Jofre Ferrer-Dalmau

Asier González

Maria Platara

Clara Navarrete

José L Martínez

Lina Barreto

José Ramos

Joaquín Ariño

Dr Antonio Casamayor
email: antonio.casamayor@uab.es

Ref2, a regulatory subunit of the yeast protein phosphatase 1, is a novel component of cation homeostasis

Keywords: type 1 protein phosphatase, cation tolerance, calcineurin, ENA1, vacuolar defects, APT complex CDRE, Calcineurin Dependent Response Element, RLU, Relative Luminiscence Units

Maintenance of cation homeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (Na + , Li + , Ca +2 ) and alkaline pH. Among eighteen candidates, only deletion of REF2 yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. A Phe-374 to Ala mutated version of Ref2, which cannot bind Glc7, is unable to rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na + -ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homeostasis may be unrelated to its previously identified function as a component of the APT complex.

Introduction

An elevated intracellular concentration of sodium ions is harmful for most eukaryotic cells, probably because it interferes with the correct functioning of certain cellular targets [START_REF] Serrano | Salt tolerance in plants and microorganisms: toxicity targets and defense responses[END_REF]. Yeasts, as well as other single-cell eukaryotic organisms, have developed mechanisms to maintain a safe cytosolic concentration of this cation even when the external concentration of sodium ions in their natural environments is high. To this end, these organisms employ three distinct strategies: a) discrimination among different alkali metal cations at the level of uptake (favouring transport of potassium vs sodium), b) triggering the efficient efflux of toxic cations, and c) sequestering the excess of cations in organelles, such as the vacuole.

The budding yeast Saccharomyces cerevisiae is a model organism used very often for cation tolerance studies. In this organism, the capacity to extrude sodium (or other toxic cations, such as lithium) represents a major mechanism to maintain low intracellular levels of these toxic cations. This is achieved by two different and complementary ways. First, the function of an H + /Na + antiporter encoded by the NHA1 gene, which is able to extrude Na + , Li + , and even K + cations by exchange with protons and, therefore, acquires biological relevance at rather acidic external pHs [2;3]. The second mechanism is based on a P-type ATPase pump encoded by the ENA system (see [START_REF] Ruiz | Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system[END_REF] for a recent review). In S. cerevisiae the ENA genes are disposed in tandem repeats with a variable number of copies (ENA1-ENA5) encoding identical or very similar proteins [START_REF] Haro | A novel P-type ATPase from yeast involved in sodium transport[END_REF][START_REF] Garciadeblas | Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae[END_REF][START_REF] Martinez | A PMR2 tandem repeat with a modified Cterminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae[END_REF]. Deletion of the entire ENA cluster in S. cerevisiae results in a dramatic phenotype of sensitivity to sodium and lithium cations or alkaline pHs [5;6;8;9]. It is widely accepted that ENA1 is the functionally relevant component of the cluster on the basis that 1) cation sensitivity is largely restored by expression of ENA1 [6;10] and, 2) whereas under standard growth conditions expression from the ENA genes is almost undetectable, ENA1 expression dramatically increases in response to osmotic, saline or alkaline pH stress [6;8;9;11].

Regulation of ENA1 expression in response to saline or alkaline stress is under the control of different pathways [START_REF] Marquez | Multiple transduction pathways regulate the sodiumextrusion gene PMR2/ENA1 during salt stress in yeast[END_REF]. These include the HOG osmoresponsive MAP kinase Hog1, the calcium-activated protein phosphatase calcineurin, the Rim101/Nrg1, the Snf1 and the Hal3/Ppz1 pathways, among others (see [START_REF] Ruiz | Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system[END_REF] and references therein). Activation of calcineurin upon saline or alkaline pH stress results in dephosphorylation and nuclear entry of the Crz1 transcription factor [START_REF] Matheos | Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae[END_REF][START_REF] Stathopoulos | Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast[END_REF][START_REF] Mendizabal | Yeast putative transcription factors involved in salt tolerance[END_REF], which binds to specific sequences, known as CDRE, in calcineurin-responsive promoters. Two CDREs have been defined in the ENA1 promoter (-821/-813 and -727/-719) the downstream element being the most relevant for induction of the gene in cells exposed to saline or high pH stress [START_REF] Mendizabal | Promoter sequences regulated by the calcineurin-activated transcription factor Crz1 in the yeast ENA1 gene[END_REF][START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF][START_REF] Viladevall | Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae[END_REF][START_REF] Ruiz | Direct regulation of genes involved in glucose utilization by the calcium/calcineurin pathway[END_REF].

