Herbert Kaltner
email: kaltner@lmu.de

Dolores Solís

Jürgen Kopitz

Martin Lensch

Michaela Lohr

Joachim C Manning

Michael Mürnseer

Martina Schnölzer

Sabine André

José Luis Sáiz

Hans-Joachim Gabius

Prototype chicken galectins revisited: characterization of a third protein with distinctive hydrodynamic behaviour and expression pattern in organs of adult animals

Keywords: homology, lectin, mitogenicity, nephron, promoter, phylogeny

come

INTRODUCTION

Divergence after gene duplication is the common route toward groups of closely related proteins. They can acquire characteristic properties by sequence alterations. However, the high degree of homology inevitably entails problems to reliably separate members of a gene family on the level of the proteins. It is thus mandatory to pinpoint problematic cases and definitively clarify them to avoid the risk of misclassification. We herein accordingly deal with a special conundrum from the realm of animal lectins, i.e. the galectins. This class of lectins is receiving increasing attention, and for good reason. Galectins are potent regulators of adhesion, cell migration, growth control and tissue invasion [START_REF] Gabius | Cell surface glycans: the why and how of their functionality as biochemical signals in lectin-mediated information transfer[END_REF]. Their large impact on cell sociology is a key factor to explain why galectins are becoming attractive models for the study of divergent evolution. Toward this end, the proto-type proteins (non-covalent homodimers or monomers) of chicken are an appealing choice, because their group size is considerably smaller than in mammals [START_REF] Gabius | Animal lectins[END_REF][START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF].

On the grounds of electrophoretic mobility or chronological order of detection, the embryonic pectoral or thigh muscle/adult liver form was referred to as C-16 or CLL-I (16 kDa; chickenlactose lectin, later named chicken galectin CG-16), the protein isolated from embryonic skin or adult intestine as C-14 or CLL-II, then CG-14 [START_REF] Den | Developmentally regulated lectin from embryonic chick pectoral muscle. Purification by affinity chromatography[END_REF][5][START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF][START_REF] Pitts | Isolation of a developmentally regulated lectin from chick embryo[END_REF][START_REF] Beyer | Quantitation of two endogenous lactoseinhibitable lectins in embryonic and adult chicken tissues[END_REF][START_REF] Oda | Purification and characterization of β-galactosidebinding lectin from chick embryonic skin[END_REF][START_REF] Levi | Isolation and characterization of chicken thymic electrolectin[END_REF]. Analysis of sequences from liver and skin chicken galectins (CGs) with an eye on phylogenesis suggested that they have likely diverged after, but not far away from the separation of the lines for birds and mammals [START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF][START_REF] Hirabayashi | Complete amino acid sequence of 14 kDa β-galactoside-binding lectin of chick embryo[END_REF][START_REF] Sakakura | Structure of chicken 16-kDa β-galactoside-binding lectin. Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin[END_REF][START_REF] Cooper | Galectinomics: finding themes in complexity[END_REF]. As a consequence of the ensuing sequence evolution, not only organ patterns of expression but also biochemical properties such as the isoelectric points acquired individual attributes. In detail, the pI value of CG-16 was determined to be between 4.1 and 4.5, one report indicating focusing in the acidic range without satisfactory reproducibility [START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF][START_REF] Barak Briles | Vertebrate lectins. Comparison of properties of β-galactoside-binding lectins from tissues of calf and chicken[END_REF][START_REF] Castagna | Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina[END_REF][START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF]. In contrast, the isoelectric point of CG-14 from embryonic chicken skin was 7.0 [START_REF] Oda | Purification and characterization of β-galactosidebinding lectin from chick embryonic skin[END_REF], that of CG-14 from intestine between 6.3 and 6.7 [START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF][START_REF] Castagna | Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina[END_REF][START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF], thus somewhat but not decisively different. Gel filtration and haemagglutination revealed only CG-16 to be completely homodimeric, what likely has a bearing on differential CG activities on cells [START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF][START_REF] Levi | Isolation and characterization of chicken thymic electrolectin[END_REF][START_REF] Castagna | Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina[END_REF][START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF][17][18][START_REF] Kakita | High-performance affinity chromatography of a chick lectin on an adsorbent based on hydrophilic polymer gel[END_REF][START_REF] Kopitz | Hippocampal neurons and recombinant galectins as tools for systematic carbohydrate structure-function studies in neuronal differentiation[END_REF][START_REF] André | Determination of structural and functional overlap/divergence of five prototype galectins by analysis of the growth-regulatory interaction with ganglioside GM 1 in silico and in vitro on human neuroblastoma cells[END_REF].

Fittingly, immunological cross-reactivity had so far not been reported between CG-16 and the CG-14 from intestine, one study on CG-14 from embryonic skin yet picking up reactivity [START_REF] Beyer | Quantitation of two endogenous lactoseinhibitable lectins in embryonic and adult chicken tissues[END_REF][START_REF] Castagna | Isolation and characterization of a soluble lactose-binding lectin from postnatal chicken retina[END_REF]22,[START_REF] Lips | Correspondence of gradual developmental increases of expression of galectinreactive glycoconjugates with alterations of the total contents of the two differentially regulated galectins in chicken intestine and liver as indication for overlapping functions[END_REF]. Reservations against using cross-reactivity as strong argument for protein classification notwithstanding, it is noteworthy that antibody preparations against CG-14 from the two tissue types generated disparate immunohistochemical staining of the neural tube and only the antiserum against intestinal CG-14 was free of binding activity against [START_REF] Didier | Different immunoreactivities of anti-soluble lactose lectin antisera to tissues from early chick embryos: a histochemical study[END_REF][START_REF] Guay | Expression of the 14 kDa and 16 kDa galactosidebinding lectins during differentiation of the chick yolk sac. Roux's[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

These data on pI and antibody reactivity raised suspicion that the assumed identity among CG-14 proteins from embryonic skin and adult intestine might not be valid. Actually, this concern was nourished by the recently noted existence of a putative gene for a third prototype galectin in the chicken genome. Separated from CG-14/-16, which are closely related to human galectin-1, this newly detected sequence was assigned to the same branch of the phylogenetic tree as mammalian galectins-2 and tentatively coined as chicken galectin-2 (CG-2) [START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF]. Whether a so far not detected CG-2 protein is really produced or whether the intestinal CG-14 form, which so far had not been sequenced, may have inadvertently been misclassified were open questions. To answer them we cloned and expressed CG-2, then set up a reliable procedure to distinguish CG-2 from the recombinant CG-14 and tested the intestinal protein. CG-2, as it turned out, is identical with the intestinal protein and shares quaternary structure with human galectin-2. Its hydrodynamic shape parameters measured by ultracentrifugation, however, are unusual for a homodimeric galectin. Next, we proceeded to examine genomic sequences of the proximal promoter regions for the three CGs as well as to measure their expression profiles by RT-PCR/Western blot analyses and immunohistochemistry. In sum, this study defines a third proto-type chicken galectin with characteristic hydrodynamic behaviour and distribution in several types of organs of adult animals. The presented data give reason to revise the current nomenclature.

EXPERIMENTAL

Cloning, Protein Expression and Purification. Total RNA from embryonic chicken kidney (developmental day 15) was isolated using the RNAeasy kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and 2.5 µg were used for first-strand cDNA synthesis. Reverse transcription was performed using a routine protocol [START_REF] Kopitz | Hippocampal neurons and recombinant galectins as tools for systematic carbohydrate structure-function studies in neuronal differentiation[END_REF], and PCR amplification to obtain full-length cDNA was based on specific primer design according to the entry no. CF251077 in the NCBI dbEST database, resulting in the following sequences: sense primer 5′-CCATGGCTAGAATGTTTGAAATGTTCAACCTGG-3′ with an internal NcoI restriction site (underlined) and antisense primer 5′-GGATCCTCACTCCACCTTGAAGGAGGTAAC-3′ with an internal BamHI restriction site (underlined). The encoded full-length protein was expressed in the E. coli strain M15

[pREP4] with the vector PQE60 (Qiagen, Hilden, Germany) at optimal conditions yielding approximately 11.5 mg CG-2 from 1.2 l TB medium (Roth, Karlsruhe, Germany) using four separate one litre flasks, induction with isopropyl-β-thio-D-galactopyranoside at a final concentration of 0.4 mM and a shift of temperature from 37 °C during initial growth to 30 °C. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Lectin purification from extracts after sonification and centrifugation was performed with affinity chromatography on lactosylated Sepharose 4B, obtained after divinyl sulfone activation and ligand conjugation, as crucial step [START_REF] André | Determination of structural and functional overlap/divergence of five prototype galectins by analysis of the growth-regulatory interaction with ganglioside GM 1 in silico and in vitro on human neuroblastoma cells[END_REF]. The recombinant forms of CG-14 and CG-16 were similarly produced after cloning of the respective cDNAs, and these proteins as well as the lectin from fresh chicken intestinal tissue were purified to homogeneity as described in detail previously [START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF][START_REF] Kopitz | Hippocampal neurons and recombinant galectins as tools for systematic carbohydrate structure-function studies in neuronal differentiation[END_REF].

