Raquel Castro Prego

Mónica Lamas Maceiras

Pilar Soengas

Isabel Carneiro

Isabel González-Siso

María Esperanza

M E Cerdán

Dpto

Keywords: IXR1 transcriptional regulation, hypoxia, oxidative stress, UPC2, ECM22, YAP1, ROX1, Saccharomyces cerevisiae CM, Complete synthetic medium, CM-Ura, CM without uracyl, ChIP, chromatin immunoprecipitaion, EMSA, electrophoresis mobility shift, PCR, polymerase chain reaction

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 (ORD1) by oxygen levels. A model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1-IXR1 cross-regulation.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 (ORD1) by oxygen levels. A model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

Raquel Castro, Mónica Lamas-Maceiras, Pilar Soengas, Isabel Carneiro,

María Isabel González Siso, María Esperanza Cerdán

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SYNOPSIS

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intra-strand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes like SRP1/TIR1, which encodes a stress-response cell wall manoprotein, or COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In this study it is shown that IXR1 mRNA levels are controlled by oxygen availability, increasing during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (-557 to -376). EMSA and ChIP assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H 2 O 2 . IXR1 expression is moderately activated by H 2 O 2 and this induction is Yap1p-dependent.. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1-IXR1 cross-regulation.

INTRODUCTION

Although Saccharomyces cerevisiae is probably the unicellular eukaryote that has been most extensively studied by traditional and genome-wide approaches, many questions about transcriptional regulation, which may throw light on more complex systems, remain unanswered. This is particularly true in regard to transcription factors that are structurally similar to other eukaryotic proteins but have been little explored in terms of function. The yeast Ixr1 protein (Ixr1p) was first characterized during screening for structure-specific recognition proteins (SSRPs) that bind to DNA containing intra-strand cross-links formed by the anticancer drug cisplatin. It was suggested that Ixr1p may have a role in mediating the cytotoxicity of cisplatin [START_REF] Brown | Ixr1, a yeast protein that binds to platinated DNA and confers sensitivity to cisplatin[END_REF]. In fact, Ixr1p blocks the excision repair of cisplatin-DNA adducts in yeast [START_REF] Mca'nulty | The HMG-domain protein Ixr1 blocks excision repair of cisplatin-DNA adducts in yeast[END_REF][START_REF] Mca'nulty | Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo[END_REF]. The gene IXR1 was also identified by complementation of trans-acting mutants defective in the aerobic repression of COX5B transcription and was renamed ORD1 [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF]. We will refer to it as IXR1 in this work because that is the first name in SGD (http://db.yeastgenome.org), and also to avoid misunderstanding: the name ORD1 has been used in the literature to refer to both IXR1/YKL032C, which encodes this DNA binding protein, and the unrelated SPE1/YKL184W, which encodes an ornithine decarboxylase.

Mutations in IXR1 cause aerobic de-repression of COX5B, which encodes the hypoxic isoform of the subunit Vb of the mitochondrial complex cytochrome c oxidase [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF]. However, these mutations do not affect the aerobic repression of other hypoxic genes such as CYC7 and ANB1 [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF], which form part of the ROX1 regulon, a wellstudied system for aerobic repression of yeast hypoxic genes during aerobiosis [START_REF] Kastaniotis | Rox1 mediated repression. oxygen dependent repression in yeast[END_REF][START_REF] Becerra | The yeast transcriptome in aerobic and hypoxic conditions: Effects of hap1, rox1, rox3 and srb10 deletions[END_REF][START_REF] Sertil | Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae[END_REF][START_REF] Klinkenberg | Combinatorial repression of the hypoxic genes of Saccharomyces cerevisiae by DNA binding proteins Rox1 and Mot3[END_REF]. Ixr1p has also been related to aerobic and anaerobic repression of SRP1/TIR1 [START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF].

Ixr1p and Rox1p both control yeast genes that are differentially expressed during aerobiosis or hypoxia, and both contain HMG motifs, which bind and bend DNA [START_REF] Mca'nulty | Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo[END_REF][START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF][10][11]. However, and in contrast to ROX1, very little is known about the conditions that influence IXR1 expression. We have found that IXR1 expression is controlled by oxygen levels, and in this work we have characterized transcriptional factors that exert control upon its transcription during aerobiosis or hypoxia. We have also demonstrated that auto-activation is necessary for full IXR1expression under hypoxic conditions and we have delimited the region in the IXR1 promoter responsible for this control. Ixr1p is also necessary for hypoxic repression of ROX1 and for resistance to oxidative stress generated by H 2 O 2 , a feature that could be related to the physiological changes necessary for adaptation from aerobiosis to hypoxia and opens new connections between hypoxic and stress-induced regulons in S. cerevisiae. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

MATERIALS AND METHODS

Strains

The S. cerevisiae strains were obtained from EUROSCARF (http://www.rz.unifrankfurt.de/FB/fb16/mikro/euroscarf): BY4741 wild-type and derivative isogenic mutants in transcriptional regulators, or from other sources [12][13][14], and are described in Table 1.

Growth conditions

Yeast cells were grown at 30ºC in YPD (2% glucose, 2% bacto-peptone, 1% yeast extract), or in complete medium (CM) prepared as previously described [START_REF] Zitomer | Yeast cytochrome c messenger RNA. in vitro translation and specific immunoprecipitation of the CYC1 gene product[END_REF], or in other media for particular applications as specified below. Hypoxic conditions (oxygen concentration < 1%.) were generated in anaerobic jars with the GasPack EZ-Anaerobe system from BD, and under these conditions the media were supplemented with 20 mg/l ergosterol and 0.5% v/v Tween 80. As a positive control for hypoxia generation, induction of the hypoxic gene HEM13 was assessed by Northern blotting in each experiment (data not shown).

Fusions to the reporter gene MEL1

The promoter of IXR1 was fused in frame to the MEL1 reporter gene, which encodes the enzyme α-galactosidase, in the centromeric plasmid pMELα [Amp r MEL1 URA3] described by [START_REF] Melcher | Zero background yeast reporter plasmids[END_REF]. The primers ECV325ED0 and ECV326E (Table 2) were used for PCR amplification. The forward primer contained a SacI site and the reverse an XhoI site for convenient cloning in pMELα, which was also digested with the same enzymes. Standard procedures for manipulation of nucleic acids were used. After ligation, the Escherichia coli strain DH10B was used for propagating the recombinant plasmids by conventional techniques. The desired recombinants were identified by PCR and restriction analysis. The selected construct was finally verified by sequencing and the recombinant vector, pIXR1::MEL1, was obtained. Other selected regions of the IXR1 promoter were also fused to the reporter gene MEL1 by the same procedure. The different promoter regions (D1-D5) were amplified by PCR using the specific forward primers described in Table 2 and the reverse ECV326E primer.

Protein extracts

Protein extracts were prepared as previously described [START_REF] Ramil | Characterization of promoter regions involved in high expression of KlCYC1[END_REF] from yeast cells incubated in CM under aerobic or hypoxic conditions and the protein concentration was determined by the method of Bradford [18].

α-galactosidase determination

The fusions to the reporter gene were used to transform several S. cerevisiae strains described in Table 1. Yeast cells were transformed following the procedure of Ito et al. [START_REF] Ito | Transformation of intact yeast cells treated with alkali cations[END_REF]. Independent transformants were randomly picked and grown in CM-Ura to an OD 600 of approximately 0.8. α-Galactosidase activity was usually determined in chemically-permeabilised cells as previously described [START_REF] Ryan | SWI-SNF complex participation in transcriptional activation at a step subsequent to activator binding[END_REF], in at least triplicate. Basically, yeast cultures (5-10 ml) were grown to an OD 600 between 0.5 and 1.5, which was recorded. A 1 ml volume of cells was centrifuged, and the pellet was resuspended in 200 µl 20 mM HEPES pH 7.5, 10 mM dithiothreitol, 0.002% sodium dodecyl sulphate. Chloroform (50 µl) was added and the sample was vortexed for 10 s. After 5 min pre-equilibration at 30°C, 800 µl 7 mM p-nitrophenyl-α-D-galactopyranoside (PNPG) from SIGMA® in 61 mM citric acid, 77 mM Na 2 HPO 4 , pH 4, was added and incubation at 30°C was continued. At various times, 100-µl aliquots were added to 900 µl 0.1 M Na 2 CO 3 to stop the reaction and develop the yellow colour characteristic of the cleaved product (which remains colourless at pH 4). The reaction products were centrifuged for 5 min in a microcentrifuge and the A 400 was determined. Units of αgalactosidase activity are given as [∆A 400 /(OD 600 × time in min x ml)] × 1,000, using samples taken at time points such that A 400 <3.0. When the strains were flocculent or low activities were expected, the activity was determined on protein extracts [START_REF] Melcher | Zero background yeast reporter plasmids[END_REF] and expressed as nmol PNPG/ min x mg protein [∆A 400 /(0.0182 × time in min x mg protein)]. Statistical analyses were carried out using the LSD (least significant difference) test included in the Statgraphics Plus Package v.2.1 with 95% confidence.