Protein phosphatase 1 is a conserved Ser/Thr protein phosphatase whose catalytic subunit is encoded in S. cerevisiae by a single, essential gene, named GLC7 [20;21]. The Glc7 protein is required for a myriad of cellular functions and specific regulation of these functions is achieved by interaction of the catalytic subunit with a variety of regulatory proteins, which influence Glc7 intracellular localization and/or substrate recognition. Many of these regulatory subunits interact with Glc7 through a conserved binding site, which usually appears as a (R/K)(V/I)X(F/W) consensus sequence [22;23] . A search of currently available S. cerevisiae databases (i.e. Saccharomyces Genome Database, http://www.yeastgenome.org/) reveals at least 119 proteins that are annotated as Glc7 interacting proteins. However, most of these relationships are based on recent high- throughput analysis, whereas compelling regulatory evidence has been only collected for a limited subset of these proteins.

Among the cellular functions attributed to the Glc7 phosphatase, a role in ion homeostasis was postulated some years ago as a result of the characterization of the glc7-109 allele [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF], which carried a mutation at Arg-260 found to be responsible for the mutant phenotypes. glc7-109 mutants displayed, among other phenotypes, a marked sensitivity to Na + cations and alkaline pH, which was remedied by inclusion of potassium ions in the medium. Interestingly, Arg-260 lies in the vicinity of Phe-256, a residue in the Glc7 hydrophobic channel that makes contact with the aromatic residue of the (R/K)(V/I)X(F/W) motif present in many regulatory subunits. Therefore, we hypothesized that the defect in ion homeostasis in the glc7-109 mutant could be due to a deficient interaction of the mutated protein with a regulatory subunit relevant for ion tolerance. To this end, we have analyzed the tolerance to diverse cations of eighteen known or putative Glc7 regulatory subunits and found that deletion of REF2 yields a rather severe and unique phenotype of sensitivity to Na + , Li + and alkaline pH. REF2 was initially described as a gene encoding a protein required for mRNA 3'-end maturation prior to the final polyadenylation step [START_REF] Russnak | REF2 encodes an RNA-binding protein directly involved in yeast mRNA 3'-end formation[END_REF]. Evidence for interaction between Ref2 and Glc7 was obtained by both two-hybrid and affinity purification techniques [START_REF] Uetz | A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae[END_REF][START_REF] Walsh | Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry[END_REF][START_REF] Gavin | Functional organization of the yeast proteome by systematic analysis of protein complexes[END_REF]. Subsequently, it has been found that Ref2 is a component of the APT (for Associated with PTa1) subcomplex of the large cleavage and polyadenylation factor (CPF)-containing complex (holo-CPF), which is required for formation of mRNA and snoRNA 3'-ends and also contains Glc7 [START_REF] Nedea | Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'ends[END_REF]. It has been shown that Ref2 is required for the permanence of Glc7 within the complex [START_REF] Nedea | The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes[END_REF]. The Ref2 protein contains a 368 RISSIKFLD 376 sequence that resembles the Glc7 binding motif and recent evidence indicates that mutation of Ref2 Phe-374 to Ala disrupts the interaction between this protein and Glc7, thus preventing incorporation of Glc7 to the APT subcomplex [START_REF] Nedea | The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes[END_REF] In this work, we highlight the phenotype of cation sensitivity of the ref2 mutant and investigate the molecular basis of this defect. Our results indicate that mutation of REF2 results in a deficient efflux of toxic cations that can be, at least in part, attributed to impaired expression of the ENA1 ATPase gene. Remarkably, this role on salt tolerance seems unrelated to the function of Ref2 in the APT complex.

Experimental

Growth of Escherichia coli and yeast strains

Escherichia coli DH5 cells were used as plasmid DNA host and were grown at 37 ºC in LB supplemented with 50 g/ml ampicillin when plasmid selection was required. Yeast cells were cultivated at 28 °C in YPD medium (10 g/l yeast extract, 20 g/l peptone, and 20 g/l dextrose) or in synthetic complete drop-out medium when carrying plasmids. Restriction reactions, DNA ligations and other standard recombinant DNA techniques were performed as described elsewhere [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. Yeast cells were transformed using the lithium acetate method [START_REF] Gietz | Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method[END_REF]. Sensitivity of yeast cells to lithium, sodium or calcium chloride and alkaline pH was evaluated by growth on plates (drop tests) or in liquid cultures as previously described [8;33]. Growth under limiting potassium concentration was carried out using an YNB-based medium (Translucent K-free medium, prepared by Formedium, UK; manuscript in preparation) formulated so that potassium content in the final medium is negligible (~15 M).