Analytical Procedures. Gel electrophoretic analysis was carried out in the one-dimensional setting using either 4% stacking/15% running gel parts or a 4-15% linear gradient system as well as in the two-dimensional system, as described previously [START_REF] Purkrábková | New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites[END_REF]. Silver staining visualized the protein spots, the measured pI values of the chicken galectins were compared computationally with the results of theoretical calculations using the respective programme of the experiments were run at 45,000 rpm and 20 °C using 400 µl samples as described [START_REF] Jiménez | Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Galectin-dependent staining of human Capan-1 pancreatic carcinoma cells with reconstituted expression of the tumour suppressor p16 INK4a , kindly provided by Dr. K. M. Detjen (Berlin, Germany), was analysed by flow cytofluorometry using streptavidin/R-phycoerythrin as fluorescent marker (1:40; Sigma, Munich, Germany) with biotinylated galectins (10 µg/ml) [START_REF] André | Tumor suppressor p16 INK4a : modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells[END_REF]. Protein labelling by the N-hydroxysuccinimide ester of biotin (Sigma, Munich, Germany) under activity-preserving conditions, assays ascertaining lectin activity after labelling and determination of extent of biotinylation followed optimized protocols [START_REF] André | Determination of structural and functional overlap/divergence of five prototype galectins by analysis of the growth-regulatory interaction with ganglioside GM 1 in silico and in vitro on human neuroblastoma cells[END_REF][START_REF] Purkrábková | New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites[END_REF][START_REF] André | Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins and wild-type and lectin-genetransfected tumor cells as targets[END_REF]. Carbohydrate-dependent lectin binding to cell surfaces was determined in the presence and absence of a mixture of lactose (75 mM) and the glycoprotein asialofetuin Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

(1 mg/ml
listed in the database. Stringency of screening was compensatorily increased by adjusting the cut-off to the most restricted value to limit occurrence of false-positive cases.

Expression Profiling by RT-PCR, Western Blotting and Immunohistochemistry. RT-PCR

analysis with cDNA preparations from tissues was performed with the primer sets given above to produce the 396 bp product in the case of CG-2 and a published set for CG-14 resulting in a 411 bp product [START_REF] Kopitz | Hippocampal neurons and recombinant galectins as tools for systematic carbohydrate structure-function studies in neuronal differentiation[END_REF]. Non-cross-reactive polyclonal antibody preparations were obtained after immunizing rabbits and removing any contaminating cross-reactive material from the immunoglobulin (Ig) G fractions by chromatographic affinity depletion with the respective galectin as matrix-conjugated ligand, as described for mammalian lectins [START_REF] Lensch | Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence[END_REF].

Complete removal of cross-reactivity was ascertained by ELISAs and Western blot assays.

The resulting IgG preparations were applied in Western blotting of tissue extracts and immunohistochemical processing using fluorescence microscopy, where rigorous specificity controls to spot any antigen-independent staining were included, as described [START_REF] Lensch | Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence[END_REF][START_REF] Lohr | Murine homodimeric adhesion/growthregulatory galectins-1, -2 and -7: comparative profiling of gene/promoter sequences by database mining, of expression by RT-PCR/immunohistochemistry and of contact sites for carbohydrate ligands by computational chemistry[END_REF].

RESULTS AND DISCUSSION

Mass profiling of CG-2. After cloning and recombinant expression of CG-2, its properties were first compared to those of CG-14 and CG-16 by one-and two-dimensional gel electrophoresis. Using running gels with either a fixed acrylamide concentration (15%) or a linear gradient (4-15%), the known difference between CG-14 and CG-16 served as internal quality control. Molecular masses of 14,030.9 Da (CG-14) and 16,639.2 Da (CG-16) with standard deviations of 3.6% and 4.4%, respectively, were determined (Figure 1). When run lane-by-lane in the same gels, the electrophoretic mobility of CG-2 was consistently slightly lower than that of recombinant CG-14 in both gel systems (Figure 1). The difference did not reach the level of statistical significance (p = 0.11). Two-dimensional gel electrophoresis could barely resolve a mixture of the two galectins. A slight disparity between the isoelectric points was measured (Figure 2). Its size reflects the theoretical difference between calculated pI values of 6.51 (CG-2) and 6.58 . This accordance excludes presence of a chargealtering posttranslational modification in the recombinant products, as also previously noted for the galectin purified from 100 g chicken intestine with a yield of 2.5 mg ± 0.5 mg (n = 6) [START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF]. If now attempting to identify the nature of the intestinal lectin on this experimental basis, any conclusion would inevitably possess ambiguity. We therefore performed tryptic fingerprinting combined with mass spectrometric peptide characterisation. Because the positions of Arg/Lys residues are not identical in the sequences of both galectins (please see and tryptophan (W) and of internal disulfide formation were encountered in the analysis as registered in Table 1. Using the proven diagnostic power of fingerprinting, the question on the nature of the intestinal lectin was answered: its mass spectrum was identical to that of recombinant CG-2, all detected peptides from the lectin purified from intestinal tissue having matching partners present in the sequence of CG-2 (Table 1; Supplementary Material, Figure 1). Of note, the previously determined mass of 14,969 Da for the intestinal protein [START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF] describes the N-terminus as acetylated alanine (theoretical value at 14,970.04 Da relative to 14,974.98 of CG-14 with acetylated serine). Except for a processed N-terminus no other sequence modification is present, in line with the protein's properties in two-dimensional gel electrophoresis regardless of the source of the protein (recombinant production or intestine).

Thus, the proto-type galectin CG-14 purified from embryonic skin and the intestinal galectin originate from two different genes. This finding is readily reconcilable with the immunological evidence for a difference between the skin and intestinal lectins, outlined in the introduction. It also defines the intestinal protein as natural product of the gene previously detected by database mining [START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF]. The implied phylogenetic vicinity to mammalian galectins-2 should be reflected in experimental data but the respective comparison, at present confined to gel filtration, revealed the following difference: human galectin-2 behaved as homodimer in gel filtration on a Superdex 75 HR 10/30 column [START_REF] Gitt | Isolation and expression of a gene encoding L-14-II, a new human soluble lactose-binding lectin[END_REF], while elution properties of a monomer were reported for the protein from chicken intestine on Sephadex G-100 [START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF]. This apparent disparity between two closely related proteins merits thorough inspection.

Quaternary structure of CG-2 and cell binding features. We first turned to gel filtration reanalysing CG-2 in a HPLC system. In order to avoid any carbohydrate-inhibitable interaction of a lectin with the resin, the analyses were also performed in the presence of 100 mM lactose.

Homodimeric CG-16/human galectin-1 were added as internal standards. Using a loading concentration of 1.2 mg/ml (estimated eluted concentration of 0.12 mg/ml), the elution time for CG-2 was 29.4 ± 0.1 min. This value corresponds to an apparent molecular mass of 16.9 G-100 column (1.5 x 30 cm) [START_REF] Beyer | Two lactose binding lectins from chicken tissues. Purified lectin from intestine is different from those in liver and muscle[END_REF]. The authors of that report had suggested that the measured M r of 14.0 ± 1.7 kDa indicated a monomeric status. If this were the case, the two galectin-2type proteins would not share quaternary structure. In order to resolve this critical issue, CG-2 was studied by analytical ultracentrifugation.

Sedimentation equilibrium data obtained for CG-2 in solutions at loading concentrations between 0.1 mg/ml and 1 mg/ml could always be fitted to a single ideal component with a weight-average molecular mass of 29.0 ± 0.85 kDa. The observed mass was virtually independent of rotor speed, indicating that the sample was homogeneous, and was not influenced by the presence of 0.1 M lactose. For comparison, apparent molecular masses of 30.6 ± 1.0 kDa and 27.3 ± 1.5 kDa were obtained for CG-16 and human galectin-1, respectively, when examined under the given conditions. Thus, in sedimentation equilibrium analysis, in the range of concentrations tested, CG-2 behaved entirely as a dimer. Its shape was studied by sedimentation velocity analysis.

These experiments at a CG-2 concentration of 0.5 mg/ml revealed a predominant peak with a sedimentation coefficient of 2.03 ± 0.1 S (2.08 S for s 0 20,w , expressed in terms of the standard solvent of water at 20 °C). Comparable results were obtained in the absence or presence of 0.1 M lactose. Respective analysis of the homodimeric CG-16 and human galectin-1 gave significantly higher sedimentation coefficients (2.6 ± 0.1 S and 2.5 ± 0.1 S, respectively) than the one measured for CG-2. An estimation of the hydrodynamic size of hydration of the proteins was enabled by using the Sednterp programme. The partial specific volume and degree of hydration of the proteins were calculated from the amino acid composition. The resulting s 0 20,w value of CG-2 was higher than the maximally possible sedimentation coefficient predicted for a particle with the mass of a monomer. On the other hand, the ratio between the experimental frictional coefficient (f ) and the minimum frictional coefficient of a compact anhydrous sphere with the mass and volume of the CG-2 dimer (f 0 ) was 1.63. For CG-16 and human galectin-1, frictional ratios were in the order of 1.3, indicating that CG-2 does not behave as a globular but as a, for a galectin, unusually extended protein under these conditions. Accordingly, approximation of the shape of CG-2 to prolate or oblate ellipsoids of revolution gave axial shape ratios of 7.7 and 9.0. These numbers are above two-fold higher than those estimated for CG-16 and human galectin-1. The apparent retardation in gel filtration might be explained by a weak interaction of CG-2 with the two types of matrix used irrespective of the presence of lactose and/or a preferential orientation of CG-2 to access the porous beads. Taken together, the sedimentation analysis proved that CG-2 is a homodimer with a peculiar hydrodynamic behaviour. Its cell-binding capacity was analyzed next.