RNA isolation and Northern blotting RNA was isolated and Northern blotting was performed as previously described [START_REF] González-Domínguez | Heme-mediated transcriptional control in Kluyveromyces lactis[END_REF]. RNA blots were hybridized overnight in a buffer containing 0.5 M Na 2 HPO 4 pH 7.2, 7% SDS, 1 mM EDTA at a temperature appropriate for the length and composition of the DNA probe. The IXR1 probe was obtained by PCR amplification of genomic DNA with ECV593M and ECV612 primers. The ROX1 probe was amplified with primers ART27 and ART28. Data were normalised for RNA loading against the signal obtained from the control probe SNR17A amplified with primers U3F and U3R. The sequences and hybridization positions of these primers are described in Table 2. After hybridization, the filters were washed twice for 10 min with 0.1% SDS and 2x SSC at the temperature selected for hybridization. Northern blots were carried out in triplicate and radioactive blots were exposed to PhosphorImager screens for quantification using the ImageQuant programme.

Ixr1p purification

The coding sequence of IXR1 was amplified with primers ECV616 and ECV617, which also contain sequences for recombining in the YEpFLAG-1™ plasmid (Sigma-Aldrich-Biotech), following the manufacturer's instructions. The protein Ixr1 fused to the FLAG™ peptide at the carboxy terminus was expressed in the yeast strain BJ3505. Cells were grown at 30°C for more than 48h in YPHSM medium (8% bactopeptone, 1% yeast extract, 3% glycerol, 1% dextrose) until they reached an absorbance of 6.0, coinciding with maximum expression of the YEpFLAG-1™ vector. They were lysed in TBS buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl). After centrifugation (15 min at 8,000 × g), the protein was purified using ANTI-FLAG® M2 Agarose™ (Sigma-Aldrich-Biotech). Ixr1 was eluted with 150 ng/µl FLAG peptide™ (Sigma-Aldrich-Biotech) in TBS buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl). Proteins were electrophoresed on 10% acrylamide gels.

EMSA assays

EMSA assays were carried out with the desired promoter fragments obtained after annealing the complementary primers ECV614M and ECV615M (IXR1 promoter from -520 to -432) or ECV651 and ECV652 (IXR1 promoter from -390 to -319). The fragments were labelled by Klenow with α-32 P-dATP. The binding reactions were carried out using protein extracts or the purified Ixr1-Flag protein as specified. With protein extracts, the best results were obtained when the binding reactions were performed at 30 ºC for 20 min in buffer A previously described [START_REF] Ramil | Characterization of promoter regions involved in high expression of KlCYC1[END_REF] with at least 10,000 c.p.m. of labelled DNA, 15 µg of protein extract and 2 µg calf thymus DNA as carrier. Binding reactions with the purified Ixr1p were performed either on ice for 20 min in a buffer described by Lambert et al. [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF] or at 30ºC for 20 min in buffer A [START_REF] Ramil | Characterization of promoter regions involved in high expression of KlCYC1[END_REF], A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

using at lest 10,000 c.p.m. of labelled DNA and 2-3 µg of protein, with similar results. In competition experiments, a large excess, 20 or 100-fold molar, of unlabelled fragment (-520 to -432) was used as specific competitor. A 100-fold molar excess of non-labelled sonicated salmon sperm DNA was used as unspecific competitor.

ChIP assays

Protein extracts were prepared from cells of the strains Z1580 and Z1465 (Table 1) grown in aerobiosis or hypoxia. These strains have Ixr1p-9cMyc and Rox1p-9cMyc modifications respectively and have been previously described [13]. The ChIP assays were carried as previously described ( [13]) with magnetic Dynabeads from Invitrogen S.A., following the instructions of the manufacturer, and anti c-Myc (9E11: sc47694 from Santa Cruz Biotechnology Inc.) for specific Ixr1p-9cMyc or Rox1p-9cMyc immunoprecipitation. The negative controls, with rabbit IgG immunoprecipitation, were included. The promoter region of IXR1, from -566 to -357, was amplified by PCR with primers ECV570M and ECV613 (Table2). The promoter region of ROX1, from -428 to -254, was amplified by PCR with primers ECV686 and ECV687 (Table 2). After optimization of PCR conditions, the percents of immunoprecipitated DNA of the specific IXR1 and ROX1 promoter regions were calculated by dividing the amount of PCR product obtained with immunoprecipitated DNA (IP) by the amount obtained with total DNA isolated from sheared chromatin and kept as input (IN) using the Quantity One 4.5.0 version from Bio-Rad. Quantifications were done in duplicate and averages and standard deviations calculated.

RESULTS

IXR1 expression is repressed by Rox1p and related factors during aerobiosis and it is increased during hypoxia

Although in the last years a certain number of genome-wide analysis on oxygen regulated genes have been performed [START_REF] Becerra | The yeast transcriptome in aerobic and hypoxic conditions: Effects of hap1, rox1, rox3 and srb10 deletions[END_REF][START_REF] Ter Linde | Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae[END_REF][START_REF] Kwast | Genomic analyses of anaerobically induced genes in Saccharomyces cerevisiae: Functional roles of Rox1 and other factors in mediating the anoxic response[END_REF][START_REF] Ter Linde | A microarray-assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae[END_REF], transcriptional regulation of IXR1 expression by oxygen levels has not been reported. Taking into account the role of Ixr1p as transcriptional regulator of several anaerobic/hypoxic genes [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF][START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF], we decided to directly test the existence of this regulation by Northern blotting and fusion of the IXR1 promoter to the MEL1 reporter gene.

Northern analyses we carried out on the S. cerevisiae strain BY4741 and a series of haploid isogenic S. cerevisiae strains obtained from the EUROSCARF collection, and with specific deletions of genes coding for transcriptional regulatory factors that have previously been related to the aerobic-hypoxic response in yeast. In these assays, IXR1 expression is detectable during aerobiosis but increases 1.8 fold during hypoxia (Fig. 1A).The proteins Ixrlp and Rox1p are related to transcriptional regulation of aerobic-hypoxic genes in S. cerevisiae although their targets are different [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF][START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF]25]. Interestingly, ROX1 and IXR1 are oppositely regulated in response to oxygen availability, ROX1 being up-regulated in aerobic conditions as previously reported [10,25] and IXR1 up regulated during hypoxia (this work).

We also confirmed the hypoxic induction of IXR1 by a second approach. To this purpose, the IXR1 promoter was fused in frame to the MEL1 reporter gene in the plasmid pMELα, as described in Materials and Methods and the S. cerevisiae strains BY4741, BWG1-7a and W303 were transformed with this construct. Cells were grown in aerobiosis and hypoxia to OD 600 ∼0.8-1.2 and harvested for determination of αgalactosidase activity. We verified that hypoxic induction was produced in all the strains tested, although the induction was strain-dependent. Using the BY4741 transformants in aerobic conditions, 0.03±0.01 α-galactosidase units were measured, which increased to 18.00±1.00 units in hypoxia. For BWG-7b the increase was from 0.02±0.01 to 1.25±0.50 α-galactosidase units and for W303 from 0.04±0.02 to 4.60±0.90 α-galactosidase units. Differences between strains are probably attributable to different genetic backgrounds, which might affect the hypoxic response. Differences between the IXR1 induction ratios obtained in the same strain, BY4741, in Northern or reporter gene analyses might be caused by the different stability of IXR1 or MEL1transcripts.

The data in Fig. 1A show that aerobic IXR1 expression was increased 2.1 fold in the ∆rox1 strain. Low expression of IXR1 under aerobic conditions is therefore attributable to repression exerted by Rox1p. Several promoters of yeast genes that are repressed by Rox1p bind the co-repressor complex Tup1p-Ssn6p [START_REF] Sertil | Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae[END_REF][START_REF] Klinkenberg | Combinatorial repression of the hypoxic genes of Saccharomyces cerevisiae by DNA binding proteins Rox1 and Mot3[END_REF]26]. The wild type strain BY4741 and its derivatives ∆tup1 and ∆ssn6 were transformed with the reporter construct pIXR1::MEL1. Aerobic expression of IXR1 (0.03±0.01 αgalactosidase units) was significantly increased in these S. cerevisiae mutant strains (1.00±0.03 α-galactosidase units in ∆tup1 and 2.00±0.04 in ∆ssn6), thus confirming the role of Rox1p in the control of IXR1 expression.