Gene disruption and plasmid construction

Single KanMX deletion mutants in the BY4741 background (MATa his3 1 leu2 met15 ura3 ) were generated in the context of the Saccharomyces Genome Deletion Project [START_REF] Winzeler | Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[END_REF]. Replacement of the REF2 coding region by the nat1 marker from Streptomyces noursei was accomplished as follows. The 1.40 kbp DNA fragment containing the nat1 gene flanked by genomic sequences corresponding to -40 to -1 and +1603 to +1643 from the REF2 ATG codon was amplified from the plasmid pAG25 [START_REF] Goldstein | Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae[END_REF] with the oligonucleotides OJFD021 and OJFD022 (Supplemental Table 1) and transformed in the wild type strains BY4741 and its cnb1::KanMX derivative to yield strains YJFD1 and YJFD7, respectively. The ref2::nat1 mutation was also introduced in the wild type DBY746 background (MATa, ura3-52 leu2-3,112 his3 1 trp1 239) to yield strain YJFD5, which was used for intracellular calcium levels measurements. Positive clones were selected in the presence of 100 nourseothricin (Werner BioAgents, Germany) and the presence of the deletions was verified by PCR. Strains YJFD17 and YJFD18 were made by co-transformation of wild type BY4741 and its ref2::KanMX derivative, respectively, with plasmid pJFD10 (containing the glc7-109 allele, see below) and a disruption cassette containing the nat1 gene flanked by genomic sequences corresponding to -41 to -1 and +1465 to +1509 from the GLC7 ATG codon (amplified from plasmid pAG25 with the oligonucleotides OJFD81 and OJFD82). Diverse tests were carried out to ensure that the only Glc7-encoding gene copy corresponded to the plasmid-borne glc7-109 allele. Strains KT1112 (GLC7), KT1935 (glc7-109), and KT2210 (glc7-F256A), a generous gift from K. Tatchell, were previously reported [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF]. Strains SB6 (YTH1) and SB7 (yth1-1) [START_REF] Barabino | The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins[END_REF]; YWK168 (SWD2) and YWK172 (swd2-3) [START_REF] Dichtl | Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation[END_REF] as well as W303 (SSU72) and YWK186 (ssu72-2) [START_REF] Dichtl | A role for SSU72 in balancing RNA polymerase II transcription elongation and termination[END_REF] were a generous gift from B. Ditchl. Strains PTA1 (PTA1) and pta1 (pta1 1-75) [START_REF] Ghazy | The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing[END_REF] were kindly provided by C. Moore.

To clone the glc7-109 allele in the YCplac33 plasmid (centromeric, URA3 marker) the Glc7-encoding ORF flanked by 2068 bp upstream and 148 bp downstream was amplified by PCR using genomic DNA from strain KT1935 and oligonucleotides Glc7-BamHI.Up and Glc7-PstI.Do, which contain artificial BamHI and PstI restriction sites, respectively (Supplemental Table 1). The amplified DNA fragment was digested by BamHI and PstI and cloned in the same restriction sites of YCplac33 to yield plasmid pJFD10 (YCplac33-glc7-109). The plasmid was isolated and sequenced.

For low-copy, centromeric expression of REF2, a 2.28 kbp fragment containing the REF2 ORF flanked by 500 bp and 200 bp from its promoter and terminator regions, respectively, was amplified using a specific pair of oligonucleotides containing artificially added BamHI (OJFD027) and SacI (OJFD026) restriction sites (Supplemental Table 1). The resulting DNA fragment was digested with BamHI and SacI and cloned in the same restriction sites of the pRS415 plasmid (centromeric, LEU2 marker) to yield pRS415-REF2 (pJFD1).

To generate the F374A allele of REF2 (pJFD2), the pRS415-REF2 construct was used as a PCR template to change the TTT codon, encoding a phenylalanine residue at position 374, to a GCT codon, encoding an alanine residue. Two overlapping DNA fragments (a and b) were generated. The 1.75 bp fragment a, spanning from nt -501 to +1140 (relative to the initiating Met) was obtained using oligonucleotides OJFD029 and M13-20 reverse. The 0.7 bp length fragment b comprises from nt +1101 to +1785 and was amplified with oligonucleotides OJFD063 and M13-20 (Supplemental Table 1). To allow in vivo DNArecombination, these two fragments were co-transformed in yeast cells together with the linearized pRS415 plasmid digested with BamHI and SacI. Cells carrying the pRS415 [F374A] construct were selected in a medium lacking leucine and the plasmid was recovered and sequenced to ensure the absence of unexpected mutations.