Compared to CG-16, a very potent cross-linker active at a minimal concentration of 10 ng/ml, CG-2 led to carbohydrate-inhibitable agglutination at 160 ng/ml, confirming its bivalency. We next tested binding to a human cell system of marked reactivity for galectins [START_REF] André | Tumor suppressor p16 INK4a : modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells[END_REF].

Concentration-dependent and carbohydrate-inhibitable binding was ascertained to validate the system (Figure 3A,B). Despite the marked contrast in hydrodynamic properties, a similarity was observed between data for CG-2 and CG-16, with CG-14 showing less reactivity (Figure 3B-D). On the level of avian immune cells, however, CG-2 preferentially bound to B cells, whereas CG-16 stained different lymphocyte populations uniformly [START_REF] Schneller | Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells[END_REF]. These results strengthen the notion of distinct functional profiles for different cell types. In this case, gene organization and expression profiles of the three proto-type galectins should have dissimilar properties.

Profiling of CG-2 gene structure. The gene for CG-2 is found on chromosome 1, approximately 42 kb upstream of the CG-14 gene, but in opposite orientation. A similar arrangement is seen in the cases of mammalian galectins-1 and -2. Remarkably, the coding sequence for CG-16 is located on chromosome 4. On the level of exons/introns the CG-2 gene is organized into four exons, a common trait of proto-type galectins. The lengths of the first and third exons, the latter one comprising the carbohydrate recognition domain, are identical or very close to those of human galectin-2 with six and 159 base pairs vs. six and 160 base pairs, respectively (Supplementary Material, Figure 2). CG-14 as well as CG-16 and human galectin-1 share the length of nine base pairs in the first exon and have 172 base pairs in the third exon. Admittedly, the corresponding data on the second and fourth exons do not entirely follow this two-group pattern. Based on sequence comparison, the level of divergence justifies assignment of CG-2 and CG-14/CG-16 to two separate branches of a phylogenetic tree [START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF].

Availability of the genomic sequence afforded comparative promoter analysis. Separately combing through the proximal promoter region of the CG-2 gene with two search algorithms to spot putative binding sites uncovered a panel of hits (Supplementary Material, Figure 3). In the same systematic manner we processed the corresponding regions of gene sequences in the cases of CG-14 and CG-16 (for detailed listing of putative sites, please see Supplementary ). This first comparative analysis of promoter sequences for non-mammalian galectins came up with further support that distinct, functionally relevant properties were acquired by sequence divergence of the proto-type galectins. Pronounced differences concern unique presence of potential binding motifs for AP-2, p53, GATA-3 and the common coactivators c-Rel and p300 in the CG-2 promoter, as well as comparatively frequent occurrence of sites for the activating homeobox protein cdx-1 and the myeloid zinc finger protein MZF-1. In contrast, CG-2's proximal promoter region lacks presence of site(s) potentially reactive with the octamer factor POU2F1/Oct-1. A similar picture emerges in the case of CG-14, its promoter region uniquely harbouring motifs for the common transactivator CP2, Oct-6, v-Maf, the co-activator YY1 (Yin and Yang 1), a glucocorticoid-responsive element and a barbiturate-inducible box (Supplementary Material, Table ).

S t a g e 2 ( a ) P O S T -P R I N T Material, Table

Expression profiling of CG-2.

To preclude false-negative results, we started at the highest level of sensitivity with RT-PCR analysis using specific primer sets. As internal controls, samples from skin with abundant expression of CG-14 and from intestine, a rich source for CG-2, were included. Indeed, strong signals in RT-PCR gels were recorded in these cases at expected positions (Supplementary Material, Figure 4). Presence of galectin-type-specific mRNA, at least in minute quantities, was also detected in extracts of other organs after running 35 amplification cycles (Supplementary Material, Figure 4). To separate spurious expression from cases with robust protein production, tissue extracts were processed by Western blotting. Because systematic ELISAs using the three IgG fractions from immunized rabbits revealed cross-reactivity, e.g. anti-CG-14 binding CG-16 and anti-CG-2 reacting both with CG-14 and CG-16, albeit at a low level, affinity depletion with resin-immobilized galectins had to be performed. Its success was rigorously controlled, using complete removal as criterion. As internal control in Western blots, the processed antibody preparations were routinely confronted with purified galectins and, additionally, we included a loading control by visualizing actin (Figure 4). As documented in the right part of Figure 4, CG-2 presence was not observed in skin and lung, a qualitative difference to CG-14. On the other hand, jejunum proved to be a rich source of CG-2. The two structurally closely related galectins are thus without doubt immunologically distinct, their expression profiles also revealing quantitative and qualitative differences.

In order to take our analysis from the level of extracts to that of individual cell types, fixed sections of organs of adult animals were immunohistochemically processed. After first defining optimal conditions for tissue preservation and staining quality, the ensuing antibody application resulted in readily assignable positivity (Table 2). Without exception, the results epidermis with already documented regional separation for CG-14 (Table 2). The same marked cell-type specificity was seen for CG-2 (Table 2, Figure 5). Epithelial lining of villi in the gut and collecting ducts in kidney were strongly positive. Examining the staining of cells more closely, cytoplasmic and nuclear lectin localizations were discerned (Table 2, Figure 5).

Encouraged by these discriminatory results we extended the scope of our analysis to CG-16.

Again, affinity depletion of the IgG fraction was required due to its reactivity with CG-2.

Even in cases of simultaneous presence of two proto-type galectins in an organ such as kidney, the individual patterns of localization were qualitatively different when examined by immunohistochemistry (Table 2). This compiled evidence identified no overlap between proto-type chicken galectins on the level of cell-type expression in organs of adult animals. Of course, presence of a fourth member in this galectin group with features deviating from this picture would quash the given inference. To address this issue we carried out extensive database mining on the current version of the chicken genome using a suitably tailored consensus sequence. No evidence for existence of a further gene for a proto-type galectin was unearthed. This result solidifies the notion that CG-2 together with CG-14 and CG-16 and no further protein comprise the group of proto-type chicken galectins.

Implications for nomenclature and perspectives. It is now timely to suggest a nomenclature, which reflects our current status of understanding phylogenetic relationships as given in [START_REF] Houzelstein | Phylogenetic analysis of the vertebrate galectin family[END_REF],

and not electrophoretic mobilities: CG-16/-14 are therefore suggested to be called CG-1A/B to signal their vicinity to galectin-1. As predicted by this concept, chemical mapping using synthetic derivatives of lactose had revealed closer similarity of CG-16 (CG-1A) to human galectin-1 than to the intestinal lectin CG-2 [START_REF] Solís | S t a g e 2 ( a ) P O S T -P R I N T Different architecture of the combining site of the two chicken galectins revealed by chemical mapping studies with synthetic ligand derivatives[END_REF]. Thus, recalling the broad range of effector functions of galectins via ligand binding [START_REF] Gabius | Cell surface glycans: the why and how of their functionality as biochemical signals in lectin-mediated information transfer[END_REF], diversity in lectin evolution has the potential to expedite establishment of a finely tuned system to turn sugar coding into specific biosignaling, and initial work on oligosaccharides and natural glycoproteins to map binding profiles of proto-type chicken galectins already tracked down differences [START_REF] Wu | Carbohydrate specificity of a galectin from chicken liver (CG-16)[END_REF][START_REF] Hirabayashi | Oligosaccharide specificity of galectins: a search by frontal affinity chromatography[END_REF][START_REF] Gabius | Chemical biology of the sugar code[END_REF][START_REF] Villalobo | A guide to signaling pathways connecting protein-glycan interaction with the emerging versatile effector functionality of mammalian lectins[END_REF][START_REF] Wu | Activity-structure correlations in divergent lectin evolution: fine specificity of chicken galectin CG-14 and computational analysis of flexible ligand docking for CG-14 and the closely related CG-16[END_REF]. The underlying structural basis will have to be defined by extending crystallographic analysis beyond CG-16 [START_REF] Varela | The 2.15 Å crystal structure of CG-16, the developmentally regulated homodimeric chicken galectin[END_REF].

On the level of gene regulation, our analysis was guided by ″the hypothesis that families of electrolectin genes could be under the control of different promoters″ [START_REF] Levi | Isolation and characterization of chicken thymic electrolectin[END_REF]. This first detailed analysis of putative sites for transcription factors in genes of avian galectins pinpointed a series of hits. Verifying the given prediction on the molecular level, a characteristic pattern is present in each proximal promoter region. How to relate the complex combinations of motifs to the actual expression profiles in organs of adult animals and also to developmental regulation will have to be addressed by functional dissection. At this stage, we took care by internal controls to ascertain the validity of the expression profiles, and previous results, e.g. on pectoral muscle [START_REF] Beyer | Localization of an endogenous lectin in chicken liver, intestine, and pancreas[END_REF], were in full accord with the presented expression profiling, in this case excluding a false-positive result. On the level of organs, zonally distinct galectin appearance determined by immunohistochemistry in organs of adult animals is a clear indication for acquisition of non-overlapping functions of the three proto-type proteins in the different cell types including nuclear and/or cytoplasmic presence. The case of epithelial kidney cells of proximal tubules with their positivity for both CG-2 and CG-16, which is limited to days 12 to 14 of incubation located at distinct subcellular sites, might even be interpreted along this line [START_REF] Stierstorfer | Temporal and spatial regulation of expression of two galectins during kidney development of the chicken[END_REF]. This finding and the need for further crystallographic data give further research on the three proto-type chicken galectins a clear direction.