Hypoxic IXR1 expression did not significantly change in the ∆sut11 or ∆sut2 strains. Therefore, IXR1 hypoxic induction is not dependent on Sut1p and Sut2p. SUT1 is a hypoxic gene encoding a nuclear protein with a Zn finger (Zn[II]2Cys-6 family) and it has been shown to be involved in the transcriptional induction of hypoxic genes (e.g. DAN1) when the cells are shifted from aerobiosis to anaerobiosis [START_REF] Ness | SUT1 is a putative zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells[END_REF][START_REF] Regnacq | SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae[END_REF]. SUT2 is homologous to SUT1 and both genes are also involved in sterol uptake [START_REF] Ness | SUT1 is a putative zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells[END_REF]. It has been proposed that the transcriptional induction of hypoxic genes acts by a physical interaction between Sut1p and Ssn6p, releasing the aerobic repression caused by Tup1p-Ssn6p [START_REF] Regnacq | SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae[END_REF]. However, this competitive mechanism does not explain the observed increase in IXR1 transcription during hypoxia, since the mRNA levels in this condition do not differ greatly between the ∆sut1 or ∆sut2 mutants and the wild type strain BY4741 (Fig. 1A).

Aerobic and hypoxic expression of IXR1 depends alternatively on the transcriptional activators Ecm22p and Upc2p

In order to determine whether the high hypoxic expression of IXR1 was mediated by other transcriptional factors that have been related to positive regulation of hypoxic or anaerobic genes in S. cerevisiae, IXR1 expression was analysed by Northern blotting in strains deficient in the regulators Upc2p and Ecm22p. Upc2p was first characterized as a hypoxic activator of the anaerobic DAN genes [START_REF] Cohen | Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites[END_REF]; UPC2 expression increases under oxygen-limited conditions [12,30]. The sequences of Ecm22p and Upc2p are similar and these factors are responsible respectively for the basal and induced expression of genes encoding enzymes of ergosterol biosynthesis in yeast (ERG genes). Synthesis of sterols and heme depend upon molecular oxygen, so their intracellular levels are potentially indicators of oxygen levels in the cellular environment [12]. Our results (Fig. 1A) indicate that Upc2p and Ecm22p act differently upon IXR1 expression under aerobic and hypoxic conditions, Ecm22p being necessary for full aerobic expression and Upc2p for full hypoxic expression. The role of Upc2 in hypoxic activation of IXR1 was also confirmed by a second experimental approach. The wild type strain BY4741 and its derivative ∆upc2 were transformed with the reporter construct pIXR1::MEL1. IXR1 hypoxic expression (18±1.00 α-galactosidase units) was significantly decreased in the ∆upc2 strain (10.00±0.03 α-galactosidase units).

Ixr1p is necessary for hypoxic repression of ROX1 and Upc2p controls the aerobic/hypoxic expression of ROX1 and IXR1 respectively Since Rox1p contributes to the transcriptional regulation of IXR1 (Fig. 1A), and transcription of ROX1 and IXR1 are oppositely regulated by oxygen, we decided to test whether Ixr1p might also control ROX1 expression. Fig. 1B shows that hypoxic ROX1

expression is increased 5.3 fold in the ∆ixr1 strain (3.2 versus 0.6).

Taking into account that Upc2p and Ecm22p are important for IXR1 expression during the change from aerobiosis to hypoxia, we included the ∆upc2 and ∆ecm22

strains in the Northern analysis of ROX1 expression (Fig. 1B). The results revealed new interconnections among the regulatory factors Upc2p Ixr1p and Rox1p. Upc2p controls the hypoxic expression of ROX1 and IXR1 in opposite ways; during hypoxia the strain ∆upc2 shows increased ROX1 expression (Fig. 1B) and decreased IXR1 expression (Fig. 1A).

Ixr1p and the hypoxic induction of IXR1 Some transcriptional factors related to the aerobic-hypoxic response in yeast exert regulatory control upon their own transcription. This is the case with Rox1p, which maintains negative retro-control under aerobic conditions [START_REF] Deckert | Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae[END_REF], and Hap1p, which in monomer form represses its promoter during anaerobiosis [START_REF] Hon | The heme activator protein Hap1 represses transcription by a heme-independent mechanism in Saccharomyces cerevisiae[END_REF]. In view of these precedents, we decided to investigate whether IXR1 expression was auto-regulated. The wild type strain BY4741 and its derivative ∆ixr1 were transformed with the pIXR1::MEL1 construct and α-galactosidase activity was measured in the transformed cells under aerobic and hypoxic conditions. The hypoxic induction of IXR1 is absent in the ∆ixr1 strain (Fig. 2A). The results indicate that Ixr1p also regulates the IXR1

promoter: it has no effect during aerobiosis (data not shown) but is necessary for hypoxic induction.

To determine more details about the mechanism of Ixr1p auto-regulation described above, we performed deletion analyses of the IXR1 promoter to identify the region necessary for hypoxic expression of Ixr1p. Serial deletions of the IXR1 promoter (D0-D5) were obtained by PCR using specific primers as explained in Materials and Methods (Table 2). The exact extension of each deletion is represented in Fig. 2B. Reporter activity was measured in the transformants and the results revealed that the region extending from -557 to -376 is the most important for hypoxic expression of IXR1. In silico analysis (Fig. 2C) of this region with RSA tools (http://liv.bmc.uu.se/rsatools/) revealed that it contains AR1 sequences for putative binding of Ecm22p or Upc2p (AR1-box) and also two sequences (underlined) with similarity to the consensus AArcmrgRAGCGGkG for Ixr1p binding [START_REF] Macisaac | An improved map of conserved regulatory sites for Saccharomyces cerevisiae[END_REF].

To test the binding of Ixr1p to the IXR1 promoter, the protein was purified using the YepFLAG system (Fig. 3A). EMSA assays showed that Ixr1p binds in vitro to the IXR1 promoter region extending from -520 to -432 and included in the previously characterized as important for hypoxic induction in the deletion analyses (Fig. 2B). The retarded band was obtained under two different assay conditions (Fig. 3B, lanes W) and was not obtained using other promoter region extending from -390 to -319 and used as negative control (Fig. 3B, lane N). The retarded band is specific, as shown by the competition experiments depicted in Fig. 3C. EMSA assays were also performed with total protein extracts obtained from wt and ∆ixr1 mutant cells grown under aerobiosis and hypoxia and a labelled DNA fragment including the IXR1 promoter region extending from -520 to -432 (Fig. 3D). The results showed a band specific for Ixr1p expression; it did not appear in the ∆ixr1 strain. This retarded band was observed under both aerobic and hypoxic conditions.

In vivo binding of Ixr1p and Rox1p to the IXR1 and ROX1 promoters

In order to give further support to the data explained above about IXR1 hypoxic autoinduction, Ixr1p binding to the IXR1 promoter and IXR1-ROX1 cross-regulation ChIP assays were carried out with the strains Z1580 (Ixr1p-tagged) and Z1465 (Rox1ptagged) grown in aerobic and hypoxic conditions, as described in Materials and Methods. The IXR1 promoter region selected to test the binding of the transcriptional regulators included the previously characterised for in vitro binding of Ixr1p (Fig. 3). The ROX1 promoter region selected in these assays (-428 to -254) was necessary for Rox1p binding and involved in aerobic auto-repression [START_REF] Deckert | Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae[END_REF].

Data from Fig. 4A show that Ixr1p binds to the IXR1 promoter and its binding is quantitatively higher during hypoxia than aerobiosis. This is in accordance with the induced IXR1 hypoxic transcription and with the role of Ixr1p as auto-inducer of the IXR1 promoter in this condition. Data from Fig. 4A also show that Rox1p binds to the IXR1 promoter, but in this case its binding is higher during aerobic conditions as expected, since ROX1 transcription is aerobically induced [START_REF] Deckert | Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae[END_REF].

Binding of Ixr1p to the ROX1 promoter also increases during hypoxia contrary to Rox1p that binds the ROX1 promoter principally during aerobiosis (Fig. 4B).

Ixr1p and the oxidative stress response mediated by peroxides

Several connections between the hypoxic and the oxidative stress responses in S. cerevisiae have previously been reported [START_REF] Dirmeier | Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes[END_REF]. In the search for new functions of IXR1, we verified that Ixr1p is related to the oxidative stress response. The wild type strain BY4741 and its derivatives ∆ixr1 and ∆yap1 (positive control for sensibility to peroxides) were grown in CM. From these initial cultures serial dilutions were prepared and cells were grown on CM plates with or without different concentrations of H 2 O 2 . The strain ∆ixr1 was less resistant to oxidative stress caused by 1 or 2 mM H 2 O 2 (Fig. 5A), so Ixr1p is important for conferring resistance to such stress.