LacZ reporter plasmids used in this work have been reported previously. ENA1 reporters were pKC201, containing the entire ENA1 promoter [START_REF] Cunningham | Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae[END_REF] and pMRK212 and pMRK213, containing the ARR1 and ARR2 regions of this same promoter [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF]. pMRK-based constructs derive from plasmid pSLF -178K [START_REF] Idrissi | Structural and functional heterogeneity of Rap1p complexes with telomeric and UASrpg-like DNA sequences[END_REF], which contains a CYC1 minimal promoter and displays virtually no reporter activity in the absence of a transcriptionally active DNA insert. Plasmid pAMS366 [START_REF] Stathopoulos | Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast[END_REF] contains four copies of a CDRE from the FKS2 promoter.

Lithium content and lithium efflux measurements.

Lithium content and efflux were measured as previously reported with minor modifications [START_REF] Ruiz | Role of protein phosphatases 2C on tolerance to lithium toxicity in the yeast Saccharomyces cerevisiae[END_REF]. For determination of the intracellular Li + , cells were grown overnight in liquid YPD medium. When cultures reached an optical density of 0.5-0.6, cells were collected and diluted (A 600 of 0.2-0.3), in fresh YPD medium containing the appropriate concentration of LiCl. Cells were loaded with lithium during 8 h and then samples were filtered, washed with 20 mM MgCl 2 , and treated with acid. Lithium was analyzed by atomic absorption spectrophotometry as previously described [START_REF] Gomez | The capacity to transport potassium influences sodium tolerance in Saccharomyces cerevisiae[END_REF]. Li + efflux was studied in cells grown overnight as described above. Cells were suspended in fresh medium and loaded with 75 mM LiCl for two hours. Yeast cells were separated from the culture medium by centrifugation, washed, and suspended in fresh lithium-free medium. At time intervals, samples were taken and treated as above for lithium determination. The different strains were transformed with the reporter plasmids pKC201, pMRK212, pMRK213 or pAMS366. Cultures were grown up to saturation in the appropriate dropout medium, then inoculated on YPD (pH 5,5) until A 660 0.6-0.7 was reached and centrifuged (5 min at 750 x g). Then cells were resuspended in fresh YPD adjusted to pH 5.5 containing the indicated concentrations of LiCl, NaCl or CaCl 2 , or adjusted to pH 8.2 in the presence of 50 mM TAPS buffer, and further incubated for 60 min, unless otherwise stated, at the specified temperatures. -galactosidase activity was measured as described previously [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF].

-Galactosidase Assays

Intracellular calcium monitoring

The determination of intracellular calcium was based in the use of pEV11/AEQ [START_REF] Batiza | Yeast respond to hypotonic shock with a calcium pulse[END_REF] which carries the APOAEQUORIN gene under the control of the ADH1 promoter. Evaluation of cytoplasmic calcium was carried out essentially as described previously [18;44]. Briefly, DBY746 wild type and ref2 mutant cells (YJFD5) were transformed with plasmid pEVP11/AEQ and grown to an A 660 of 1.0. Aliquots of 30 µl of culture were incubated in luminometer tubes for 20 min at room temperature. One-tenth volume of 592 µM coelenterazine (Sigma) in methanol was added to each sample and further incubated at room temperature for 20 min. Basal luminescence was recorded every 0.1 s for 5 s. using a Berthold LB9507 luminometer. The relative luminescence units (RLU/s) were normalized by the number of cells in each sample.

Vacuolar staining and visualization

Vacuole morphology was assessed basically as described previously [START_REF] Vida | A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast[END_REF]. Five ml of yeast cultures at A 660 of 0.8-1.0 were harvested, washed with YPD, and resuspended in 0.1 ml of fresh YPD. The lypophilic fluorescent dye FM4-64 (Invitrogen) was added at a final concentration of 20 µM and cells were incubated for 15 min at 30 °C. The cells were then washed, resuspended in 3 ml of YPD, and incubated for 15-45 min to allow the internalization by endocytosis and accumulation of the dye within the vacuole. Cells were visualized with a FITC filter using a Nikon Eclipse E800 fluorescence microscope (400×). Digital images were captured with an ORCA-ER 4742-80 camera (Hamamatsu) using the Wasabi software.

