type) and C-terminal (y-type) fragments were used to interpret the spectra. Note that PIP1;1, PIP1;2, PIP1;3 and PIP1;4 isoforms share a perfectly conserved Met1-Arg9 sequence. Analysis was performed on the [M+2H] 2+ species of 567.76 Da that corresponds to the [M+H] + species of 1134.52 Da, as described in Table2. The whole sequence can be deduced from the spectrum.

Figure 2

 2 Figure 2 Representative sequence analysis of N-terminal PIP2;1 tryptic peptides from root PM. Ala2-Arg16 peptides of PIP2;1 were analyzed by ESI-MS/MS and N-terminal (b-type), Cterminal (y-type) and internal fragments were used to interpret the spectra. Fragmented peptides are indicated according to either b and y series, or by their presumed sequence in the case of internal fragments. (A) Analysis was performed on the [M+2H] 2+ species of 816.40 Da that corresponds to the [M+H] + species of 1631.80 Da, as described in Table3. The whole sequence can be deduced from the spectrum. The initiating Met is cleaved. Lys3 is dimethylated ( 2me K). Z = K + 28 Da.(B) Analysis was performed on the [M+3H] 3+ species of 549.27 Da that corresponds to the [M+H] + species of 1645.80 Da, as described in Table3. The whole sequence can be deduced

Fig 2 A

 2 Fig 2

7 A

 7 Relative intensity (%)Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060569

13 Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060569 Copyright 2006 Biochemical Society or

  membrane associated proteins, including subunit c of mitochondrial ATP synthase [19] or cytochrome c

Table 2 Characterization by MALDI-TOF MS and ESI-MS/MS of N-terminal PIP1 peptides from a root PM extract

 2

	Measured					Position in
	mass (Da)	Predicted peptide	Determined peptide	Modification	Isoform	the
	[M+H + ]	sequence	sequence			sequence
	1017.55	VGANKFPER		none	PIP1;1/PIP1;2/	10-18
					PIP1;3/PIP1;4
	1031.55	VGANKFPER		1me	PIP1;1/PIP1;2/	10-18
					PIP1;3/PIP1;4
	1134.52*	MEGKEEDVR + 42 Da	MEGKEEDVR	ac M1	PIP1;1/PIP1;2/	1-9
					PIP1;3/PIP1;4
	1537.75	QPIGTSAQSDKDYK		none	PIP1;1/PIP1;2	19-32
	1638.80	QPIGTSAQSTDKDYK		none	PIP1;4	19-33
	1660.82	FPERQPIGTSAQSDK		none	PIP1;1/PIP1;2	15-29
	1960.97	DYKEPPPAPFFEPGELK	tag APFFE	none	PIP1;5	31-47
	2313.14	EPPPAPLFEPGELASWSFWR		none	PIP1;2	33-52
	2363.12	EPPPAPFFEPGELSSWSFWR	EPPPAPFFEPGELSSWSFWR	none	PIP1;1	33-52

Table 3 Characterization by MALDI-TOF MS and ESI-MS/MS of N-terminal PIP2 peptides from a root PM extract

 3

	Measured					Position in
	mass (Da)	Predicted peptide	Determined peptide	Modification	Isoform	the
	[M+H + ]	sequence	sequence			sequence
	886.46	KWSFYR		none	PIP2;1 /	34-39 /
					PIP2;6	33-38
	1234.56	DVEGPEGFQTR		none	PIP2;2	4-14
	1340.65	DLDVNESGPPAAR		none	PIP2;4	4-16
	1404.70	DVEAVPGEGFQTR	DVEAVPGEGFQTR	none	PIP2;1	4-16
	1418.70	DVEAVPGEGFQTR + 14 Da		1me	PIP2;1	4-16
	1433.70	AKDVEGPEGFQTR	tag PEGFQTR	M1 cleaved	PIP2;2	2-14
	1447.70	AKDVEGPEGFQTR +14 Da		M1 cleaved	PIP2 ;2	2-14
				+ 1me
	1461.70	AKDVEGPEGFQTR +28 Da		M1 cleaved	PIP2 ;2	2-14
				+ 2me
	1475.70**	AKDVEGPEGFQTR	AKDVEGPEGFQTR	M1 cleaved	PIP2;2	2-14
		+ 42 Da		+ 2me K3 + 1me
	1539.78	AKDLDVNESGPPAAR		M1 cleaved	PIP2;4	2-16
	1603.80	AKDVEAVPGEGFQTR	AKDVEGPEGFQTR	M1 cleaved	PIP2;1	2-16
	1617.80	AKDVEAVPGEGFQTR		M1 cleaved	PIP2;1	2-16
		+ 14 Da		+ 1me
	1631.80*	AKDVEAVPGEGFQTR	AKDVEAVPGEGFQTR	M1 cleaved	PIP2;1	2-16
		+ 28 Da		+ 2me K3
	1645.80*	AKDVEAVPGEGFQTR	AKDVEAVPGEGFQTR	M1 cleaved	PIP2;1	2-16
		+ 42 Da		+ 2me K3 + 1me E6
	1869.93	DYVDPPPAPLLDMGELK		none	PIP2;7	16-32
	1872.90	DYQDPPPAPFIDGAELK	DYQDPPPAPFI/LDGAELK	none	PIP2;1	17-33
	2000.99	DYQDPPPAPFIDGAELKK	DYQDPPPAPFI/LDGAEL/IKK none	PIP2;1	17-34
	2066.96	DYKDPPPAPFFDMEELR		none	PIP2;4	17-33
	2096.94	DYEDPPPTPFFDADELTK	DYEDPPPTPFFDADEL/ITK	none	PIP2;2	15-32

Table 4 Characterization by MS of N-terminal peptides of WT and mutated forms of PIP2;1

 4

		Measured
	Form expressed	peptide mass Predicted sequence	Abundance (%)
		(Da)		roots	Suspension cells
	WT	1603.80*	AKDVEAVPGEGFQTR	64 9	49 27
		1617.80	AKDV 1me EAVPGEGFQTR	3 2	1 7 3
		1631.80*	A 2me KDVEAVPGEGFQTR	7 4	9 6
		1645.80*	A 2me KDV 1me EAVPGEGFQTR 26 6	2 5 15
				n = 9	n = 4
	E6A	1545.80*	AKDVAAVPGEGFQTR		100
					n = 2
	K3A	1546.75	AADVEAVPGEGFQTR		13 5
		1588.75*	ac AADVEAVPGEGFQTR		82 1
		1602.75*	ac A 1me ADVEAVPGEGFQTR		4 4
					n = 3
	K3R	1631.81	ARDVEAVPGEGFQTR		94 6
		1645.81 £	ARDVEAVPGEGFQTR + 1me		4 4
		1673.81*	ac ARDVEAVPGEGFQTR		2 4
					n = 4

Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060569 Copyright 2006 Biochemical Society

supported in part by Genoplante (AF 2001093).