The wild type strain BY4741 and its derivative ∆yap1 were transformed with the pIXR1::MEL1 construct and grown in CM-Ura. α-galactosidase activity was determined before and after addition of 1 mM H 2 O 2. Data, presented in Fig. 5(B), show that there was a 3-4 fold induction by H 2 O 2 , statistically significant with a 95% confidence, and absent in the ∆yap1 strain. Since Yap1p is a transcriptional activator of genes regulated by oxidative stress [START_REF] He | Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae[END_REF], this suggests that the induction of IXR1 by H 2 O 2 is mediated by Yap1p.

DISCUSSION

Knowledge about regulation of Ixr1p transcription is of general interest not only for the yeast community but also for clinical researchers because Ixr1p mediates the cytotoxicity of the anticancer drug cisplatin in yeasts [START_REF] Brown | Ixr1, a yeast protein that binds to platinated DNA and confers sensitivity to cisplatin[END_REF]. Besides, the HMG domains present in Ixr1p have similarities to other HMG proteins from various species, including Rox1p from S. cerevisiae and the human high mobility group proteins B1 (P09429) and B2 (P26583). In humans, a nuclear protein complex containing B1, B2 and other factors is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues [START_REF] Krynetski | A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues[END_REF]. Moreover, the high mobility group protein B1 interacts with sterol regulatory element-binding proteins to enhance their DNA binding [START_REF] Najima | High mobility group protein-B1 interacts with sterol regulatory element-binding proteins to enhance their DNA binding[END_REF], and it has recently been suggested that it functions as a new cofactor of the hepatocyte nuclear factor HNF-1α [START_REF] Yu | Proteomic screen defines the hepatocyte nuclear factor 1alpha-binding partners and identifies HMGB1 as a new cofactor of HNF1alpha[END_REF]. The study of mechanisms involved in yeast IXR1 transcriptional regulation opens new clues to understand similar processes in its human homologues.

The present study shows that IXR1 mRNA levels in S. cerevisiae are controlled by oxygen availability, increasing during hypoxia. During aerobiosis, low levels of IXR1 expression are maintained by Rox1p (Fig. 1A). In this work we have also found that Ixr1p is necessary for the expression of IXR1 during hypoxia. The IXR1 promoter region from -557 to -376, necessary for hypoxic expression (Fig. 2), contains two sequences that resemble the degenerate consensus proposed for Ixr1p binding [START_REF] Macisaac | An improved map of conserved regulatory sites for Saccharomyces cerevisiae[END_REF], and purified Ixr1p specifically binds to the region containing those sequences (Fig. 3B and3C). Using whole cell extracts we found that the same band is formed during both aerobiosis and hypoxia (Fig. 3D). ChIP assays indicate that Ixr1p binds in vivo to the IXR1 promoter in aerobiosis and binding increases during hypoxia (Fig. 4A) The fact that Ixr1p also binds to the IXR1 promoter during aerobiosis may suggest that a structural modification in the regulator and/or its interactions with other proteins are necessary to produce the change to an activating hypoxic form, but more data will be necessary to confirm this mechanism.

Since Rox1p represses IXR1 during aerobiosis (Fig. 1A) and Ixr1p represses ROX1 during hypoxia (Fig. 1B), these results suggest a model of transcriptional adaptation from aerobiosis to hypoxia that involves cross-regulation between ROX1 and IXR1. This cross-regulation is also supported by the reported in vivo binding to their respective promoters (Fig. 4A and4B). It is interesting to remark that Ixr1p and Rox1p are two HMG proteins, which regulate transcription of specific groups of aerobic and hypoxic target genes, and therefore their cross-regulation has wide consequences in the adaptation of yeast cells from aerobiosis to hypoxia.

The shift between transcriptional regulation mediated by Rox1p or Ixr1p might be related to other signals in the transition from aerobiosis to hypoxia. We have found that Ecm22p and Upc2p activate IXR1 transcription during aerobiosis and hypoxia respectively (Fig. 1A) and that Upc2p represses directly or indirectly ROX1 transcription during hypoxia (Fig. 1B). Upc2p and Ecm22p regulate the expression of many of the ergosterol biosynthesis (ERG) genes [START_REF] Vik | Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cerevisiae[END_REF]40] as well as the transcription of several hypoxically expressed genes, including genes involved in anaerobic cell wall reorganization and anaerobic sterol uptake [30,[START_REF] Abramova | Reciprocal regulation of anaerobic and aerobic cell wall mannoprotein gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Wilcox | Transcriptional profiling identifies two members of the ATPbinding cassette transporter superfamily required for sterol uptake in yeast[END_REF]. Although Upc2 expression increases during hypoxia, this increase is reportedly a consequence of sterol depletion rather of heme decrease [12]. It has been suggested that Upc2p and Ecm22p share similar functions but they act at different times. Ecm22p is generally more abundant at target promoters; however, when sterols are depleted, Ecm22p levels drop and Upc2p replaces it [START_REF] Davies | Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms[END_REF].

A summary of the different regulators that control IXR1 expression during aerobiosis and hypoxia is depicted in Fig. 6. In aerobiosis, it has been previously reported that transcription of IXR1 is necessary to preclude expression of hypoxic genes [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF][START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF]. According to data presented in our work, this low level IXR1 expression depends on a balance between repression, caused by Rox1p, acting on the IXR1 promoter, and activation, caused by Ecm22p. During transition from aerobic to hypoxic growth, Ecm22p could be replaced by Upc2p as previously reported ( [START_REF] Davies | Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: Similar activation/regulatory domains but different response mechanisms[END_REF]). This, together with Ixr1p auto-activation, would increase IXR1 expression, ROX1 repression and adaptation to hypoxia. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Until now, knowledge of the function of Ixr1p as a transcriptional regulator was mostly limited to a repressor effect over hypoxic genes under aerobic conditions, preventing their function [START_REF] Lambert | The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae[END_REF][START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF]. The fact that IXR1 expression increases during hypoxia suggests new functions for it. This hypoxic function of IXR1 is not essential for the cells because the null strain ∆ixr1::kanMX4 is able to grow in hypoxia with an ergosterol supplement (data not shown). It has been reported [START_REF] Becerra | The yeast transcriptome in aerobic and hypoxic conditions: Effects of hap1, rox1, rox3 and srb10 deletions[END_REF][START_REF] Dirmeier | Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes[END_REF][START_REF] Ter Linde | Transcriptional regulation of YML083c under aerobic and anaerobic conditions[END_REF] that the mRNA levels of several stress response genes in S. cerevisiae also increase during hypoxia. Searching for a growth-related phenotype of IXR1 knock-out, we found that Ixr1p is also necessary for resistance to oxidative stress generated by H 2 O 2 (Fig. 5A). A target of Ixr1p, the hypoxic gene SRP1, repressed by Ixr1p under aerobic conditions, has also been found to be regulated by Yap1p during hypoxia [START_REF] Bourdineaud | A Rox1independent hypoxic pathway in yeast. antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene[END_REF]. We found in the α-galactosidase assays (Fig. 5B) that IXR1 expression is moderately activated by H 2 O 2 and this induction is Yap1pdependent.

In conclusion, the IXR1 promoter is controlled directly or indirectly by different regulators that have previously been related to transcriptional control by changes in oxygen concentration. As a consequence of changes in oxygen availability, heme, sterol and ROS cellular levels are altered. The IXR1 promoter may serve as a relay sensing the global input of these individual changes, integrating the different signals: heme through Rox1p, sterols through Ecm22p and Upc2p and ROS through Yap1p.