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419

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  locate genomic sequences upstream to the site for initiation of transcription. Corresponding sequence information for genes of CG-14 (based on M11674) and CG-16 (AY553270) was retrieved from the same database, the promoter region of CG-16 still containing a stretch of unknown sequence between the positions -233 and -110. DNA sequences were edited using the EditSeq sequence analysis software version 4.0 (DNAstar Inc., Madison, WI, USA) as well as the Reformat and Map algorithms included in the GCG (Genetics Computer Group Inc. Sequence Analysis Software Package) programmes available from the HUSAR biocomputing service of the German Cancer Research Center (Heidelberg, Germany; http://genius.embnet.dkfz-heidelberg.de/). In order to scour proximal promoter regions for putative transcription-factor-binding sites and compare obtained results systematically the online versions of the programmes Match ™ (http://www.gene-regulation.com/cgibin/pub/programs/match/bin/match.cgi) and P-Match ™ (http://www.gene-regulation.com/cgibin/pub/programs/pmatch/bin/p-match.cgi) were selected, both using the latest update of the TRANSFAC ® transcription factor database as source for weight matrices and consensus sequences. Presettings were deliberately chosen to include "low quality" matrices to avoid excluding important factors (such as Sp1) for which only "low quality" weight matrices are S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

± 0. 1

 1 kDa for a globular protein. For comparison, the homodimeric CG-16 and galectin-1 had S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society elution times of 27.4 ± 0.1 min and 27.5 ± 0.1 min, respectively, corresponding to an apparent molecular mass of approximately 30 kDa. Further experiments with CG-2 concentrations of 0.5 mg/ml and 2.5 mg/ml to exclude an influence of concentration confirmed the marked deviation of CG-2 properties from those of the homodimeric galectins (not shown). All obtained results are in accord with the initial evidence from gel filtration on a Sephadex

S t a g e 2

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 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society of immunohistochemistry and Western blotting are in full accord. As examples for cell-type specificity, lung and skin presented strong signals in the respiratory epithelium and in

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  methionine or tryptophan by single oxidation (+16) or double oxidation (+32), S-S bond formation between internal cysteine residues. g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

1

 1 Figure 1 Electrophoretic mobility of the three proto-type chicken galectins Purified proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions in the presence of β-mercaptoethanol in a 15% running gel (A) or a 4-15% linear gradient gel (B). Positions of relevant marker proteins are given in the upper panels. Molecular masses were determined by the plots given in the bottom panels. Insets show the quantitative relationship between relative migration distance and known molecular mass of the six standard proteins (β-galactosidase -116 kDa, bovine serum albumin -66 kDa, ovalbumin -45 kDa, carbonic anhydrase -29 kDa, β-lactoglobulin -18.4 kDa, lysozyme -14.2 kDa).

Figure 2

 2 Figure 2Isoelectric points of the proto-type chicken galectins CG-2 and CG-14The relevant section of a gel after two-dimensional gel electrophoresis is shown, experimental assignment of pI values was performed using human galectins of known properties. The given section shows the two spots for CG-2 and CG-14 together with those of CG-16 (pI = 4.95)and human galectins-1 (pI = 5.34), -2 (pI = 5.93), and -7 (pI = 7.00).

Figure 3

 3 Figure 3 Cell surface staining by the three proto-type chicken galectins Semilogarithmic representation of fluorescent surface staining of p16 INK4a -reconstituted human Capan-1 pancreatic carcinoma cells by biotinylated chicken galectins. Concentration dependence is documented for CG-14 showing fluorescent staining after incubation with 2 µg/ml, 5 µg/ml, 10 µg/ml and 20 µg/ml biotinylated lectin (A). Staining profiles in the absence (solid line) and presence of glycoinhibitors (dotted line) are given for CG-2 (B), CG-14 (C) and CG-16 (D) at a constant lectin concentration of 10 µg/ml. The characteristics of background staining when using the fluorescent indicator without prior incubation of cells with a biotinylated marker are illustrated as reference in each panel as shaded area. Quantitative data on percentage of positive cells (%) and mean channel fluorescence are inserted into each panel.

Figure 4

 4 Figure 4Expression profiling of proto-type chicken galectins CG-2 and CG-14 byWestern blottingLectin presence was determined in tissue extracts (30 µg protein per lane) using non-crossreactive IgG fractions for CG-2 (A; 0.5 µg/ml) and CG-14 (B; 0.5 µg/ml). Specificity controls were run with 25 ng purified lectin per lane, and actin presence was probed as internal loading S t a g e 2 ( a ) P O S T -P R I N T

Figure 5

 5 Figure 5 Localization of proto-type chicken galectin CG-2 in tissue sections by immunohistochemistry and the comparison to CG-14 staining Microphotographs of a cross-section through a medullary cone of a kidney of a six-month old chicken at two levels of magnification (A-D). Control without incubation step using antiserum ascertained lack of antigen-independent staining (A). Application of the non-cross-reactive antibody preparation at 1 µg/ml revealed presence of CG-2 in epithelial cells of the collecting ducts, whereas sections of Henle's loop of mammalian type nephrons I were free of staining (B). On the cellular level, epithelial cells of collecting ducts (CD) were present with signal in cytoplasm and nuclei as well as at the membrane, in stark contrast to the negative cells in Henle's loops (LH) (C). The application of anti-CG-14 antibody under identical conditions yielded no signal (D). Corresponding processing of cross-section through jejunum of a sixmonth old chicken resulted in microphotographs, which are also shown at two levels of magnification (E-J). Lectin presence (CG-2) was confined to epithelial cells of villi and intestinal glands (E). Intense cytoplasmic staining characterizes the epithelial cells of villi (F) and intestinal glands (G). In contrast, CG-14 was localized in the connective tissue (lamina propria mucosae), as shown in a serial section to allow direct comparison (H-J). Bar lengths: 20 µm: A, C, D, F, G, I, J; 50 µm: B; 250 µm: E, H. S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 S

 1 Figure 1

Figure 2 S

 2 Figure 2

Figure 3 S

 3 Figure 3

Figure 4 S

 4 Figure 4

Figure 5 S

 5 Figure 5

Table 1 ,

 1 bottom), this method should allow to definitively decide on the nature of the intestinal galectin. Indeed, the data obtained bear out this expectation: two clearly different profiles were recorded (Table1; please see Supplementary Material, Figure1, for spectra). Sequence coverage by the detected peptides, as illustrated by shading the respective sequence stretches in the bottom part of Table1, was 67% or 63%, respectively. The individual characteristics of these peptides are compiled in Table1. Cases of oxidative modifications of methionine (M)

Table 1 Characteristics of the galectin-derived peptides identified by mass spectrometry

 1 For peptide numbering, please see Figure1, Supplementary Material. Detected sequences for each of the two galectins from recombinant production are highlighted. The identified peptide sequences of the intestinal galectin are pointed out by underlining them in the sequence part below, revealing an identical pattern to that of CG-2.

	Peak	m/z	Modifications* Missed	Database Sequence	Position	Position	Intestinal
	No.			Cleavages		in	in	Galectin
						rCG-14	rCG-2
	1	911.62	M+16	0	SFVMNLGK	30-37
	2	995.52	0	0	ETVFPFQK	75-82	-	-
	3	1165.50	0	0	MEEWGTEQR	66-74	-	-
	4	1181.47	M+16	0	MEEWGTEQR	66-74	-	-
	5	1293.55	0	1	KMEEWGTEQR	65-74	-	-
	6	1309.52	M+16	1	KMEEWGTEQR	65-74	-	-
	7	1393.64	0	0	DSTHLGLHFNPR	38-49	-	-
	8	2103.02	0	0	LGLSVFDYFDTHGDFTLR	112-129	-	-
	9	2270.16	0	1	SFVMNLGKDSTHLGLHFNPR	30-49		-
	10	2286.11	M+16	1	SFVMNLGKDSTHLGLHFNPR	30-49	-	-
	11	2691.27	0	1	LGLSVFDYFDTHGDFTLRSVSWE	112-134	-	-
	12	3191.59	0	0	GAPIEITFSINPPSDLTVHLPGHQFSFPNR 83-111	-	-
	13	1224.92	0	0	ISYLNILGGFK	-	113-123	+
	14	1256.88	0	0	SSDLALHFNPR	-	38-48	+
	15	1360.81	0	0	MFEMFNLDWK	-	3-12	+
	16	1376.79	M+16	0	MFEMFNLDWK	-	3-12	+
	17	1392.78	2 x M+16	0	MFEMFNLDWK	-	3-12	+
	18	1409.90	0	0	LPDGHEVEFPNR	-	97-108	+
	19	1791.08	0	0	GHISEDAESFAINLGCK	-	22-38