Daniel Scherzinger

Sandra Ruch

Daniel P Kloer

Annegret Wilde

Salim Al- Babili

Salim Al-Babili
email: salim.albabili@biologie.uni-freiburg.de

Retinal is formed from apo-carotenoids in Nostoc sp. PCC7120: In vitro characterization of an apo-carotenoid oxygenase

Keywords: Carotenoid Cleavage, Carotenoid Oxygenase, Cyanobacteria, Opsin, Retinal DTT, dithiothreitol, GC-MS, gas chromatography-mass spectrometry, SynACO, Synechocystis apo-carotenoid oxygenase, NosACO, Nostoc apo-carotenoid oxygenase

The sensory rhodopsin from Anabaena (Nostoc) sp. PCC7120 is the first cyanobacterial retinylidene protein identified. Here, we report on NosACO, encoded by the ORF all4284, as the candidate responsible for the formation of the required chromophore, retinal. In contrast to enzymes from animals, NosACO converts β-apo-carotenals instead of β-carotene into retinal in vitro. The identity of the enzymatic products was proven by HPLC and GC-MS. NosACO exhibits a wide substrate specificity with respect to chain lengths and functional end-groups converting β-apo-carotenals, (3R)-3-OH-β-apo-carotenals and the corresponding alcohols into retinal and 3(R)-3-OH-retinal, respectively. However, kinetic analyses revealed very divergent K m and V max values. Based on the crystal structure of SynACO, a related enzyme from Synechocystis sp. PCC6803 showing similar enzymatic activity, we designed a homology model of the native NosACO. The deduced structure explains the absence of β-carotene-cleavage activity and indicates that NosACO is a monotopic membrane protein. Accordingly, NosACO could be readily reconstituted into liposomes . To localize SynACO in vivo, a Synechocystis knock-out strain was generated expressing SynACO as the sole carotenoid oxygenase. Western blot analyses showed that the main portion of SynACO occurred in a membrane-bound form.

INTRODUCTION

Retinal represents the chromophore of the widespread family of retinylidene proteins, commonly called rhodopsins. These light-responsive seven-helix transmembrane proteins are common in archaea, algae and animals. In addition, they have recently been discovered in Eubacteria and Fungi. The basic reaction catalyzed by rhodopsins is the light-dependent cis-trans isomerization of retinal, attached in a Schiff base linkage to a lysine residue in the seventh helix, across one of the double bonds in the polyene chain [for review see 1, 2].

Cyanobacteria employ different receptors for sensing light [for review see 3]. It was shown that they contain phytochromes, cryptochromes and, as recently reported, rhodopsin [START_REF] Jung | Demonstration of a sensory rhodopsin in Eubacteria[END_REF]. There have been earlier indications for the occurrence of retinylidene receptors in cyanobacteria like Calothrix [START_REF] Hoff | Rhodopsin(s) in eubacteria[END_REF] and Leptolyngbya [START_REF] Albertano | A complex photoreceptive structure in the cyanobacterium Leptolynbya sp[END_REF] and direct evidence was delivered by Jung et al. [START_REF] Jung | Demonstration of a sensory rhodopsin in Eubacteria[END_REF], who identified a sensory rhodopsin from Anabaena (Nostoc) sp. PCC7120 (hereafter Nostoc sp. PCC7120). The Anabaena Sensory Rhodopsin (ASR) has been a subject of comprehensive analyses including its photochemical characteristics and structure at atomic level. ASR shows unique characteristics regarding its interacting transducer, its photocycle and the isomeric state of the bound chromophore upon light and dark adaptation. In addition, it was shown that ASR exhibits light-induced reversible interconversion between [START_REF] Redmond | Expression, and Substrate Specifity of a Mammalian β-Carotene 15,15´-Dioxygenase[END_REF]-cis and all-trans retinal leading to a wavelength-dependent isomer ratio [START_REF] Jung | Demonstration of a sensory rhodopsin in Eubacteria[END_REF][START_REF] Sineshchekov | Light-induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells[END_REF][START_REF] Sineshchekov | Photochromicity of Anbaena sensory rhodopsin, an atypical microbial receptor with cis-retinal light adapted form[END_REF][START_REF] Vogeley | Anabaena Sensory Rhodopsin: A photochromic Color Sensor at 2.0 Å[END_REF].

Therefore, ASR may act as a sensor for light-regulated, color-sensitive processes such as chromatic adaptation [START_REF] Grossman | Tracking the light environment by cyanobacteria and the dynamic nature of light-harvesting[END_REF].

The chromophore of rhodopsins, retinal, is a C 20 -isoprenoid which is usually synthesized as a cleavage product of carotenoids. In animals, retinal is formed through symmetrical oxidative cleavage of β-carotene (C 40 ) at the central C15-C15´ double bond. The corresponding enzyme 15,15´ BCO (ββ-Carotene-15,15´-Oxygenase), also named BCO I, is a member of the widespread carotenoid oxygenase family and has been identified from Drosophila melanogaster [START_REF] Von Lintig | Filling the gap in vitamin A research[END_REF], chicken [START_REF] Wyss | Cloning and expression of beta,beta-carotene 15,15'dioxygenase[END_REF] and mammals [START_REF] Redmond | Expression, and Substrate Specifity of a Mammalian β-Carotene 15,15´-Dioxygenase[END_REF][START_REF] Yan | Cloning and characterization of a human beta,beta-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium[END_REF]. Biochemical studies of human BCO I, expressed in insect cells and purified to homogeneity, suggested that at least one unsubstituted β-ionone ring half-site was compulsory for effective cleavage of the carotenoid substrate [15]. It has also been shown that the 15,15´ BCO I acts as a monooxygenase [START_REF] Leuenberger | The reaction mechanism of the enzyme catalyzed central cleavage of β-carotene to retinal[END_REF]. However, recent investigations of AtCCD1 (A. thaliana Carotenoid Cleavage Dioxygenase 1) suggested a dioxygenase mechanism [START_REF] Schmidt | The carotenase AtCCD1 from Arabidopsis thaliana is a Dioxygenase[END_REF].