LEGENDS TO THE FIGURES

CCCATTCGTTCTCTCACCAA IXR1 W --- -566 ECV570M GGGGAGCTCCCATAAAACATACACATCGTGCT IXR1 C --- -357 ECV614M CCGTGGAGGAATGGGTGGAACGGTTACTGACGACGGCCGCGCGGGG TTTTGTGTTGGGTGTGATGTCCGGATGCAACAGCAGCAAAGGA IXR1 W --- -520 ECV615M TCCTTTGCTGCTGTTGCATCCGGACATCACACCCAACACAAAACCCC GCGCGGCCGTCGTCAGTAACCGTTCCACCCATTCCTCCACGG IXR1 C --- -432 ECV616 ctatatcgtaatacaccaagctcgacctcgcgATGAACACCGGTATCTCGCC IXR1 W --- +1 ECV617 ggtcgacgggcccggatccatcgatagatcttcacttgtcatcgtcatccttgtagtcTTCATTTTTTATGA TCGAACCATT IXR1 C --- +1791 ECV593M AAGCCCAGCCTCAACAACAA IXR1 W --- +161 ECV612 TGGTGAGTCAATTGAGGTTG IXR1 C --- +860 ECV651 TTTTCCACCTCCCCTTGGTGAGAGAACGAATGGGAACAAAAAAAAAATGCA CACATCAGCTGGTGG IXR1 W --- -390 ECV652 tTTGGCTCCACCAGCTGATGTGTGCATTTTTTTTTTGTTCCCATTCGTTCTCTC ACCAAGGGGAGGTGG IXR1 C --- -319 ECV686 AACTTGGCGATTGCTGACA ROX1 W --- -428 ECV687 GACCGTTACATTACGCAAAGTG ROX1 C --- -254 ART27 GACCCAAGAACGCATTTATT ROX1 W --- +35 ART28 TTGATTTGAATTGTTGATACTG ROX1 C --- +858 U3F CGACGTACTTCAGTATGTAA SNR17A W --- +3 U3R ATTTGTACCCACCCATAGAG SNR17A

  Castro, Mónica Lamas-Maceiras, Pilar Soengas, Isabel Carneiro, María Isabel González Siso, et al.. Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 (ORD1) by oxygen levels. A model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation. Biochemical Journal, 2009, 425 (1), pp.235-243. 10.1042/BJ20091500. hal-00479262

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500

  Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500

Fig. 1

 1 Fig. 1 Transcriptional regulation of IXR1 and ROX1 expression A) Effect of ROX1, SUT1, SUT2, ECM22 and UPC2 disruption on IXR1 expression. B) Effect of IXR1, ECM22 and UPC2 disruption on ROX1 expression. Northern blots were carried out in triplicate and a representative picture is shown. After loading normalization with reference to the SNR17A signal, and in order to compare expression under different conditions, the intensity of the wild type strain in aerobiosis was considered to have the value 1 arbitrary unit and was used as the reference to calculate the relative values of the other signals. Given values are average calculated from three independent blots. DNA probes are described in Materials and Methods.

Fig. 2

 2 Fig. 2 Analysis of the IXR1 promoter region involved in Ixr1p hypoxic activation IXR1 expression during aerobiosis and hypoxia was measured using MEL1 as reporter gene. Transformed yeast cells were grown in CM-Ura media under hypoxic conditions as defined in Materials and Methods, until OD 600 reached 0.8-1.0. α-galactosidase activity was assayed in permeabilised cells. (A) Effect of IXR1 disruption on the activity of the IXR1 promoter fused to the reporter gene MEL1 in hypoxic conditions. (B) Hypoxic expression of MEL1 under the control of different regions of the IXR1 promoter. The extension of the promoter in the reporter constructs (D0 to D5) is indicated in brackets. (C) Sequence of the IXR1 promoter that is important for hypoxic expression. Putative AR1 (box) and IXR1-binding sequences (underlined) are marked in the figure.

Fig. 3

 3 Fig. 3 In vitro binding of Ixr1p to the IXR1 promoter (A) Purification of Ixr1p with the Yep-FLAG1 system. M, size marker, PAGE Ruler Plus™ (Fermentas). 1, first enriched eluate; 2, second purified eluate. (B) EMSA assays carried out with 3 or 2 µg purified Ixr1p factor as indicated. C, control without protein; W, binding reaction carried with the labelled DNA fragment extending from -520 to -432 in the IXR1 promoter. N, negative control: binding reaction carried with the labelled DNA fragment extending from -390 to -319 in the IXR1 promoter rt, room temperature. (C) Competition experiments: W, binding without competitor; U, binding in the presence of unspecific competitor; S x20 , binding in the presence of specific competitor in x20 molar excess; S x100 , binding in the presence of specific competitor in x100 molar excess. Unspecific and specific competitors are defined in Materials and Methods (D) EMSA assays carried out with protein extracts from wild type (wt) or ∆ixr1 yeast cells grown in aerobiosis or hypoxia and the labelled DNA fragment extending from -520 to -432 in the IXR1 promoter. The retarded band obtained in each experiment is indicated by an arrow.

Fig. 4

 4 Fig. 4 In vivo binding of Ixr1p and Rox1p to the regulated IXR1 and ROX1 promoters (A) Binding to the IXR1 promoter. (B) Binding to the ROX1 promoter. ChIP experiments were done as explained in Materials and Methods. Strains used for immunoprecipitation are: I, Z1580, expressing the tagged Ixr1p; R, Z1465, expressing the tagged Rox1p. Culture conditions were: A, aerobiosis; H, hypoxia. Addition/omission of IgG or the specific c-Myc antibody (Ab) to the immunoprecipitation reactions is indicated by +/-. IN, PCR amplification from Imput ; IP, PCR amplification from immunoprecipitates.

Fig. 5

 5 Fig. 5 IXR1 expression and oxidative stress caused by peroxides (A) Resistance to H 2 O 2 in the wild type and ∆ixr1 strains. Serial dilutions of the cells (1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ) were obtained from an initial culture of OD 600 = 0.05 and 10 µL were grown at 30 ºC for three days on CM plates or CM plates with different concentrations of H 2 O 2 as specified. (B) Expression of the reporter gene MEL1 under the IXR1 promoter after H 2 O 2 treatment and dependence of this expression on Yap1p. Once the culture reached OD 600 = 0.8, an aliquot was saved for further analysis and H 2 O 2 was added to the medium to a final concentration of 1 mM. New aliquots were collected 15, 30, 45 and 60 min later and αgalactosidase activity determined.

Fig. 6

 6 Fig. 6 Model for the aerobic and hypoxic control of IXR1 expression Factors that directly or indirectly control IXR1 transcription in aerobiosis or hypoxia and IXR1-ROX1 cross-regulation.

  Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited wt ∆ixr1 ∆ecm22 ∆upc2 wt ∆ixr1 ∆ecm22 ∆upc2 wt ∆ixr1 ∆ecm22 ∆upc2 Castro et al., Fig1 Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limitedα w t / p I X R 1 : : M E L 1 ∆ p M E L α ∆ i x r 1 / p I X R 1 : : M E L 1Castro et al., Fig2Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Castro et al., Fig3 Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited I I R R I I II R R R R strain A H A H A A H H A A H H condition + -+ -+ -+ -IgG -+ -+ -+ -+ AbIN IP I I R R I I I I R R R R strain A H A H A A H H A A H H condition + -+ -+ -+ -IgG -+ -+ -+ -+ AbBiochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Castro et al., Fig5 Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500

Table 1

 1 Strains used in this work

	Strain	Genotype

Table 2

 2 Oligonucleotides used in this study

	Name	Sequence

(W, Watson; C, Crick). Lower case, sequences added for restriction or recombination. Underlined, restriction sites

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 06 Oct 2009 as manuscript BJ20091500 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091500 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We thank BioMedES (http://www.biomedes.co.uk/) for revision of English language. We thank to Pfr. Richard A Young (Massachusetts Institute of Technology, Cambridge, USA) for the S. cerevisiae strains Z1580 and Z1465. Emilie Dambroise, foreign student from the Erasmus exchange program, initially helped in the construction of pIXR1::MEL1. This research was supported by grants BFU2006-03961 from MCYT (Spain) and BFU2009-08854 from MICINN (Spain) co-financed by FEDER (CEE). General support to the lab during 2008-09 was funded by "Programa de axudas para a consolidación e a estruturación de unidades de investigación competitivas do sistema galego de I+D+i" (Xunta de Galicia). RC received a pre-doctoral fellowship 2007 from Xunta de Galicia. PSF was supported with charge to Parga Pondal Program-2006 (Xunta de Galicia). M.E.C. was sponsored during 2007 by "Programa de promoción da intensificación da actividade investigadora no sistema universitario de Galicia" (Xunta de Galicia).