Table 2 Immunohistochemical profiling of presence of the proto-type chicken galectins CG-2, CG-14 and CG-16 in various organs of adult animals

 2 The intensity of staining is grouped into categories: -no staining, + weak staining, ++ medium staining, +++ strong staining. a only cytoplasmic, b cytoplasmic and nuclear,

	c MNT-I: mammalian-type nephron I (juxtamedullar), MNT-II: mammalian-type nephron II
	(midcortical), RTN: reptilian-type nephron (superficial).
	type of organ	type of galectin
		CG-2 CG-14 CG-16
	Larynx
	respiratory epithelium	-	-	++ a
	l. propria mucosae	-	+	-
	Trachea
	respiratory epithelium	-	-	++ a
	l. propria mucosae	-	++	-
	Lung
	respiratory epithelium	-	+++ a	-
	connective tissue	-	+	-
	Esophagus
	l. propria mucosae	-	++	-
	Gut
	epithelial lining of villi	+++ a	-	-
	and intestinal glands
	l. propria mucosae	-	+	-
	Liver
	hepatocytes (parenchyma)	-	-	+++ a
	Kidney
	epithelium
	collecting ducts (medulla)	++ b	-	-
	proximal/distal tubules	-	-	+++ a
	(MTN I, MTN II, RTN) c
	Skin
	epidermis
	stratum corneum	-	-	-
	stratum intermedium	-	+++ b	-
	stratum basalis	-	+ a	-
	dermis	-	++	-
	subcutis	-	+	-

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Sep 2007 as manuscript BJ20070419 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070419 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We are grateful to Prof. Dr. K.-i. Kasai for his expert advice and approval of the suggested nomenclature, to Dr. B. Friday and Dr. S. Namirha for inspiring discussions, to the reviewers of our manuscript for their valuable input, to A. Helfrich, B. Hofer, L. Mantel and S. Fiedler for skilful technical assistance as well as to the Spanish Dirección General de Investigación (BQU2003-03550-C03-03 and BFU2006-10288/BMC), to the research initiative LMUexcellent, to the Verein zur Förderung des biologisch-technologischen Fortschritts in der Medizin e.V. and to an EC Marie Curie Research Training Network grant (contract no. MRTN-CT-2005-019561) for generous financial support.


Silke Schilling

Christine Oesterhelt

Structurally reduced monosaccharide transporters in an evolutionary conserved red alga

Keywords: molecular biology, gene regulation, sugar transport Cyanidiaceae, Galdieria, polyol transport, Rhodophyta, sugar transport Biochemical

The unicellular red alga Galdieria sulphuraria is a facultative heterotrophic member of the Cyanidiaceae, a group of evolutionary highly conserved extremophilic red algae. Uptake of various sugars and polyols is accomplished by a large number of distinct plasma membrane transporters. We have cloned three transporters (GsSPT1, GsSPT2, GsSPT4), followed their transcriptional regulation and assayed their transport capacities in the heterologous yeast system. SPT1 is a conserved type of sugar / H + symporter with twelve predicted transmembrane spanning domains, while SPT2 and SPT4 represent monosaccharide transporters, characterized by only nine hydrophobic domains. Surprisingly, all three proteins are functional plasma membrane transporters, as demonstrated by genetic complementation of a sugar uptake-deficient yeast mutant. Substrate specificities were broad and largely redundant, except for glucose which was only taken up by SPT1. Comparison of SPT1 and truncated SPT1( 1-3) indicated that the N-terminus of the protein is not required for sugar transport or substrate recognition. However, its deletion affected substrate affinity as well as maximal transport velocity and released the pH dependency of sugar uptake. In line with these results, uptake by SPT2 and SPT4 was active but not pH-dependent, making a H + symport mechanism unlikely for the truncated proteins. We postulate SPT2 and SPT4 as functional plasma membrane transporters in G. sulphuraria. Most likely, they originated from genes encoding active monosaccharide / H + symporters with twelve transmembrane spanning domains.

INTRODUCTION

Unicellular algae are ideal candidates to study transport processes across the plasma membrane. The hexose uptake machinery (HUP1, HUP2, HUP3) of the green alga Chlorella kessleri was the first to be characterized within the green plant lineage and it still serves as a model for conserved hexose transport in higher plants [1 -6]. However, sugar transport within the branch of non-green plants is not well characterized. Cyanidiaceae, a group of evolutionary conserved, unicellular acidophilic red algae, have been described as the oldest extant members of the red lineage and are most likely located at the basis of secondary endosymbiosis [START_REF] Yoon | The single, ancient origin of chromist plastids[END_REF][START_REF] Yoon | A molecular timeline for the origin of photsynthetic eukaryotes[END_REF], an event that was dated back some 1260 +/-30 million years and gave rise to the highly diverse group of chromalveolates [START_REF] Bhattacharya | Photosynthetic eukaryotes unite: endosymbiosis connects the dots[END_REF]. Following current taxonomy, three genera are classified as Cyanidiaceae: Cyanidium, Cyanidioschyzon, and Galdieria [START_REF] Albertano | The taxonomic position of Cyanidium, Cyanidioschyzon and Galdieria: an update[END_REF][START_REF] Ciniglia | Hidden biodiversity of the extremophilic Cyanidiales red algae[END_REF].

These algae have a cosmopolitan distribution but are restricted to habitats with pH values between 0.05 and 3 and temperatures not above 56°C. Due to a high sensitivity towards desiccation, cells often grow endolithically and, as a result, are subjected to severe light limitation, hampering autotrophic growth [START_REF] Gross | Cryptoendolithic growth of the red alga Galdieria sulphuraria in volcanic areas[END_REF][START_REF] Gross | Ecophysiological studies on the red alga Galdieria sulphuraria isolated from South-West Iceland[END_REF]. G. sulphuraria can survive periods of light limitation by metabolizing carbohydrates (disaccharides, hexoses, pentoses, deoxysugars), polyols (hexitols, pentiols, tetriols and triols), amino acids and organic acids [14 -16]. Many sugars are taken up and metabolized in the D-as well as in the L-configuration [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF][START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]. This large spectrum of substrates for heterotrophic growth is unique among eukaryots and, therefore, G. sulphuraria is well suited as a model for sugar transport in the non-green plant lineage, comprising such diverse groups as the cryptomonades, haptophytes, heterokonts, dinoflagellates and apicomplexa [START_REF] Archibald | Recycled plastids: a 'green movement' in eukaryotic evolution[END_REF].

In vivo studies with intact cells indicated at least 14 different sugar and polyol transporters with a pronounced pH dependency and a partly overlapping spectrum of substrates [START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]. However, up to now, a detailed characterization of the uptake machinery for sugars and polyols in G. sulphuraria has been hampered by a lack of genomic sequence information and thus access to individual transporters. The recent initiation of genome projects for Galdieria sulphuraria [START_REF] Weber | EST-analysis of the thermoacidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts[END_REF] (http://genomics.msu.edu/galdieria/index.html) and the obligate autotrophic Cyanidioschyzon merolae [20] (http://merolae.biol.s.u-tokyo.ac.jp/) has now made it possible to identify putative sugar transporters encoded by these red algal genomes. Comparative genomics of the physiologically very different Cyanidiaceae revealed surprisingly few differences. One of them is a large number of putative sugar transporters, likely up to 28, encoded only by G. sulphuraria [START_REF] Barbier | Comparative genomics of two closely related unicellular thermoacidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of G. sulphuraria and significant differences in carbohydrate metabolism of both algae[END_REF]. In this paper, we report the cloning of three monosaccharide and polyol transporters of G. sulphuraria. We follow their transcriptional regulation, and characterize the functions of these proteins in the heterologous yeast system.

Biochemical

EXPERIMENTAL

Cultivation of algae

Galdieria sulphuraria strain 074G (Mt. Lawu, Java, Indonesia [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF]) was cultivated at ambient temperature in inorganic salt medium at pH 2 [START_REF] Ford | Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga Cyanidium caldarium Geitler. Purification, characterisation and thermostability of the enzyme[END_REF]. Autotrophic cultures were illuminated with white incandescant light (120 µE m -2 s -1 ) and supplied with sterile air enriched with 2% CO 2 from the bottom of the flask. Heterotrophic cultures were kept in the dark on a rotary shaker (120 rpm) and supplied with 25 mM organic substrate. Cell growth was monitored by following the optical density of the culture at 800 nm. For quantification, cells were diluted to give a reading between 0.05 and 0.15.

Extraction of RNA

Cells were harvested by centrifugation at 3.000 g and washed twice with 20 mM Tris-HCl, pH 7.5. The cell pellet was either stored at -20°C or immediately used for extraction. Cells (1 g fw) were disrupted in 4 ml of lysis buffer (100 mM Tris-HCl, pH 8.6, 2% (w/v) Sarkosyl, 25 mM EDTA, 25 mM EGTA, 100 mM ß-mercaptoethanol) using a bead beater (Biospec.