The identification of BCO I has become possible through its sequence homology to Viviparous14 (VP14) from maize, the first carotenoid cleavage enzyme characterized. VP14 represents a non-heme iron oxygenase that catalyzes the oxidative cleavage of 9-cis-violaxanthin and 9´-cis-neoxanthin, leading to xanthoxin, the precursor of the phytohormone abscisic acid, ABA [START_REF] Schwartz | Specific Oxidative Cleavage of Carotenoids by VP14 of Maize[END_REF]. This cleavage reaction seems to be key regulatory in the biosynthesis of ABA [START_REF] Qin | The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean[END_REF][START_REF] Thompson | Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid[END_REF][START_REF] Iuchi | Regulation of drought tolearance by gene manipulation of 9-cis-epoxycarotenoids dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis[END_REF][START_REF] Qin | Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance[END_REF]. Database information for sequenced genomes reveals the occurrence of homologous enzymes in all taxa [for review see [START_REF] Guiliano | Carotenoid oxygenases: cleave it or leave it[END_REF][START_REF] Camara | Oxidative remodeling of plastid carotenoids[END_REF] indicating that VP14 is a member of a widespread oxygenase family involved in Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

Copyright 2006 Biochemical Society different physiological processes. For instance, the genome of A. thaliana encodes 9 putative carotenoid oxygenases [START_REF] Tan | Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family[END_REF] catalyzing different cleavage reactions [START_REF] Camara | Oxidative remodeling of plastid carotenoids[END_REF]. The cloning of VP14 also led to the identification of carotenoid cleavage enzymes responsible for the synthesis of well known apocarotenoid pigments, such as bixin in Bixa orellana [26] and saffron in Crocus sativus [27]. It was also shown that the VP14-homolog, Carotenoid Cleavage Dioxygenase 1 (CCD1), catalyzes the cleavage of several carotenoids leading to the formation of C 13 volatile compounds like β-ionone in A. thaliana [START_REF] Schwartz | Characterization of a Novel Carotenoid Cleavage Dioxygenase from Plants[END_REF], tomato [29] and petunia flowers [START_REF] Simkin | Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission of beta-ionone, a fragrance volatile of petunia flowers[END_REF]. Recent studies revealed that members of the carotenoid oxygenase family are involved in plant development mediating the synthesis of a yet unknown, carotenoid-derived compound, which regulates the apical dominance [START_REF] Ward | Shoot branching[END_REF][32][START_REF] Booker | MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule[END_REF][START_REF] Auldridge | Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family[END_REF].

Recently, we have described an Apo-carotenoid Cleavage Oxygenase (ACO) from Synechocystis sp. PCC6803 (SynACO, formerly named Diox1) as the first retinal-forming enzyme of eubacterial origin. In contrast to the related BCO I from animals, SynACO does not utilize β-carotene, but several carotenoid-cleavage products named apo-carotenoids, e.g. 3-OH-apo-carotenoids, to form retinal and retinal-like compounds [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. As the first member of the widespread carotenoid oxygenase family, the crystal structure of SynACO was recently solved at 2.4 Å resolution, revealing the reaction center geometry and establishing a solid base for modeling of other family members [START_REF] Kloer | The structure of a retinal-forming carotenoid oxygenase[END_REF].

The occurrence of a functional sensory rhodopsin in Nostoc sp. PCC7120 implies the ability of this cyanobacterium to synthesize the chromophore, retinal. The genome of this filamentous cyanobacterium encodes 3 members of the carotenoid oxygenase family (ORFs: all4284, all4895 and all1106) which may catalyze retinal-synthesis. Here, we report that the ORF all4284-encoded polypeptide (here named NosACO) is an apo-carotenoid cleaving, retinal-forming enzyme showing similar enzymatic activity to SynACO from Synechocystis sp. PCC6803. In addition, reconstitution of NosACO and localization of SynACO indicated that both polypeptides are membrane-bound. The obtained experimental data were corroborated by a model of NosACO deduced from the crystal structure of SynACO. Our data indicate that the cleavage of apo-carotenoids to form retinal and retinal-like compounds is probably a widespread reaction in cyanobacteria.

EXPERIMENTAL

Cultures of cyanobacteria

Nostoc sp. PCC7120 cells were cultured in liquid BG11 0 medium [START_REF] Barua | β-Carotene is Converted Primarily to Retinoids in Rats in vivo[END_REF] supplemented with 10 mM NaHCO 3 . The Synechocystis sp. PCC6803 SynDiox2 knock-out strain was grown in BG11 medium [START_REF] Barua | β-Carotene is Converted Primarily to Retinoids in Rats in vivo[END_REF] containing 15 µg/ml chloramphenicol. Cultures were grown in Erlenmeyer flasks under constant illumination of 15 µmol photons/m 2 •s at 30 °C.

Cloning of Nostoc carotenoid oxygenases

The gene all4284 coding for NosACO (accession number: NP_488324) was amplified using the primer pair NosACO-for: 5'-ATGCAAAGTTACAAATATCAACA-3' and NosACO-back: 5'-TTATACAAATACCTGGGGAGTAA-3'. The ORFs all4895 and all1106, encoding the other two carotenoid oxygenases from Nostoc, were amplified using the primer pairs ND2F/ND2B (5'-ATGCAGATAGTTGATAAAAGGTCA-3';

Protein expression and purification

NosACO was expressed as a glutathione-S-transferase (GST) fusion protein. For this purpose, NosACO was excised from pNosACO-CR2.1 using EcoRI, and ligated into EcoRI/CIP treated pGEX-5X-1 (Amersham Biosciences, NJ, USA) to yield the vector pGEX-5X-NosACO. Subsequently, BL21 E. coli cells were transformed with pGEX-5X-NosACO, grown at 37 °C in 2X YT-medium and induced at an OD 600 of 0.5 with 0.2 mM IPTG. After incubation for additional 4 h at 28 °C, cells were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS) and lysed in a French press. The fusion protein was then purified using glutathione-sepharose 4B (Amersham Biosciences, NJ, USA), and NosACO was released by overnight treatment with the protease factor X a in PBS containing 0.1% (v/v) Triton X-100 at room temperature, according to the instructions of the manufacturer (Amersham Biosciences, NJ, USA). For the reconstitution assays, NosACO was released in PBS containing 1% (w/v) octyl-β-glucoside. Purification steps and protein expression were analyzed by SDS-PAGE.