Elmar Jaenicke
email: elmar.jaenicke@uni-mainz.de

Kay Büchler

Jürgen Markl

Heinz Decker

Thomas Rm Barends

The Cupredoxin-like Domains in Hemocyanins

Keywords: hemocyanin, cupredoxin, evolution, copper protein, mollusca

AcH

-Aplysia californica hemocyanin CBP -cucumber basic protein CopC -copper carrier protein FU -functional unit HtH1, HtH2

-isoform 1 and 2 of Haliotis tuberculata hemocyanin KLH -keyhole limpet (Megathura crenulata) hemocyanin KLH1, KLH2

-isoform 1 and 2 of KLH KLH1-h -functional unit h of KLH1 KLH1-h-D1 (-D2, -D3) -domains 1, 2 and 3 of KLH1-h NpH1, NpH2

-isoform 1 and 2 of Nucula nucleus

Introduction

Hemocyanins are giant oxygen transport proteins found in the hemolymph of many arthropods and molluscs. They reversibly bind oxygen, often with very high cooperativity, at binuclear type 3 copper centers (types 1 and 2 are mononuclear, respectively) [START_REF] Van Holde | Hemocyanins and invertebrate evolution[END_REF]2]. The copper centers of all hemocyanins are very similar, with the two copper atoms being bound to a four -helix bundle and ligated by three histidines each [START_REF] Cuff | Crystal structure of a functional unit from Octopus hemocyanin[END_REF][START_REF] Gaykema | 3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus haemocyanin[END_REF][START_REF] Magnus | Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences[END_REF][START_REF] Perbandt | The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity[END_REF]. As type 3 copper proteins, hemocyanins are structurally related to the phenoloxidases, tyrosinases and catecholoxidases; all these proteins bind oxygen in a side-on (µ-2 : 2 ) coordination between the two copper atoms of the active site [START_REF] Matoba | Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis[END_REF].

In spite of their common identity as type 3 copper proteins, the hemocyanins of arthropods and molluscs have been considered as two distinct protein families, based on differences in their amino acid sequences and quaternary structures. [START_REF] Burmester | Molecular evolution of the arthropod hemocyanin superfamily[END_REF][START_REF] Decker | Recent progress in hemocyanin research[END_REF][START_REF] Lieb | Structures of two molluscan hemocyanin genes: significance for gene evolution[END_REF]. Molluscan hemocyanins are semi-hollow cylinders 35 nm in diameter representing decamers, didecamers or multidecamers of a 350-400 kDa subunit. The latter is a concatenation of 7 or 8 paralogous functional units (FU) of ~50 kDa each, termed FU-a to FU-h. Thus, 160 oxygen binding sites exist in a molluscan hemocyanin didecamer such as isoform 1 of keyhole limpet hemocyanin (KLH1) [START_REF] Gatsogiannis | Keyhole limpet hemocyanin (KLH): 9 Å cryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units[END_REF]. KLH is a mixture of two isoforms, KLH1 and KLH2, isolated from the hemolymph of the giant keyhole limpet Megathura crenulata, and widely used as an immunological tool [START_REF] Harris | Keyhole limpet hemocyanin: Molecular structure of a potent marine immunoactivator[END_REF]. In contrast, arthropod hemocyanins occur as hexamer (1x6mer) or oligo-hexamer (2x6mer, 4x6mer, 6x6mer, 8x6mer) assembled from several paralogous subunit types, each of molecular mass ~75 kDa and with a single active site [13]. The largest arthropod hemocyanin type occurs in horseshoe crabs such as Limulus polyphemus; it is a cubic 8x6mer of 25 nm length, with 48 oxygen binding sites; it has recently been analysed structurally in detail [START_REF] Martin | Limulus polyphemus hemocyanin: 10 Å structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers[END_REF]. From a functional viewpoint, the arthropod hemocyanin subunit corresponds to the molluscan hemocyanin FU, each showing a single active site.

Representative crystal structures of the molluscan hemocyanin FU and the arthropod hemocyanin subunit are available [START_REF] Cuff | Crystal structure of a functional unit from Octopus hemocyanin[END_REF][START_REF] Gaykema | 3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus haemocyanin[END_REF][START_REF] Magnus | Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences[END_REF][START_REF] Perbandt | The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity[END_REF], but questions about the evolutionary connection between the two hemocyanin families remain. The arthropod hemocyanin subunit folds into three structural domains: The N-terminal domain #1 is predominantly -helical and shields the entrance to the active site. The central domain #2 contains the active site in a 4--helix bundle motif. The C-terminal domain #3 folds into a 7-stranded anti-parallel -barrel, a feature previously interpreted as being immunoglobulin-like [15]. Whether in the basic hexamer domain #3 has a role other than structural is unknown, but in oligo-hexamers it certainly contributes to allosterism [START_REF] Martin | Limulus polyphemus hemocyanin: 10 Å structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers[END_REF]. Most molluscan hemocyanin FUs fold into only two structural domains: The N-terminal core domain (D1) contains the oxygen binding site in a 4--helix bundle and therefore corresponds to domain #2 in arthropod hemocyanin, although their remaining tertiary structure elements are very different. The C-terminal domain (D2) is dominated by a 6-stranded -sandwich and shields the entrance to the active site. Hence, its role in molluscan hemocyanin is comparable to that of domain #1 in arthropod hemocyanin [START_REF] Decker | Recent progress in hemocyanin research[END_REF].

There is one exception from the standard architecture of molluscan hemocyanin FUs, namely the C-terminal FU-h. Rather than 50 kDa, its molecular mass is 60 kDa due to a C-terminal extension of ~100 amino acids which marks the very end of the 400 kDa polypeptide [START_REF] Lieb | The sequence of a gastropod hemocyanin (HtH1 from Halotis tuberculata)[END_REF]. The sequence of this peculiar tail is unrelated to any other protein in the databases, and therefore its fold remained unknown as yet. In this paper we report the 4 Å crystal structure of KLH1-h, and discuss the structural and evolutionary implications of its tail domain. Thereby, we will substantially reinterprete the phylogenetic origin and functional role of the structural domains found in molluscan and arthropod hemocyanins.

Experimental

Purification. KLH1 was purified from Megathura crenulata hemolymph serum (Biosyn, Fellbach, Germany) by selective dissociation of KLH2 as described elsewhere [START_REF] Harris | Keyhole Limpet Hemocyanin (KLH): Purification of Intact KLH1 Through Selective Dissociation of KLH2[END_REF]. Briefly, overnight dialysis against 2 % ammonium molybdate (pH 5.7) at 4 °C results in selective dissociation of KLH2 into its subunits, while KLH1 remains intact as multimers. KLH isoforms were then separated by pelleting the intact KLH1 multimers at 30,000 g for 4 h at 4 °C, while leaving KLH2 subunits in the supernatant. The pellet was resolved in stabilizing buffer (150 mM NaCl, 50 mM Tris/HCl, 5 mM MgCl 2 , 5 mM CaCl 2 , pH 7.4) and then dialyzed overnight against dissociation buffer (130 mM glycine/NaOH, pH 9.6) to dissociate KLH1 multimers into their subunits. Purified KLH1 subunits were subjected to limited proteolysis by Staphylococcus aureus V8 protease type XVII (Sigma-Aldrich, Taufkirchen). KLH1 subunits (10 mg/ml final concentration) dissolved in proteolysis buffer (100 mM NH4HCO3, pH 8.0) were mixed with V8 protease (2 % (w/w) final concentration) and incubated at 37 °C for 5 h. Proteolytic cleavage was terminated by adding proteolysis inhibitor (Pefabloc®, Roth, Karlsruhe, Germany) to a final concentration of 10 mM. KLH1-h was purified from the resulting protein fragments by anion exchange chromatography on a Q-Sepharose column (V = 25 ml, Pharmacia, Freiburg, Germany). To this end about 50 mg of protein fragments were applied to the column equilibrated with 20 mM Tris/HCl (pH 8.0). Fragments were then eluted by a linear sodium chloride gradient from 160 mM to 400 mM NaCl in the same buffer at a flow rate of 0.5 ml/min (Fig. S1,A). The KLH1-h content and purity of the eluting fractions was assayed by SDS-PAGE on 10 % polyacrylamide gels stained with Coomassie brilliant blue [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the bacteriophage T4[END_REF] and by immunoelectrophoresis using rabbit antibodies against dissociated KLH1 [START_REF] Markl | Subunit-specific monoclonal antibodies to tarantula hemocyanin, and a common epotope shared with calliphorin[END_REF]20] (Fig. S1,BC). Absorption spectra were measured with a U-3000 spectrophotometer (Hitachi, Japan) in quartz cuvettes with 1 cm pathlength. For obtaining deoxy spectra KLH1-h was deoxygenated by addition of sodium dithionite.