Products, Bartlesville, USA) operated for one minute at 4°C. Cell debris and membranes were removed at 35,000 g (15 min). RNA was isolated according to Sheen and Bogorad [START_REF] Sheen | Differential expression of the ribulose bisphosphate carboxylase large subunit gene in bundle sheath and mesophyll cells of developing maize leaves is influenced by light[END_REF]. It was resuspended in a suitable volume DEPC-H 2 O, precipitated over night at 4°C with 0.5 volume 6 M LiCl and collected at 14,000 g (20 min, 4°C), followed by a second precipitation with 2.5 volumes 96% ethanol (1 h, -20°C) and centrifugation at 14,000 g. The pellet was washed twice in 70% ethanol, dried and resuspended in DEPC-H 2 O.

Northern blot analysis

30 µg of RNA were separated on 1.4% (w/v) agarose-formaldehyde gels and transferred to Hybond-N+ nylon membrane by capillary blotting. Hybridization was carried out in 0.12 M Na 2 HPO 4 (pH 7.2), 0.25 M NaCl, 7% (w/v) SDS and 50% formamide at 42°C. Filters were washed twice for 15 min in 2x SSC, 0.1% SDS at 65°C, then twice for 20 min in 0.1x SSC, 0.1% SDS at 65°C.

Biochemical

Construction of cDNA libraries

cDNA libraries of G. sulphuraria (strain 074G) were constructed as ZAP cDNA libraries following the instructions of the provider (Strategene, La Jolla, USA). Autotrophic and heterotrophic (grown in the dark with glucose as carbon source) cells were harvested during exponential growth.

Heterologous expression of transporters in yeast

For heterologous expression in Saccharomyces cerevisiae, the coding sequences of the three sugar and polyol transporters GsSPT1, GsSPT2 and GsSPT4 were cloned into the yeast expression vector pAUR123, under the control of the alcohol dehydrogenase promotor (TAKARA Bio Inc, Japan). Resistance agaist aureobasidine was used as selective marker for transformands. Transporter GsSPT1 was also cloned as a 5' deletion construct (omitting the first 444 nucleic acids), termed GsSPT1( 1-3). As expression system, we used the sugar uptake-deficient yeast strain EBY.VW4000 (MAT a hxt1-17 gal2 stl1 agt1 mph2 mph3 leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8 c SUC2), kindly provided by Prof. E. Boles (Johann Wolfgang Goethe University, Frankfurt/Main, Germany) [24]. Cells were routinely cultivated in yeast minimal medium (YNB), supplemented with leucine, uracile, tryptophane and histidine, containing 2% maltose as carbon source unless indicated otherwise.

Transformation of yeast cells was done as described [START_REF] Dohmen | An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera[END_REF]. After transformation, cells were plated onto minimal selective medium containing 2% maltose and aureobasidine A (3 µg / ml) and incubated for 3-4 days at 30°C. Transformation of colonies was verified by PCR with transporter-and vector-specific primers. Transformed cells were used for complementation studies of yeast growth on minimal selective medium containing 2% sugars and polyols, respectively, and also for uptake of radiolabeled substrates (see below).

Uptake studies with radiolabeled substrates

Yeast cells were harvested during exponential growth at an optical density between 0.5 -0.8 (600 nm), washed twice and resuspended in distilled water to give a final concentration of 100 mg fw ml -1 . 20 µl of the cell suspension (2 mg fw) were mixed with one volume of 100 mM Tris/citrate buffer, pH 5.0. After 2 min preincubation at 30°C, uptake of radiolabeled substrate was initiated by rapid addition to give a final volume of 50 µl. As labelled substrates we used L-[C-6- C]glucose (10.7 GBq mmol -1 ), D-[U- 14 C]fructose (11.2 GBq mmol -1 ), D-[U- 14 C]sorbitol (9.5 GBq mmol -1 ), D-[U-14 C]mannitol (1.9 GBq mmol -1 ) (Moravek Biochemicals and Radiochemicals, Brea, CA, USA). The final sugar concentration was 0.1 mM with a specific activity of 18.5 MBq mmol -1 for fucose, rhamnose, ribose, arabinose and glucose and 9.25 MBq mmol -1 for fructose, mannose and sorbitol. Incubation was done for 20 s at 30°C unless indicated otherwise. Uptake was stopped by the addition of 1 ml ice-cold distilled water. Cells were harvested on a glass-fiber filter (GE Healthcare, Little Chalfont, England) and washed 4 times with 1 ml cold distilled water. Radioactivity was monitored using 3 ml scintillation cocktail and a Beckman liquid scintillation counter (Beckman Coulter, Fullterton, CA, USA).

Assays were performed in doublicates and repeated at least twice in independent experiments. Untransformed cells were used as a blank. Their intake as well as binding of radioactive material to the surface was subtracted from the samples in all experiments.

RESULTS

Cloning of a novel type of sugar transporter

We have cloned three sugar (monosaccharide) and polyol transporters from the acidophilic red alga Galdieria sulphuraria (strain 074G). They were termed GsSPT1, GsSPT2, and GsSPT4 and their coding sequences were deposited into the NCBI GenBank as EF166070, EF166071, and EF166072, respectively. SPT1 was isolated from cDNA libraries of autotrophic as well as of glucose-grown heterotrophic cells. SPT2 could only be isolated from cDNA of autotrophic cells, while SPT4 was specific for cDNA of glucose-grown cells.

Attempts to amplify PCR fragments of SPT2 and SPT4 from the respective other type of cDNA yielded no results, suggesting a sugar-dependent expression of transporters. The cDNA of SPT1 comprises a coding sequence of 1671 bp. cDNA sequences of SPT2 and SPT4 were found to contain stop codons upstream of the first ATG. SPT2 contains three stop codons at -120, -147, and -204 bp, leaving a 1206 bp coding sequence of the transporter. SPT4 contains only one stop codon at -381 bp, resulting in a 1236 bp coding sequence. These stop codons were confirmed by multiple and independent RT-PCR. The gDNA sequences of SPT1, SPT2, and SPT4 were identical to the corresponding cDNA sequences and did not contain introns. Secondary structures of the deduced protein sequences were predicted using the public DAStransmembrane prediction server for putative membrane proteins. Apparently, SPT1 follows the highly conserved structure of sugar transporters, exhibiting twelve transmembrane spanning domains with a large cytosolic loop in the middle of the protein (predicted 6+6 structure). SPT2 and SPT4 were predicted to consist of only nine transmembrane spanning domains: three at the N-terminus, six at the C-terminus. They are separated by a cytosolic middle loop, resulting in a 3+6 structure of the transporters. Going back into the putatively non-translated 5' region of the cDNA sequences of SPT2 and SPT4, we found the three 'missing' N-terminal domains present in both sequences. However, as stop codons separate the corresponding nucleotides from the putative start of translation, the proteins have to be regarded as N-terminally deleted. The new N-termini of both 3+6 transporters were predicted on the outer side of the plasma membrane, with 8 -10 (SPT2) and 10 (SPT4) amino acids reaching into the periplasmatic space.

Transcriptional regulation of transporters

The fact that SPT2 could only be isolated from cDNA of autotrophic and SPT4 only from glucose-grown heterotrophic algae, indicated a sugar-dependent transcriptional regulation of protein expression. We used Northern blot analysis to investigate this regulation in more detail (Fig. 1). Algae were grown autotrophically as well as heterotrophically on hexoses (glucose, fructose), pentoses (xylose, L-arabinose), polyols (mannitol, sorbitol, dulcitol, xylitol, arabitol), and deoxysugars (L-fucose). They were harvested and extracted during early exponential growth. Transporter-specific probes were used for RNA-hybridization. SPT1-and SPT2-transcripts were detected in autotrophic as wells as in heterotrophic cells. Transcript levels, however, were much higher in heterotrophic cells. The induction of SPT1 was independent of the type of substrate provided for heterotrophic growth, while induction of SPT2 was highly specific for dulcitol. Transcript levels of SPT4 were generally very low. In autotrophic cells, no signal was detected. Under heterotrophic conditions, only glucose-and arabitol-grown cells exhibited a faint signal. We conclude that the regulation of monosaccharide and polyol transporters takes place -at least in part -on the transcriptional level in G. sulphuraria. Transcription of all three transporters was upregulated under heterotrophic conditions. For SPT2 and SPT4, this induction was substrate-specific, while

Complementation of yeast cell growth

To determine the functionality and substrate specificity of both, the predicted 6+6 and 3+6 type of transporters, we expressed SPT1, SPT2 and SPT4 in a sugar uptake-deficient yeast strain [24]. We assessed transformands for cell growth on various substrates and determined their uptake capacities for radiolabeled substrates. To evaluate the importance of the first three domains for transport, we deleted the corresponding 148 amino acids from SPT1 to obtain the artificial 3+6 transporter SPT1( 1-3).

For complementation of growth, yeast cells were pregrown on maltose before spotting them onto minimal selective plates, supplemented with different carbon sources. We assessed cell growth of transformands on hexoses (glucose, galactose, mannose, fructose), polyols (mannitol, dulcitol, arabitol), pentoses (arabinose, xylose) and deoxysugars (fucose, rhamnose). All carbon sources were used in the D-configuration as yeast is not known to metabolize L-sugars. Heterologous expression of the algal transporters in uptake-deficient yeast, did not complentement growth on any of the substrates tested (data not shown).