Analyses of the membrane association

All steps were performed in 100 mM Bistris/HCl, pH 6. with the liposomes and incubated on ice for 10 min. Liposomes were pelleted at 100.000 g for 30 min, washed with 3.6 ml buffer, pelleted again and resuspended in the same volume of the buffer.

For the salt-treatment, 1.8 ml liposomes suspension, containing NosACO and prepared as described above, was adjusted to a final concentration of 0.2 M KCl and incubated on ice for 10 min. Liposomes were then harvested by centrifugation at 100.000 g for 30 min, washed in 1.8 ml buffer, pelleted again and resuspended in 1.8 ml buffer.

Samples corresponding to equal portions of each fraction were subjected to SDS-PAGE and analyzed by western blotting using the ECL TM Western Blotting Analysis System (Amersham Biosciences, NJ, USA). NosACO was detected using mice polyclonal anti-SynACO-antibodies raised against overexpressed and purified enzyme [START_REF] Kloer | The structure of a retinal-forming carotenoid oxygenase[END_REF]. Synechocystis SynDiox2 knock-out cultures were harvested, washed and resuspended in PBS. The cells were broken by four passages through a French press cell at 18.000 psi. Cell debris was removed by repeated centrifugation for 10 min at 4.000 g. The obtained supernatant was then separated into a membrane and a cytoplasmic fraction by centrifugation for 4 h at 130.000 g. Membranes were washed with PBS, pelleted by centrifugation at 130.000 g for 2 h and resuspended in PBS. Samples containing equal protein amounts from each fraction were analyzed by SDS-PAGE and western blotting using the ECL TM Western Blotting Analysis System (Amersham Biosciences, NJ, USA) and mice polyclonal anti-SynACO-antibodies.

Enzyme assays

Substrates were purified according to [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. Synthetic apo-carotenals were kindly provided by BASF (Ludwigshafen, Germany). The corresponding alcohols were obtained by reduction of the aldehydes with NaBH 4 in EtOH. Enzyme assays were performed according to [26] with some modifications.

Substrates were mixed with an EtOH solution of octyl-β-glucoside, dried using a vacuum centrifuge and then resuspended in water. The final concentration of the apo-carotenoid substrates was 40 µM, the concentration of octyl-β-glucoside 1% (w/v). The incubation buffer contained 1 mM DTT and 1 mg/ml catalase (Sigma, Deisenhofen, Germany) in 200 µl of 100 mM Bistris/HCl, pH 6.8. Purified

NosACO was then added to obtain a concentration of 100 ng/µl. Assays were incubated at 27 °C for 2 h. For GC-MS identification of the cleavage products the assay volume was 2 ml with incubation for 2.5 h. The assays were stopped by adding one volume of acetone and extracted using petroleum benzene/diethylether (1:4, v/v). The analysis was carried out as given below.

Analytical methods

Substrates and products were quantified spectrophotometrically at their individual λ max using extinction coefficients calculated from E1% as given by Barua and Olson [START_REF] Barua | β-Carotene is Converted Primarily to Retinoids in Rats in vivo[END_REF] or as determined with Copyright 2006 Biochemical Society the synthetic compounds (BASF, Ludwigshafen, Germany). Protein concentration was determined using the BioRad protein assay kit (BioRad, CA, USA). For HPLC, a Waters system (Eschborn, Germany) equipped with a photodiode array detector (model 996) was used. The separation was performed using a C 30 -reversed phase column (YMC Europe, Schermbeck, Germany) with the solvent systems B: MeOH/tert-butylmethyl ether/water (120:4:40, v/v/v) and A: MeOH/tert-butylmethyl ether (500:500, v/v). The column was developed at a flow-rate of 1 ml/min with a gradient from 100%

B to 43% B within 45 min, then to 0% B within 1 min, maintaining the final conditions for another 14 min at a flow-rate of 2 ml/min.

GC-MS analyses

To remove detergents present in the extracts, products were pre-purified by TLC using RP-18 F 245s plates (Merck, Darmstadt, Germany). All plates were washed in a chromatographic step with chloroform prior to use. The plates were developed in MeOH/water (100:1, v/v). Products were then scraped off and eluted with chloroform, evaporated and redissolved in acetone for analyses. GC-MS analyses were performed according to [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]

Kinetic analyses

Measurements were carried out photometrically at 20 °C using a UV-2501PC spectrophotometer (Shimadzu, Japan). The assays contained 100 mM Bistris/HCl, pH 6.8, 1% (w/v) octyl-β-glucoside, 1 mM DTT, 200 ng/ml catalase and purified NosACO at a concentration of 30 ng/µl in a final volume of 700 µl. The concentrations of the apo-carotenoid substrates used ranged from 5 to 80 µM. The formation of the C 20 cleavage products was determined during an incubation of 5 to 15 min. Time intervals with maximum velocity were used for further calculations. The enzyme kinetics were calculated using the solver function of MS-Excel. This function fits the data to the Michaelis-Menten equation using non-linear regression analysis.

Homology modeling

A homology model of NosACO was built using the published coordinates of SynACO (PDB code 2BIX) as template. Model building and energy minimization was performed using SWISS-MODEL [START_REF] Schwede | SWISS-MODEL: an automated protein homology-modeling server[END_REF] in first approach mode. Solvent-accessible surfaces were generated with MSMS [START_REF] Krissinel | Detection of Protein Assemblies in Crystals[END_REF]

RESULTS

β-carotene is not cleaved by any of the three Nostoc carotenoid oxygenases

To determine the enzyme catalyzing the formation of retinal, the three Nostoc ORFs all4284 (here NosACO), all4895 (NosDiox2) and all1106 (NosDiox3) encoding putative carotenoid oxygenases were cloned and expressed in β-carotene accumulating E. coli cells. However, none of the expressed enzymes was capable of converting β-carotene as determined by the absence of cleavage products in HPLC analyses (data not shown).

NosACO converts apo-carotenals into retinal

Since no cleavage activity was observed in carotenoid-accumulating E. coli cells, in vitro assays were performed using total lysate of cells expressing NosACO, NosDiox2 and NosDiox3, respectively.