Crystallization. Preliminary crystallization conditions were determined for batch-under-oil by high-throughput screening at the Hauptman-Woodward Institut (Buffalo, NY, USA) and then refined for the sitting-drop setup [21]. Crystals were grown on microbridges in Linbro plates covered with cover slides (Hampton Research, Aliso Viejo, CA, USA). Briefly, 5 µl of a 15 mg/ml KLH-h solution were mixed with 5 µl reservoir solution (9.0 % (w/v) PEG 1000, 50 mM K2HPO4, 50 mM sodium citratebuffer, pH 4.0) and left to equilibrate in the sealed well at 20 °C. Single, non-birefringent crystals were obtained after 10 days which were shaped like rhombic dodecahedra and had a maximum dimension of 0.1 mm (Fig. S1,D). statistics are shown in Table 1. Molecular replacement with PHASER [START_REF] Mccoy | Likelihood-enhanced fast translation functions[END_REF] using the structure of the homologous FU-g from Octopus dofleini hemocyanin (OdH-g ) (pdb code: 1JS8 [START_REF] Cuff | Crystal structure of a functional unit from Octopus hemocyanin[END_REF]) as the search model identified the space group as I213 and showed the asymmetric unit to contain a dimer, indicating a solvent content of 83 %. Between the two monomers of the search model, clear additional electron density indicating -sheet topology could be seen for the C-terminal domain that is not present in the search model. Prime-and-switch density modification using RESOLVE [23,24] starting from the phases calculated by PHASER resulted in an electron density map in which the fold of the C-terminal domain could be traced with high confidence. C atoms for the C-terminal domain were placed using COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and XFIT [START_REF] Mcree | XtalView/Xfit -A versatile program for manipulating atomic coordinates and electron density[END_REF] and were submitted to the DALI server [START_REF] Holm | Protein structure comparison by alignment of distance matrices[END_REF][START_REF] Holm | Mapping the protein universe[END_REF] to find similar folds. Strikingly, the closest match (Z-score 7.4) was the structure of cucumber basic protein (pdb code: 2cbp [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF]) which is a type I copper protein. Based on the similarity of KLH1-h with the folds of FU-g of Octopus hemocyanin (OdH-g) and cucumber basic protein, a homology model of the KLH1-h was built using the SWISS-MODEL server [START_REF] Arnold | The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling[END_REF][START_REF] Schwede | SWISS-MODEL: an automated protein homology-modeling server[END_REF] to aid in obtaining a tentative assignment of the sequence to the C trace (Fig. S2).

Results and Discussion

C -trace of KLH1-h. Functional unit KLH1-h was released from the KLH1 subunit by limited proteolysis, preparatively purified, crystallized and its three-dimensional structure solved to a resolution of 4 Å. This allowed tracing its C -backbone, encompassing 490 amino acids (Fig. 1, Fig 2A .1). It folds into three well defined structural domains: The N-terminal core domain (KLH1-h-D1) and the central -sandwich domain (KLH1-h-D2) exhibit the canonical molluscan hemocyanin fold already known from the crystal structures of two other FU types, FU-e and FU-g [START_REF] Cuff | Crystal structure of a functional unit from Octopus hemocyanin[END_REF][START_REF] Perbandt | The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity[END_REF]. The additional C-terminal domain (KLH1-h-D3) contains mainly -strands (Fig. 1, Fig. 2A.1). It shares a striking and completely unexpected structural similarity with cucumber basic protein (CBP) which has a type 1 copper site (Fig. 1C, Fig. 2A). CBP is a member of the cupredoxin protein family and belongs to the sub-group plantacyanins [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF]. The cupredoxin-like fold of KLH1-h-D3 resembles a Greek key -barrel, except that, as in CBP, the barrel is open at one side and thus might better be described as -sandwich or " -taco" [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF]. The rms deviation between KLH1-h-D3 and CBP is 2.5 Å. Moreover, KLH1-h-D3 and CBP show a disulphide bridge at identical positions, with the two cysteines separated by 20 amino acids, accurately. On the other hand, their sequence identity is only ~10%, and consequently, they show specific physico-chemical differences (Fig. 2, A.2). In particular, the isolelectric point (pI) of KLH1-h-D3 is 6.3 (predicted from the sequence), while the pI of CMP is 11 [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF]. Moreover, this low degree of sequence identity prohibits using CBP as template for molecular modeling of the KLH1-h-D3 sequence. Therefore, the present considerations are restricted to the C backbone.

Copper active sites. As CBP contains a type 1 copper site [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF], this was also to be expected for the cupredoxin-like fold of KLH1-h-D3 and would mean a second copper center in FU-h, in addition to the canonical type 3 copper site. In blue copper proteins such as cupredoxins, the copper (I) ion form in the type 1 copper site is stabilised by a constrained His 2 Cys coordination environment. Unfortunately 4 Å resolution as presently achieved did not resolve the binuclear copper center in KLH1-h-D1, and therefore, the chance of detecting a putative single copper in KLH1-h-D3 is very low. However, the amino acid sequence of KLH1-h-D3 lacks any histidine or cysteine residues in appropriate position that could allow copper binding to the protein (a His 2 Cys coordination environment in CBP and other coppercontaining cupredoxins). Consequently, we assume that KLH1-h-D3 is devoid of a type 1 copper center which might have been lost at an early stage of molluscan hemocyanin evolution (Fig. 1C). A similar situation is encountered in the murine ephrin ectodomain, which also shares the cupredoxin fold with CBP, but lacks the copper binding site [START_REF] Toth | Crystal structure of an Ephrin Ectodomain[END_REF]. In this context, the presence of a disulphide bridge in KLH1-h-D3 and the ephrin ectodomain at a conserved position is remarkable, because in CBP it is thought to stabilize the type 1 copper center and is a typical feature of phytocyanins [START_REF] Guss | The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 Å resolution[END_REF]. Therefore, since no type 1 copper center is present, it might be required for the structural integrity of the entire cupredoxin-like fold. In the present context, this disulphide bridge is a strong argument in favour of our interpretation of KLH1-h-D3 as a cupredoxin-like protein.

Oxygenated KLH1-h has a blue/purple color due to a broad absorption peak in the visible range with a maximum at 554 nm (Fig. S3). This peak disappears completely upon deoxygenation of KLH1-h which is typical for all hemocyanins and has been thoroughly characterized as resulting from the * v Cu(II) charge transfer transition, where * v is the peroxide orbital orthogonal to the Cu 2 O 2 plane of the type 3 copper center [33]. Consequently it can be ruled out that the cupredoxin-like domain of KLH1-h contains any additional ligands or active sites, which absorb light in the visible range.

Cupredoxin-like domains in hemocyanin. The unexpected presence of two copper protein folds within a molluscan hemocyanin functional unit raises the question as to whether the cupredoxin-like domain is a structural feature only encountered in FU-h of molluscan hemocyanins. Moreover, the structural and possibly functional role of this peculiar tail domain of the molluscan hemocyanin subunit should be addressed. A recent database search for structural correlates of the C-terminal domain #3 of arthropod hemocyanin unearthed another member of the cupredoxin family, the bacterial CopC protein [34] (Fig. 2B). CopC is one of four proteins encoded by the copABCD operon of Pseudomonas syringae pv tomato and confers resistance to the pathogen against high copper levels [START_REF] Cha | Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins[END_REF]. The CopC protein has a -barrel structure and features two distinct mono-copper binding sites. It has been considered to represent a link between copper-trafficking proteins and cupredoxins [34,[START_REF] Cha | Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins[END_REF].

From an evolutionary point of view, it is remarkable that the cupredoxin fold motif can be found in both molluscan and arthropod hemocyanins (Fig. 2, Fig. 3), because as already mentioned, there are convincing arguments that both hemocyanin families evolved convergently from tyrosinase/phenoloxidase-like ancestors [START_REF] Burmester | Molecular evolution of the arthropod hemocyanin superfamily[END_REF]. Indeed, hemocyanins can be considered as permanently inactivated phenoloxidases [START_REF] Van Holde | Hemocyanins and invertebrate evolution[END_REF][START_REF] Decker | Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins[END_REF]. It now appears that both hemocyanin families evolved from proteins containing a type 3 and a type 1 copper site. In principal, the combination of several copper centers is not unusual. There exist blue oxidases such as laccase, ascorbate oxidase or ceruloplasmin which combine a mixed type 2 and type 3 copper center along with a type 1 copper center [START_REF] Davies | Laccases. In Handbook of Metalloproteins[END_REF][38][39]. However, the combination of a pure type 3 copper center and a type 1 copper center in a single protein as it is suggested here for ancestral hemocyanins is unprecedented. Moreover, it is remarkable that the copper 1 site seems to have been lost in both recent hemocyanin families.

Possible role(s) of the cupredoxin-like hemocyanin domains.

If in fact a functional type 1 copper site in ancestral hemocyanins did exist, one might suppose that its cooperation with the type 3 copper center could significantly improve oxidative processes. However, the large distance between the two copper centers (more than 30 Å in arthropod hemocyanin and FU-h, A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. respectively) does not support this scenario: In blue oxidases such as laccase, ascorbate oxidase or ceruloplasmin, in which an electron transfer between copper centers is documented, their maximum distance is ~13 Å [39]. Thus, a similar coupling of the two sites in the framework of an ancient enzymatic function seems unlikely in case of both hemocyanins.