Uptake of radiolabeled substrates

To determine the substrate specificities of transporters independent of cell growth, we followed uptake of radiolabeled substrates in the heterologous expression system. Cells were pregrown on maltose, and subsequently assessed for their uptake capacities. Results are summarized in figure 2. In contrast to the above results obtained from complementation studies, all three algal transporters, SPT1, SPT2 and SPT4, mediated uptake of hexoses, polyols, pentoses, and deoxysugars. Glucose was specifically taken up by SPT1, while fructose transport was also mediated by SPT2 and SPT4. Obviously, the growth defect of yeast transformands on hexoses was not due to a general lack of hexose uptake. We therefore assessed hexose phosphorylation capacities of our yeast strain. We confirmed the presence of glucokinase I, hexokinase I and hexokinase II in the sugar-uptake deficient host by PCR and by following enzyme activities in crude cell extracts (data not shown). Complementation of yeast growth on glucose and fructose should thus be feasible and the negative results can only be explained if uptake as mediated by the algal transporters was not sufficient to sustain yeast cell growth. For comparison, glucose uptake of wild type yeast was about 15 times higher than in transformands expressing GsSPT1 (Fig. 2).

In contrast to yeast, Galdieria sulphuraria can take up and metabolize a number of substrates in the L-configuation [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF][START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]. In the heterologous yeast system, uptake of these substrates by SPT1, SPT2, and SPT4 could only be assessed by using radiolabelled compounds. As demonstrated in figure 2, all three algal transporters mediated high uptake of L-fucose, while another deoxysugar, L-rhamnose, was not taken up. Transport of L-arabinose was very poor and only mediated by SPT4. In general, substrate specificities of SPT1 and its deletion construct SPT1( 1-3) were identical, while uptake capacities differed significantly. This effect was most pronounced for L-fucose and D-ribose. Their uptake by SPT1( 1-3) was twice that mediated by SPT1. For mannitol, the effect was inverse. Uptake of glucose and fructose was only slightly affected by the deletion (Fig. 2). We conclude, that the N-terminus of SPT1 does not determine the transporter's substrate specificity, i.e. take part in substrate recognition, while, on the other hand, it does seem to be involved in determining transport capacities.

Uptake kinetics

Uptake kinetics were determined for L-fucose, as uptake of this deoxysugar was highest for all transporters (Fig. 3). Substrate affinity was highest for SPT1( 1-3) (K m = 17 µM) and lowest for SPT1 (K m = 36 µM), indicating a significant influence of the 148 N-terminal amino acids of SPT1 on substrate binding. SPT2 and SPT4 exhibited similar K m values of 27 and 29 µM, respectively. Maximal uptake velocity was highest for SPT1( 1-3) (417 nmol x g -1 fw x 20 s -1 ). Apparently, the N-terminal truncation of SPT1 resulted in a reduced sterical hindrance of L-fucose uptake. V max values for SPT1 and the predicted 3+6 transporter SPT4 were identical (357 nmol x g -1 fw x 20 s -1 ), that for SPT2 was lowest (303 nmol x g -1 fw x 20 s -1 ) (Fig. 3).

In order to distinguish between active and passive transport, we incubated yeast cells expressing the algal transporters with saturating concentrations of L-fucose (0.1 mM) until a steady state was reached (Fig. 4). Intracellular L-fucose concentrations were calculated, estimating an average yeast cell volume of 20 fl and 10 x 10 9 cells x g -1 fw [START_REF] Seliber | Der Einfluss der Zusammensetzung des Nährbodens auf das Gewicht und den osmotischen Wert der Hefezelle[END_REF][START_REF] Tyson | Dependency of size of Saccharomyces cerevisiae cells on growth rate[END_REF]. In SPT1 as wells as in SPT1( 1-3) expressing cells, uptake was saturated after 2 min at a level of 750 nmol x g -1 fw. SPT4 expressing cells reached the same steady state after 4 min, while SPT2 mediated uptake of only 600 nmol x g -1 fw. Intake of 600 -750 nmol L-fucose x g -1 fw gives an intracellular concentration of 3.0 -3. As there are different types of active transporters (e.g. H + or Na + cotransporters, ABCtransportes), we followed the pH-dependency of sugar uptake in yeast (Fig. 5). Only SPT1 exhibited a pronounced pH-dependency of L-fucose transport with maximal uptake at pH 4.5.

The predicted 6+6 transporter is therefore likely to function as an active sugar / H + symporter.

Deletion of the three N-terminal transmembrane spanning domains resulted in a significant reduction of this pH-dependency in SPT1( 1-3) expressing cells, while uptake mediated by SPT2 and SPT4 was completely independent of external pH in the range of pH 4.5 -8.0. The 3+6 transporters are therefore unlikely to function as H + symporters and have to rely on alternative energy sources for active transport.

DISCUSSION

Heterotrophy of Galdieria sulphuraria is mediated by transporters in the plasma membrane which are specifically encoded in the Galdieria-genome but absent from that of the closely related but obligate autotrophic red alga Cyanidioschyzon merolae [START_REF] Barbier | Comparative genomics of two closely related unicellular thermoacidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of G. sulphuraria and significant differences in carbohydrate metabolism of both algae[END_REF]. We have identified two types of transporters in G. sulphuraria -one that follows the conserved structure of twelve transmembrane spanning domains with a large cytosolic central loop (predicted 6+6 structure), the other one consisting of nine transmembrane spanning domains with a cytosolic loop between the third and the fourth hydrophobic domain (predicted 3+6 structure). GsSPT2 and GsSPT4 are two examples of transporters with a putative 3+6 configuration. Their coding regions are significantly shorter than that of GsSPT1, a conserved 6+6 type of transporter, while the cDNA sequences as well as the algal genome still encode the missing N-terminal domains within the putatively non-translated 5' regions. We suggest that the 3+6 transporters were generated by mutations within the conserved 6+6 type of transporters. The introduction of stop codon(s) has left the resulting proteins with nine instead of twelve transmembrane spanning domains. Based on molecular clock estimates, Cyanidiaceae are probably more than 1.5 billion years old and thus likely some of the oldest extant eukaryotic organisms [START_REF] Yoon | The single, ancient origin of chromist plastids[END_REF][START_REF] Yoon | A molecular timeline for the origin of photsynthetic eukaryotes[END_REF]. This phylogenetic age increases the chance for an accumulation of mutations -as long as they do not impair the cell's viability and evolutionary fitness. From a comparison of SPT1 with its deletion construct SPT1( 1-3) -both expressed in the heterologous yeast system -we conclude that loss of the three N-terminal transmembrane spanning domains does not impair the basic function of active sugar uptake. It has to be kept in mind, though, that uptake specificity as well as kinetics could be affected by heterologous expression. In yeast, both proteins mediated an intracellular accumulation of monosaccharides and polyols and had the same spectrum of substrates. On the other hand, transport kinetics (K m /V max ) and especially the pH-dependency of uptake were significantly altered in SPT1( 1-3) expressing cells. H + symport has previously been suggested as the mechanism for sugar and polyol uptake in Galdieria [START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]. And while this still holds true for SPT1, N-terminal truncation released the pH dependency of sugar uptake and therefore appears to significantly impair H + symport. At the same time, intracellular accumulation of sugars was still possible in SPT1( 1-3) expressing yeast cells, and transport therefore had to be active. In line with these results, the predicted 3+6 transporters SPT2 and SPT4 were functional and active transporters when expressed in yeast. Their spectrum of substrates did not differ significantly from that of SPT1, yet uptake was pH-independent and therefore unlikely to be mediated by H + symport. Our attempts to complement the growth defect of an hexose uptake-deficient yeast strain by expressing the algal transporters, however, were not successful. This is quite surprising as we could clearly demonstrate uptake of radiolabeled hexoses by the corresponding yeast transformands.

Apparently, hexose uptake rates as mediated by the algal transporters were not sufficient to support yeast cell growth. A second explanation is based on concurrent activity of a sugar exporter in the uptake-deficient yeast strain. EBY.VW4000, deleted in at least 20 transporters

[24], still encodes a putative hexose exporter (E. Boles, pers. com.). Low hexose uptake by the algal transporters would thus be nullified by this endogenous exporter. We conclude that the inability of the Galdieria transporters, GsSPT1, GsSPT2, and GsSPT4, to complement cell growth of the uptake-deficient yeast strain on hexoses, is most likely due to endogenous features of the yeast strain in combination with low uptake rates of the algal transporters in the heterologous system.