Subsequent analyses revealed that none of the expressed oxygenases is capable of β-carotene cleavage. Therefore, apo-carotenals with different chain lengths were applied. Subsequent HPLC analyses showed that NosACO converted apo-carotenoids with different chain lengths into a compound resembling retinal (C 20 ), while the products obtained from NosDiox2 and NosDiox3 were different in chain lengths depending on the substrates applied (data not shown). This indicated that NosACO is the candidate for retinal formation. For a more comprehensive investigation of its enzymatic activity, NosACO was expressed as a GST fusion, purified using glutathione-sepharose and released by the protease factor X a (supplemental Fig. 1). Here again, in vitro assays using the As can be seen in Fig. 1, a striking activity was observed with β-apo-8´-carotenal which was converted by NosACO into a compound that resembled retinal in its UV/Vis spectrum and its chromatographic behavior on HPLC. To prove its identity, GC-MS analyses were carried out using retinal as a standard, which showed an identical retention time. The mass spectrum obtained was identical with published data [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF][START_REF] Robinson | Retinal identification in Pelvetia fastigiata[END_REF] and with spectra in the NIST database, including the presence of the correct molecular ion of m/z = 284 (Fig. 2). Thus, NosACO converted the C was detected and proven by HPLC (Fig. 1) and GC-MS analyses (Fig. 2) as a second product showing the chromatographic characteristics and the EI mass spectrum of the authentic reference compound (kindly provided by BASF, Ludwigshafen, Germany). Taken together, the nature of the products identified demonstrates the cleavage of the used apo-carotenoids at the C15-C15´ double bond (Fig. 3).

Substrate specificity of NosACO

The Synechocystis enzyme SynACO was shown to cleave a wide range of different apo-carotenoids [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. To investigate the end-group specificity of NosACO, (3R)-3-OH-β-apo-8´-carotenal formally derived from zeaxanthin was used in vitro as described. As shown by HPLC analysis (Fig. 1), this substrate was converted by the enzyme into (3R)-3-OH-retinal and apo-8´,15´-apo-carotene-dial. The nature of the C 20 -product obtained was further confirmed by GC-MS-analysis (Fig. 2) revealing a correct molecular ion of m/z = 300 and a mass spectrum identical to that of the authentic reference compound (kindly provided by BASF, Ludwigshafen, Germany). Further, we tested β-apo-8´- were not cleaved. The substrates and the corresponding chemical structures are given in Fig. 3.

Kinetic analyses

The data given above suggest a wide substrate specificity. However, the cleavage reactions exhibited very divergent initial reaction velocities depending on the substrate employed. For instance, apo-8´-lycopenal was converted obviously at a slow rate as compared to β-apo-8´-carotenal. Copyright 2006 Biochemical Society analyses were performed with the substrates β-apo-8´-carotenal, β-apo-10´-carotenal, β-apo-8´carotenol and (3R)-3-OH-β-apo-8´-carotenal exhibiting different chain lengths and functional groups.

Table 1 gives the K m and V max values determined in the biphasic detergent-containing in vitro incubation system used. The lowest K m values were obtained for (3R)-3-OH-β-apo-8´-carotenal, followed by β-apo-8´-carotenal, β-apo-10´-carotenal and finally by β-apo-8´-carotenol. However, βapo-8´-carotenal showed lower V max values when compared to the corresponding alcohol, i.e., NosACO exhibited a higher affinity to aldehydes but converted alcohols faster. In addition, β-apo-8´carotenal showed also a significantly higher V max than (3R)-3-OH-β-apo-8´-carotenal indicating that the enzyme converted unsubstituted substrates faster than (3R)-3-OH-β-derivatives. Although, the determined K m values for β-apo-8´-carotenal (C 30 ) and β-apo-10´-carotenal (C 27 ) were similar, the C 30substrate was converted much faster, as indicated by the V max values.

Structure alignment with SynACO and modeling

The observed enzymatic activity of NosACO resembles the one described for SynACO from Synechocystis [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. In addition, both enzymes belong to the same family and share about 53% identity at amino acid level. The recently solved crystal structure of SynACO [START_REF] Kloer | The structure of a retinal-forming carotenoid oxygenase[END_REF] allowed the construction of a homology model of NosACO. In the structure-based sequence alignment used, there are no gaps within secondary structure elements (supplemental Fig. 2). Two small insertions of two residues each occur in NosACO as compared to SynACO. Both are located in a loop between β-strands 18 and 19 and were modeled with good stereochemistry. All other residues of the NosACO sequence were exactly superimposable onto the template. Given the high degree of sequence identity between SynACO and NosACO, the resulting homology model is expected to be exact within a 1.5 Å root mean square deviation with respect to C α positions [START_REF] Chothia | The relation between the divergence of sequence and structure in proteins[END_REF]. All residues known to be important for substrate binding and catalysis in SynACO are found at corresponding positions in the NosACO homology model. NosACO consists of a single domain and adopts a β-propeller fold with seven propeller blades (Fig. 4). Long loops on the top side of the β-propeller form a dome covering the active site, which is located in the center of the protein at the propeller axis. The catalytic center is formed by four histidine residues that are strictly conserved in all carotenoid cleavage oxygenases and coordinates a ferrous iron ion. Interestingly, all residues lining the hydrophobic substrate tunnel in SynACO, up to 10 Å away from the catalytic center, are either identical or conservatively replaced by residues with the same steric requirements in NosACO, giving rise to a substrate binding cavity with an almost identical shape (Fig. 4). Assuming the same substrate binding geometry as was previously observed in SynACO [START_REF] Kloer | The structure of a retinal-forming carotenoid oxygenase[END_REF], the homology model clearly predicts that C 40 substrates will not be cleaved by NosACO. On the surface of NosACO, a large nonpolar patch consisting of leucine, tryptophane, phenylalanine and isoleucine residues lies at the same position as in SynACO (Fig. 4), while the remaining surface of the molecule is predominantly polar. It was presumed that the corresponding region in SynACO dips into the membrane in order to provide access to the unpolar substrate. Copyright 2006 Biochemical Society hydrophobic patch in NosACO is even larger (889 Å 2 as compared to 800 Å 2 in SynACO), leading to the prediction that NosACO may also be a monotopic membrane protein.

NosACO and SynACO are membrane proteins

The lipophilic nature of the substrates as well as the predicted model indicate that NosACO is a membrane protein. Therefore, we performed reconstitution assays into artificial membranes using the purified enzyme. NosACO could be readily reconstituted as shown in the Western blot analysis (Fig. 5). Additionally, the membrane binding was stable upon high-salt treatment. These data coincide with the structural model and indicate that NosACO is a membrane-bound enzyme.