A more plausible explanation for the presence of the cupredoxin-like domain in hemocyanins may be deduced from Streptomyces tyrosinase that has a hemocyanin-like type 3 copper site. The structure of this tyrosinase complexed with its "caddie" protein (MelC1) was recently solved (Fig. 2C) [START_REF] Matoba | Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis[END_REF]. The small copper binding caddie protein is coexpressed with tyrosinase and apparently responsible for correct copper loading to the active site of the latter [START_REF] Matoba | Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis[END_REF]. In other words, the "caddie" protein appears to serve as copper chaperone of the tyrosinase.

Copper insertion into proteins is an incompletely understood phenomenon and little is known about copper loading to type 3 copper proteins. Typically copper concentrations within the cell are so low that most copper proteins need copper chaperones to help load the active site [START_REF] Poulos | Helping copper find a home[END_REF]. We therefore assume that in the case of the hemocyanins, the ancestral tyrosinases that gave rise to arthropod and molluscan hemocyanin, respectively, combined with their copper chaperones by gene fusion resulting in a more efficient copper loading onto their active sites.

If so, then why did the copper binding site of the cupredoxin-like domain in both hemocyanin families degenerate? In tyrosinase, the active site is solvent accessible, allowing bulky substrates to approach the type 3 copper site for catalysis [START_REF] Decker | The first crystal structure of tyrosinase: all questions answered?[END_REF]. Thus, loss of copper atoms could easily occur during the lifetime of the tyrosinase molecule which would make it necessary to reload copper with the help of the "caddie" protein. In hemocyanins as oxygen carriers, the type 3 copper site is protected from the solvent because it is covered by additional domains (domain D2 in molluscan and domain #1 in arthropod hemocyanin), leaving only a narrow oxygen diffusion channel [START_REF] Decker | Recent progress in hemocyanin research[END_REF]. Thus, the probability for hemocyanins to lose copper during their lifetime is very low and the necessity to reload lost copper atoms would have faded. In such an instance, we assume that copper loading during hemocyanin biosynthesis was outsourced to dedicated copper chaperones and consequently, selective pressure to conserve the copper center decreased in the cupredoxin-like domains of hemocyanins.

The further evolution of hemocyanin as an extracellular, highly allosteric oxygen carrier was then dominated by the process of forming large macromolecular assemblies. In arthropods, the basic quaternary structure is the hexamer (1x6mer). For this, domain #3 (the cupredoxinlike domain) was probably not directly required, apart from generally stabilizing the individual subunits, because it is not involved in subunit/subunit bonding [START_REF] Gaykema | 3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus haemocyanin[END_REF]. However, in 4x6mer/8x6mer chelicerate hemocyanins, domain #3 is directly involved in a variety of interhexamer interfaces that also might transfer allosterism [START_REF] Martin | Limulus polyphemus hemocyanin: 10 Å structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers[END_REF]. Even in the "simple" case of a spider 2x6mer formed from two hexamers by a single disulfide bridge, domain #3 is directly involved [START_REF] Ballweber | Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders[END_REF]. Consequently, domain #3 is an indispensable structural and functional element in the architecture and allosterism of oligo-hexameric arthropod hemocyanins.

In molluscan hemocyanins, domain D3 was abandoned in seven of the eight types of functional unit, namely in FU-a to FU-g [START_REF] Lieb | The sequence of a gastropod hemocyanin (HtH1 from Halotis tuberculata)[END_REF][START_REF] Miller | Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications[END_REF]. Indeed, a detailed analysis of the FU topology of the cephalopod hemocyanin decamer revealed that for spatial reasons such a component would not be compatible with the architecture of the cylinder wall [START_REF] Gatsogiannis | Nautilus pompilius hemocyanin: 9 Å cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units[END_REF]. However, in the internal collar of the gastropod hemocyanin decamer, the characteristic dipentameric ring made from ten copies of FU-h clearly requires domain D3 as a connecting element [START_REF] Gatsogiannis | Keyhole limpet hemocyanin (KLH): 9 Å cryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units[END_REF].

In this context it should be noted that according to a previous hypothesis based on gene sequence data, the molluscan hemocyanin quaternary structure originated from a primordial dipentameric FU-h ring [START_REF] Lieb | Structures of two molluscan hemocyanin genes: significance for gene evolution[END_REF]. This is further support for the present idea that the cupredoxinlike domain is an ancestral feature of hemocyanins.

In summary we describe cupredoxin-like domains encountered in arthropod and molluscan hemocyanins. Today they specifically function, within the various quaternary structures, as connecting elements and notably in arthropods they apparently contribute to allosteric force transfer. With respect to evolution we suggest that their original function was associated with copper loading of the active site. Thus, the cupredoxin-like domains open new insights in the adaptive evolution and function of hemocyanins.

Fig. 3. Putative phylogenetic origin of the hemocyanin domains

The oxygen binding domain (white) of arthropod and molluscan hemocyanin presumably evolved independently from ancestral tyrosinases. To guarantee efficient copper loading to the active site in the oxygen binding domain a cupredoxin-like domain (dark grey) was added, endowing the subunit with an additional monocopper center. A high specificity for oxygen was established by an additional domain (grey), which shields the binuclear copper site from regular tyrosinase substrates, but still allows entry and exit of oxygen molecules. Moreover, this established allosteric regulation via slight tilting movements of the shielding domain which opens and closes the narrow oxygen diffusion channel [START_REF] Decker | Recent progress in hemocyanin research[END_REF]. Later in evolution of hemocyanin, the cupredoxin-like domain assumed new functions in assembly and allosterism of the hemocyanin multimers, and its monocopper center became obsolete and was lost. Today the cupredoxin-like domain is still ubiquitous in the 75 kDa arthropod hemocyanin subunits, whereas in molluscan hemocyanin it is restricted to FU-h where it marks the tail of the 400 kDa subunit. Nevertheless, this means that each molluscan hemocyanin didecamer carries 20 copies of this peculiar domain [START_REF] Gatsogiannis | Nautilus pompilius hemocyanin: 9 Å cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Structure determination .

 determination The crystals were cryocooled in liquid nitrogen after being cryoprotected by soaking them in reservoir solution containing 30% (v/v) glycerol. The crystals diffracted to 4.0 Å resolution at the PX-II beamline of the Swiss Light Source, Villigen, Switzerland. Data processing indicated I-centered cubic symmetry, and data Biochemical Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501

Biochemical

  Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 . 1 , 2 , 1 , 1 ,

 21211 Fig. 2. Cupredoxin-like domains in hemocyanins A.1, Superposition of KLH1-h (red, domain D3) and CBP (pdb code: 2CBP, yellow). A.2, Superposition of KLH1-h-D3 and CBP. B.1, Superposition of Limulus hemocyanin subunit II (pdb code: 1LLA; red, domain #3) and CopC (pdb code: 2C9Q, yellow). B.2, Superposition of domain #3 of Limulus hemocyanin subunit II and CopC (with several loops of #3 omitted for clarity). C.1, Streptomyces tyrosinase (pdb code: 1WX4, blue) associated with the caddie protein (pdb code: 1WX4, yellow). Orange spheres represent copper atoms; CopC binds a second copper atom if present as a subunit dimer (not shown).

Biochemical 18 Fig. 1 :

 181 Fig. 1: Jaenicke et al, Cupredoxin like domains in hemocyanins

Fig. 2 :Fig. 3 :

 23 Fig. 2: Jaenicke et al, Cupredoxin like domains in hemocyanins

Table 1 . Data collection statistics

 1

	Biochemical Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501
	Tables
	Wavelength	0.97941 Å
	Space group	I2 1 3
	Unit cell	a=b=c=251.0 Å
	Resolution limits	30 -4.2 Å (4.2-4.0 Å)
	No. unique reflections	21995 (2893)
	R merge	0.189 (0.847)
	I/ I	14.2 (3.5)
	Completeness	96.4 % (96.4 %)
	FOM after
	PHASER	0.51
	RESOLVE	0.61

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgments

We wish to thank Prof. Dr. Ilme Schlichting (Max-Panck Institute for Medical Research, Heidelberg) for providing access to X-ray sources. Also, we thank Prof. Dr. Joseph Bonaventura (Duke University, USA) for discussion and revision of the manuscript.

The structure of KLH1-h was deposited in the Protein Data Bank with accession code 3EU2.

Funding

Granted by "GK 1043 -International Graduate School of Immunotherapy" (DFG) and "Research Center Immunology" of the Johannes Gutenberg University. Biochemical Journal Immediate Publication. Published on 21 Dec 2009 as manuscript BJ20091501 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091501

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.