With as many as 28 distinct sugar and polyol transporters present in the plasma membrane of Galdieria [START_REF] Barbier | Comparative genomics of two closely related unicellular thermoacidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of G. sulphuraria and significant differences in carbohydrate metabolism of both algae[END_REF], a highly coordinated regulation of their activity has to be applied. Based on in vivo 14 C/ 3 H-uptake studies with native cells, we have previously reported a sugar-dependent regulation of transporter activity in the alga [28]. We now corroborate these previous results by Northern blot analyses and conclude that regulation is, at least in part, achieved on the transcriptional level. Transcripts of SPT1 and SPT2 are detectable in autotrophic algae but massively up-regulated under heterotrophic conditions. SPT1 was nonspecifically induced by all substrates tested in our study, SPT2 on the other hand was specifically induced by dulcitol. SPT4 transcript was hardly detectable under any of the conditions tested, implying either very high turnover rates or very low transcription of the transporter. For several sugar transporters from higher plants, a stress-related induction has been reported [START_REF] Truernit | The sink-specific and stress-regulated Arabidopsis STP4 gene: enhanced expression of a gene endoding a monosaccharide transporter by wounding, elicitors, and pathogen challenge[END_REF][START_REF] Meyer | Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues[END_REF]. It may well be that SPT4 serves a similar purpose in G. sulphuraria and we did not apply the appropriate conditions for maximal gene induction. Protein phosphorylation as a way of regulating transporter activity [START_REF] Roblin | Regulation of a plant plasma membrane sucrose transporter by phosphorylation[END_REF] was not investigated in this study, but it should not be excluded as a regulatory level in G. sulphuraria.

The presence of low transcript levels of SPT1 and SPT2 in autotrophic cells is in agreement with previous data indicating marginal sugar uptake already in autotrophic cells [START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]28]. Several explanations for this early gene activation come to mind: one is a direct role for SPT1 and SPT2 in sugar sensing, i.e. signaling the presence of extracellular substrates to the cell. Rgt2 and Snf3 are two signaling transporter-homologues known from the yeast system [START_REF] Özcan | Two glucose transporters in Saccharomyces cervisiae are glucose sensors that generate a signal for induction of gene expression[END_REF][START_REF] Özcan | Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae[END_REF]. They are no longer active in the uptake of substrates but involved in the regulation of the high-and low-affinity hexose transporters (Hxt1-7) of yeast. Rgt2 and Snf3 exhibit extended cytosolic C-termini which are needed for interaction with the signal transducing proteins Std1 and Mth1 [START_REF] Schmidt | Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Lafuente | Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae[END_REF]. Neither SPT1 nor SPT2 have such putative signaling domains (data not shown). In addition, they proved to be functional transporters when expressed in yeast. A primary role in sugar sensing is therefore unlikely. However, an indirect role of SPT1 and SPT2 in sugar sensing by mediating early uptake of sugars and polyols prior to full activation of the cell's heterotrophic machinery appears possible. In a third model, the presence of SPT1 and SPT2 transcript in autotrophic cells reflects a 'ready-togo' status of the alga. This could speed up the generation of mature transporters upon the arrival of organic carbon.

Until recently, sugar uptake in G. sulphuraria could only be studied in intact cells with an unknown set of transporters obscuring the results. The cloning of SPT1, SPT2 and SPT4 made it possible to assess transporters seperately for the first time, to distinguish between active transport and facilitated diffusion, and to determine substrate specificities and uptake kinetics. In the heterologous yeast system, substrate specificities of GsSPT1, GsSPT2 and GsSPT4 were largely redundant, except for glucose which was only transported by SPT1.

These results corroborate previous in vivo studies on intact Galdieria cells, indicating transporters with a broad and partly overlapping spectrum of substrates [START_REF] Oesterhelt | Characterization of a sugar/polyol-uptake system in the red alga Galdieria sulphuraria[END_REF]. However, in contrast to previous models, transporters were not specific for a certain class of substrates, e.g. polyols or hexoses. Furthermore, they mediated transport of substrates in the Dconfiguation (e.g. fructose) as well as in the L-configuration (e.g. L-fucose). This is in accordance with the spectrum of substrates used for heterotrophic growth of G. sulphuraria [START_REF] Gross | Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria[END_REF]. Fucose uptake rates were very high for all three Galdieria transporters, suggesting an important role for this deoxysugar as an in vivo substrate for the alga. K m values ranged between 27 and 36 µM, corresponding to about 5 mg fucose l -1 . In general, the level of organic compounds is very low at the natural habitat [START_REF] Gross | Cryptoendolithic growth of the red alga Galdieria sulphuraria in volcanic areas[END_REF][START_REF] Gross | Ecophysiological studies on the red alga Galdieria sulphuraria isolated from South-West Iceland[END_REF]. Living in endolithic cell mats, it has been suggested that G. sulphuraria draws substrates from dead cell material of neighboring Cyanidiaceae and other species. Dissolved organic carbon is highly concentrated by the dense packing of mats and directly available to heterotrophic cells. If fucosylated cell walls should be abundant in these communities, the deoxysugar could be a major source of organic material for Galdieria. N-terminal truncation of conserved 6+6 transporters apparently leads to increased uptake velocity and substrate affinity (as shown for SPT1 and SPT1( 1-3)). Expressing these truncated transporters, cells would thus be able to rapidly deplete organic substrates from the extracellular space.

Comparing the monosaccharide and polyol transporters of G. sulphuraria to that of the green plant line, substrate specificities appear broader in the red alga. The Chlorella kessleri hexose transporters HUP1 and HUP3 are specific for hexoses (glucose, fructose and mannose), while HUP2 takes up glucose, galactose and xylose [START_REF] Sauer | Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe[END_REF][START_REF] Stadler | Subcellular localization of the inducible Chlorella HUP1 monosaccharide-H + symporter and cloning of a co-induced galactose-H + symporter[END_REF]. In higher plants, sugar transporters for glucose, fructose, xylose, arabinose, mannose and galactose [36 -40] as well as some polyol-specific transporters [41 -45] have been described. To our knowledge, only one transporter from higher plants (Arabidopsis thaliana) exhibits a spectrum of substrates that is comparable to that of G. sulphuraria transporters: AtPLT5 takes up a large number of polyols in addition to hexoses and pentoses [START_REF] Klepek | Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H + symport of numerous substrates, including myo-inositol, glycerol, and ribose[END_REF]. None of the transporters mentioned has been described as active also after truncation of the three N-terminal transmembrane spans. In this respect, the red algal transporters are unique in the plant kingdom.

While higher green plants and red algae are only distantly related, our studies on the monosaccharide and polyol transporters of G. sulphuraria will widen the understanding of sugar transport and heterotrophic capacities within the red line of evolution. Galdieria sulphuraria was grown autotrophically or heterotrophically on different substrates, as indicated. RNA was isolated during early exponential growth. Ribosomal RNA was used as loading control for total RNA.

Figure 2. Uptake of radiolabeled substrates by yeast cells expressing transporters from

Galdieria sulphuraria. GsSPT1 (black), GsSPT2 (dark grey), GsSPT4 (white), and the deletion construct GsSPT1( 1-3) (light grey) were expressed in the sugar uptake-deficient yeast strain EBY.VW4000. Transformands were assessed for their uptake capacities for radiolabeled substrates. Uptake of cells expressing the empty vector (below resolution, last column position) as well as of wild type cells (striped) are given as reference. Assays were done in triplicate as described.

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  3 H]fucose (3152.4 GBq mmol -1 ), D-[C-1-3 H]ribose (740 GBq mmol -1 ), L-[C-1-3 H]arabinose (111 GBq mmol -1 ), L-[C-1-3 H]rhamnose (185 GBq mmol -1 ), D-[U-Biochemical Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

14

 14

SPT1

  translates into a mature protein of 557 amino acids. SPT2 translates into 402, and SPT4 into 412 amino acids. A protein alignment indicates an overall identity of 25% for all three transporters. Closest homologs to the algal proteins are myo-inositol transporters from yeast. Biochemical Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  SPT1-transcription was unspecifically upregulated in the presence of monosaccharides or polyols. Biochemical Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society Functional characterization of transporters in the heterlogous system

Biochemical

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  75 mM, roughly corresponding to a 30 -40-fold Biochemical Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society enrichment as compared to the medium. This intracellular accumulation of sugars has to be based on active transport, and we therefore classify SPT1, SPT2 and SPT4 as active transporters.

Biochemical

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. Northern Blot analysis for GsSPT1, GsSPT2, and GsSPT4.

Figure 3 .

 3 Figure 3. Michaelis-Menten (A) and Lineweaver-Burk (B) plots of uptake kinetics for Lfucose. The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (crosses), GsSPT4 (closed squares), and the deletion construct GsSPT1( 1-3) (open circles), respectively, was incubated with L-fucose concentrations ranging between 0 and 0.2 mM. Assays were done in triplicate as described. K m and V max values are given in the text.

Figure 4 .

 4 Figure 4. Time-dependent uptake of L-fucose. The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (crosses), GsSPT4 (closed squares), and the deletion construct GsSPT1( 1-3) (open circles), respectively, was incubated with saturating L-fucose concentrations (0.1 mM) for up to 6 min. Assays were done in triplicate as described.

Figure 5 .

 5 Figure 5. pH-dependet uptake of L-fucose. The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (crosses), GsSPT4 (closed squares), and the deletion construct GsSPT1( 1-3) (open circles), respectively, was incubated

Biochemical Journal Immediate Publication. Published on 11 May 2007 as manuscript BJ20070448 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

The authors would like to thank Prof. Dr. Andreas Weber for his support, for critical reading of the manuscript and for making unpublished sequence data available to us. We are grateful to Prof. Dr. Eckhard Boles for providing the yeast strain EBY.VW4000, sharing essential

This study was funded by the Deutsche Forschungsgemeinschaft (Emmy Noether grant to C.O.).