The genome of Nostoc encodes three members of the carotenoid oxygenase family which may not be distinguished by polyclonal antibodies. In contrast, Synechocystis contains only two members of this family (SynACO and ORF: slr648, hereafter SynDiox2). To localize SynACO in vivo, we generated a knock-out strain by interrupting SynDiox2. Thus, this strain encodes only SynACO and allows, therefore, the localization of SynACO by using polyclonal, anti-SynACO-antibodies avoiding crossreactions. As can be seen in Fig. 6, only traces of SynACO could be detected in the cytoplasmic fraction, while the main portion of the enzyme occurred in a membrane-bound form.

DISCUSSION

The Nostoc sensory rhodopsin was the first of its kind found in cyanobacteria. Its occurrence implies the biosynthetic capacity for the chromophore, retinal. According to the current state of knowledge, retinal is formed either by a member of the widespread carotenoid-oxygenase family (for review see: [START_REF] Moise | Related enzymes solve evolutionarily recurrent problems in the metabolism of carotenoids[END_REF][START_REF] Bouvier | Oxidative tailoring of carotenoids: a prospect towards novel functions in plants[END_REF] or by the very recently described enzyme Blh from marine proteobacteria [START_REF] Sabehi | New insights into metabolic properties of marine bacteria encoding proteorhodopsins[END_REF] and its formerly identified halobacterial homologs [START_REF] Peck | brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum[END_REF]. Searching the genome of Nostoc for putative carotenoid oxygenases revealed the occurrence of three members of this protein family, ORFs: all4284, all4895 and all1106, while no sequence with significant similarity to Blh was found. This led us to suggest that retinal is formed through one of the encoded carotenoid oxygenases. However, in vivo and in vitro studies showed that none of these three enzymes is capable of cleaving β-carotene to yield retinal (data not shown) excluding a central cleavage mechanism of β-carotene, as commonly found in animals. Based on our recent finding that the Synechocystis-enzyme SynACO forms retinal and retinal-like compounds from apo-carotenoids and not from β-carotene [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF], we assumed that Nostoc may utilize similar substrates for retinal-formation. Accordingly, we performed in vitro-enzymatic analyses using apo-carotenoids as substrates. Among the three carotenoid oxygenases, NosACO, encoded by the ORF all4284, was the one to form retinal and derivatives from apo-carotenals with different chain lengths indicating its involvement in retinal supply. Albeit the significance of the sequence homology between SynACO and NosACO (53% identity), it could not necessarily be expected that both enzymes exhibit the same activity and, thus, that both cyanobacteria utilize apo- Copyright 2006 Biochemical Society carotenals to form retinal. Generally, orthologous carotenoid oxygenases show more pronounced homology. For instance, the carotenoid cleavage dioxygenases 1 (CCD1) from A. thaliana [START_REF] Schwartz | Characterization of a Novel Carotenoid Cleavage Dioxygenase from Plants[END_REF] and L. esculentum [29] share about 79% identity at amino acid level. The sequence divergence between SynACO and NosACO could be explained by the evolutionary distance between the unicellular Synechocystis sp. PCC6803 and the axenic, nitrogen fixing Nostoc sp. PCC7120.

To characterize the substrate specificity, in vitro studies were performed using the purified enzyme. NosACO cleaved β-apo-carotenals with different chain lengths, (3R)-OH-β-apo-carotenals as well as the corresponding alcohols. Depending on the nature of the end-groups, the cleavage led to the formation of retinal and (3R)-3-OH-retinal. However, the values determined for K m and V max differed markedly (Table 1). Given that low substrate concentrations prevail in vivo, one would assume the preferred natural substrate to be a C3-hydroxylated apo-carotenal with a C 27 or C 30 chain length. However, the identity of the natural substrate and the reaction leading to its formation are still to be elucidated. Like in Synechocystis [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF], HPLC analyses of carotenoid extracts from Nostoc revealed the occurrence of small amounts of compounds which resembled (3R)-3-OH-β-apocarotenals in their UV/Vis spectra as well as in their elution characteristics (data not shown), but these were too low in abundance to allow an investigation of their molecular structure. Such precursor molecules may be formed through the action of a different enzyme. The two additional members of the carotenoid oxygenase family found in the genome of Nostoc are good candidates. Although, our preliminary analyses indicate that none of these enzymes cleaves β-carotene, it cannot be excluded that a (3R)-3-OH-substrate may be supplied by one of these oxygenases through an initial cleavage of myxoxanthophylls which are common in Nostoc [START_REF] Takaichi | Myxol and 4-Ketomyxol 2'-Fucosides, not Rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and Proposal for the Biosynthetic Pathway of Carotenoids[END_REF]. However, carotenoids may be also cleaved by enzymes, like peroxidases and lipoxygenases, which do not belong to the carotenoid oxygenase family. For instance, Zorn et al. [START_REF] Zorn | A peroxidase from Lepista irina cleaves beta,beta-carotene to flavor compounds[END_REF] reported on a fungal peroxidase from Lepista irina which cleaved β-carotene to form certain flavor compounds. In addition, the genome of Nostoc encodes a putative lipoxygenase (ORF all8020) with unknown function. Alternatively, apo-carotenoids may arise from photo-destruction processes mediated by reactive oxygen species (ROS), increased under light or salt stress. It was reported that the transcript level of the homologous enzyme SynACO from Synechocystis was increased by 4 fold within 15 min after exposure to high light [START_REF] Hihara | DNA microarray analysis of cyanobacterial gene expression during acclimation to high light[END_REF] and by 21 fold within 30 min after treatment with 684 mM NaCl [START_REF] Marin | Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803[END_REF]. Considering the similarity in the catalytic activity and the amino acid sequence, it appears conceivable that NosACO may be subjected to a similar regulation. In this case, NosACO may convert a non-enzymatically originated, inhomogeneous population of apo-carotenoids into homogeneous C 20 compounds, namely retinal, retinol and its in-ring hydroxylated derivatives.

The enzymatic activity of NosACO, the substrate specificity for apo-carotenoids, and the kinetic characteristics resemble those of SynACO [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. However, NosACO showed more specificity with respect to the substrate´s chain lengths and did not convert β-apo-4´and β-apo-12´-carotenal, which were both cleaved by SynACO [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF].

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

Copyright 2006 Biochemical Society

The crystal structure of the latter enzyme allowed to explain and thus supported the experimentally shown absence of β-carotene cleaving activity [START_REF] Ruch | Retinal Biosynthesis in eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp[END_REF]. SynACO exhibits a substrate cavity with a bottleneck at the entrance arresting the β-ionone ring, thus placing the substrate into the correct position for retinal formation. In other words, the substrate enters the cavity with its openchain end (Fig. 4), since the β-ionone ring cannot pass. The NosACO model deduced from the structure-based alignment with SynACO showed a very similar geometry of the substrate cavity explaining the observed absence of β-carotene cleaving activity.

Consistent with the lipophilic nature of substrates and products, a further prediction drawn from the NosACO-model is the attribute of being a monotopic membrane protein. In the model, the surface of NosACO exhibits a large nonpolar patch consisting of leucine, tryptophane, phenylalanine and isoleucine residues at the same position as in SynACO. It was presumed that the corresponding region in SynACO dips into the membrane. The reconstitution experiments and localization studies

shown here indicate that both enzymes are membrane proteins. In both experiments, only traces of soluble enzymes were detected. Similarly, the xanthoxin-forming enzyme PvNCED (Phaseolus vulgaris nine-cis-epoxy-carotenoid dioxygenase) was found only in the thylakoid fraction of bean leaves [START_REF] Qin | The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean[END_REF]. In contrast, it was shown that only about 35% of VP14 from maize are associated with the thylakoid membrane upon import. In addition, Western blot analyses of maize chloroplasts revealed a stromal and a membrane-bound VP14-fraction [START_REF] Tan | Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family[END_REF]. It has been speculated that the degree of membrane association may be regulated in intact chloroplasts and that the soluble fraction is inactive [START_REF] Tan | Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family[END_REF], as reported for the phytoene desaturase from Narcissus pseudonarcissus [START_REF] Al-Babili | A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70-complexed and competent for flavinylation, membrane association and enzymatic activation[END_REF]. The membrane association of VP14 is mediated by an N-terminal amphipathic helix [START_REF] Tan | Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family[END_REF]. A more complicated pattern was reported for RPE65 (Retinal Pigment Epithelium 65), an animal member of the carotenoid oxygenase family involved in the visual cycle. RPE65 occurs in a triply palmitoylated, membraneassociated (mRPE65) and in a soluble, un-palmitoylated form (sRPE65). It appears that the palmitoylation determines whether RPE65 binds all-trans-retinyl ester (mRPE65) or all-trans retinol (sRPE65), and thus, controls the ligand binding selectivity [START_REF] Xue | A palmitoylation switch mechanism in the regulation of the visual cycle[END_REF]. Taken together, it might be speculated that the soluble traces of SynACO may represent an inactive form.

The model also indicates that the product formed is deposited into membranes through a tunnel and not passed to a cytoplasmic protein. To test this prediction, we conducted in vitro assays using artificial membranes containing reconstituted NosACO. The subsequent analyses revealed a clear retinal formation (data not shown). However, the amount of the product was low, compared to the amounts obtained from the detergent-based assays and, therefore, did not allow a clear conclusion about the destination of retinal.

TABLES Table 1

The OH-β-apo-8´-carotenal (C). The structure of the products is given above the corresponding UV/Vis spectra, see also Fig. 2 and Fig. 3.

Figure 2 GC-MS analyses of the in vitro assay products

The products obtained from in vitro assays (Fig. 1) were subjected to GC-MS analyses. The EI mass spectrum of product I was identical to that obtained with synthetic apo-8´,15´-apo-carotene-dial (C 10 O 2 H 11 ) and showed the expected molecular mass of m/z 164. The EI mass spectrum of product II showed identity with the reference spectrum of retinal and the expected molecular ion of m/z 284. The EI mass spectrum of product III showed identity with the reference spectrum and the expected molecular ion of m/z 300 as well as the typical fragment of 162 m/z, corresponding to a mass of 300 minus 138, indicative for the loss of the 3-OH-β-ionone ring as described for zeaxanthin [START_REF] Enzell | Carotenoids[END_REF].

Figure 5 Western blot analysis of the reconstitution assay

Total reconstitution assay containing soluble and liposome-bound NosACO (1), isolated and washed liposomes (2), the corresponding supernatant containing unreconstituted enzyme (3), liposomes after treatment with 0.2 M KCl (4) and the corresponding supernatant [START_REF] Hoff | Rhodopsin(s) in eubacteria[END_REF]. NosACO was detected using polyclonal anti-Syn-ACO-antibodies.

  using a Finnigan Trace DSQ mass spectrometer coupled to a Trace GC gas chromatograph. Identification of compounds was done by chromatographic comparison with the authentic references and by comparing the mass spectra with the NIST Mass Spectral Search Program Version 2.0 (National Institute of Standards and Technology).

  carotenol and the corresponding alcohols of the above mentioned substrates. Here again, retinal or the corresponding C 20 compounds were produced. To determine the chain length specificity, we performed in vitro assays with β-apo-4´-carotenal (C 35 ), β-apo-10´-carotenal (C 27 ), β-apo-12´carotenal (C 25 ) and (3R)-3-OH-β-apo-12´-carotenal. The subsequent analysis showed that NosACO cleaved β-apo-10´-carotenal and (3R)-3-OH-β-apo-12´-carotenal forming retinal and (3R)-3-OHretinal, respectively (data not shown). In addition, the enzyme also cleaved apo-10´-lycopenal into acycloretinal (data not shown). In contrast, β-apo-12´-carotenal (C 25 ) and β-apo-4´-carotenal (C 35 )

Figure 1 HPLC

 1 Figure 1 HPLC analyses of the in vitro assay products obtained from β-apo-8´-carotenal (A), βapo-10´-carotenal (B) and (3R)-3-OH-β-apo-8´-carotenal (C) and the corresponding UV/Vis spectra (I-III) After 2.5 h incubation with purified NosACO (ACO), a striking cleavage acitivity was observed, indicated by color changes compared to the controls (con). The reactions led to the formation of the products apo-8´,15´-apo-carotene-dial (I) and retinal (II) from β-apo-8´-carotenal (A), retinal (II) from β-apo-10´-carotenal (B) and (3R)-3-OH-retinal (III) and apo-8´,15´-apo-carotene-dial (I) from (3R)-3-

Biochemical Journal Immediate Publication. Published on 7 Figure 3

 73 Figure 3 Structure of applied substrates and determined products (I-III)The degree of cleavage was estimated by the ratio of cleavage product compared to the substrate. ++ cleavage; + low cleavage; n.d. not detected. The substrates β-apo-8´-carotenal (C 30 ), its corresponding alcohol and β-apo-10´-carotenal (C 27 ) were cleaved into retinal (C 20 , II). (3R)-3-OH-β-apo-8´carotenal, its corresponding alcohol and (3R)-3-OH-β-apo-12´-carotenal (C 25 ) were converted to (3R)-3-OH-retinal (III). Apo-8´,15´-apo-carotene-dial (C 10, Ι) was produced from the cleavage of the β-apo-8´-carotenoids (C 30 ) as the second product. Substrates were cleaved at the C15-C15´ double bond, as indicated. With exeption of apo-12´-lycopenals, the enzyme converted all applied apolycopenals into the corresponding C 20 -compound acycloretinal (not shown). The incubation with βapo-4´-carotenal and β-apo-12´-carotenal did not lead to detectable products.

Figure 4

 4 Figure 4 Model of NosACO based on homology to SynACO A: Ribbon representation of NosACO. β-strands and α-helices are drawn in orange and green, respectively. The eight residues forming the large hydrophobic patch at the top of the enzyme are shown in stick representation and labeled. The axis of the β-propeller runs vertically. B: Surface representation showing the solvent-accessible surface in grey. The entrance to the substrate binding tunnel is drawn in blue and the hydrophobic patch of 889 Å 2 in yellow. C: Geometry of the substrate binding tunnel. Surface representation of NosACO rotated by 90° along the propeller axis and cut through the middle of the enyzme. The substrate binding tunnel runs horizontally and is shaded in blue. The position of the catalytic iron is indicated in red. In order for cleavage to occur, the substrate must enter the tunnel far enough to bring the scissile double bond in close proximity to the catalytic iron. A constriction in the tunnel, marked by arrows, prevents entry of bulky substrates. D: Close-up view of the constriction together with a β-ionone ring for size comparison. The constriction is too small to allow substrates carrying β-ionone rings on both termini such as β-carotene to enter the tunnel.

Figure 6

 6 Figure 6 Localization of SynACO in a Synechocystis SynDiox2 knock-out strain A: Coomassie stained SDS-gel of membrane (1) and cytoplasmic fraction proteins (2). B: The corresponding Western blot analysis. The localization was performed using 25 µg protein of each fraction.

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592 Copyright 2006 Biochemical Society

	of 1 mg/ml. After incubation for 1 h on ice, liposomes were obtained by sonication on ice. 720 µl
	purified NosACO (about 1.2 µg/µl) in PBS containing 1% (w/v) octyl-β-glucoside was diluted (1:5)
	8, buffer containing 1 mM DTT. For
	preparation of liposomes 1,2-diacyl-sn-glycero-3-phosphocholine from soybean (Sigma, Deisenhofen,
	Germany) was solubilized in chloroform/MeOH (2:1, v/v), β-apo-8´-carotenal was then added to a
	final concentration of 30 µM. After drying, the buffer was added to obtain a final lipid concentration

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592 Generation of the Synechocystis SynDiox2 knock-out strain A

  557-bp fragment (delta5), representing a stretch of the 5′-non-coding region and a part of SynDiox2, and a 448-bp fragment (delta3), corresponding to the 3′-non-coding region and a part of SynDiox2, were amplified from 25 ng Synechocystis sp. PCC6803 genomic DNA using Advantage ® cDNA EcoRV site, after XhoI/HindIII (for delta5) and EcoRI/XbaI (for delta3) digestion. The correctness of the resulting suicide vector pDiox2Knock was proofed by DNA sequencing. pDiox2Knock, bearing a chloramphenicol resistance cassette between delta5 and delta3 in antisense orientation, was then transformed into wild-type cells of Synechocystis sp. PCC6803 according toErmakova et al. [41]. The absence of the wild-type allele in fully

	Polymerase Mix (BD Biosciences, CA, USA) in the buffer provided. The PCR reactions were
	performed using the primer pair I/II and III/IV for the amplification of delta5 and delta3, respectively
	(I: 5´-GGGCCGCTACTCGAGCCAGTCTG-3´; II: 5′-TCCTCTGCGTAAGCTTGGGTTTGC-3′; III:
	5′-ACCGGCGGAATTCCTTGGTTC-3′; IV: 5′-TGCTTAATGAGCTCTCCAGTTTCA-3′). Both
	fragments were then cloned sequentially into pKSCm2, a pBluescript KS with a cat (chloramphenicol
	transferase) gene cassette integrated into the segregated mutants was verified by	PCR	using	the	primers	Co1:	5'-
							and
	analyzed with PISA [40].

AGTTTGTTGACCCAGACCGATGTA-3' and Co2: 5'-ATAGTTGACCATCCGTGGAGGTTT-3'.

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592 Copyright 2006 Biochemical Society purified

  enzyme and major carotenoid compounds of Nostoc, e.g. β-carotene, echinenone and myxoxanthophyll, did not show any cleavage activity, while apo-carotenals were converted readily.

  30 substrate into retinal (C 20 ). The catalytic activity of NosACO should also lead to a second product(s) with a chain length of C 10 or shorter. Indeed, apo-8´,15´-apo-carotene-dial (2,6-dimethyl-octa-2,4,6-trien-dial, C 10 O 2 H 11 )

Kinetic Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

The Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

Biochemical Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592

K m and V max values of NosACO for different substrates

	Each value represents the average ± S.D. of three independent experiments.
	Substrate	K m (µM)	V max (nmol/min)
	β-8´apo-carotenal	16.3±1.6	1.0±0.3
	β-10´apo-carotenal	17.2±1.8	0.3±0.1
	(3R)-3-OH-β-8´apo-carotenal	2.1±0.3	0.1±0.04
	β-8´apo-carotenol	24.5±3.1	3.3±1.7

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Grant AL892-1 and by the HarvestPlus programme (www.harvestplus.org). We are indebted to Dr. Peter Beyer for valuable discussions. We thank Dr. Hansgeorg Ernst for providing the apo-carotenoids and Dr. Andreas Seidler for his help in constructing the knock-out plasmids.

Biochemical

Journal Immediate Publication. Published on 7 Jun 2006 as manuscript BJ20060592 Copyright 2006 Biochemical Society