Results

Lack of Ref2 causes defects in cation tolerance

The observation that Glc7 point mutations lying in the vicinity of residues relevant for binding diverse regulatory subunits may cause alterations in ion homeostasis prompted us to examine tolerance to NaCl, LiCl, alkaline pH and calcium in eighteen strains carrying a deletion of functionally well-documented regulatory subunits of Glc7. Figure 1 shows a selected panel of conditions. As it can be observed, mutation of most regulatory subunits does not result in significantly altered tolerance to the indicated treatments, with the exception of the ref2 and shp1 mutants. However, deletion of shp1 caused a rather severe growth defect even in the absence of stress, as previously described [START_REF] Zhang | The Saccharomyces SHP1 gene, which encodes a regulator of phosphoprotein phosphatase 1 with differential effects on glycogen metabolism, meiotic differentiation, and mitotic cell cycle progression[END_REF], making difficult to assess the actual effect of the mutation under the conditions tested. In contrast, lack of Ref2, which only caused a slight growth defect under standard culture conditions, prevented growth in the presence of lithium, sodium and calcium cations, as well as under alkaline conditions. ref2 cells were also markedly sensitive to caesium ions (not shown). The intensity of the phenotype for Na + and Li + stress was comparable to that of a trk1 trk2 mutant (not shown) and it was not caused by the inability to adapt to hyperosmotic stress, since 1) growth was not affected by inclusion of 1 M sorbitol or KCl in the medium and 2) an obvious growth defect was already observed at a lithium concentration as low as 30 mM, which does not represent an osmotic stress condition (data not shown). Increased sensitivity to Na + and Li + cations is often observed in strains with defective potassium uptake (i.e. trk1 mutants). This prompted us to test the requirements for potassium in the ref2 mutant, by using a recently developed YNB-based K-free medium. As observed in Figure 2, ref2 cells display a slow-growth phenotype at limiting potassium concentrations (0.5-1.5 mM) that was alleviated by increasing the amount of potassium in the medium. This suggests that ref2 cells might have a decreased high-affinity potassium uptake, although direct measurement of rubidium influx (an analogue commonly used to assess for potassium uptake capacity) failed to reveal a significant decrease in Rb + uptake (data not shown). In any case, the ref2 strain showed increased sensitivity to several organic toxic cations, such as hygromycin B, spermine and tetramethylammonium (TMA), a phenotype which is commonly observed in strains with altered cation homeostasis (Figure 3A).

Ref2 contains a RISSIKFLD sequence (residues 368-376) that closely matches the (R/K)(V/I)X(F/W) Glc7-binding consensus sequence. We considered it necessary to test whether or not the salt-related phenotypes derived from the absence of Ref2 could be explained by its role as putative Glc7-regulatory subunit. To this end, Phe-374 was changed to Ala and the mutated version of the gene (including its own promoter) was cloned in a centromeric plasmid. As shown in Figure 3A, introduction of the wild type version of REF2 (pJFD1) in a ref2 mutant was able to increase tolerance to sodium, lithium and alkaline pH, as well as to diverse toxic organic cations. In contrast, mutation of Phe-374 (pJFD2) resulted in complete inability to do so. It must be noted that this mutation does not affect Ref2 cellular levels but it is known to disrupt the interaction with Glc7 [START_REF] Nedea | The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes[END_REF]. Expression of the mutated form of Ref2 from a high-copy number plasmid, which should lead to overexpression of the protein, was also unable to rescue the ref2 defects (not shown). These evidences suggest that blocking interaction of Ref2 with Glc7 results in loss of Ref2 function in salt tolerance. A similar effect was observed when the growth deficiency at limiting external potassium concentrations of the ref2 mutant was tested (Figure 3B). In this case, expression of the Phe-374 mutated version of REF2 (pJFD2) even aggravated the growth defect at low potassium of the strain lacking the chromosomal copy of the gene. We then considered the possibility that the cation-related defects of the ref2 strain could be attenuated or even suppressed by a surplus of Glc7. However, expression of Glc7 on a multicopy plasmid did not increase at all tolerance to LiCl or NaCl of a ref2 strain (Supplementary Figure 1). In contrast, the same plasmid was able to abolish the salt sensitive phenotype of specific Glc7 mutants (Supplementary Figure 1), indicating proper expression of Glc7.

Increased sensitivity to Na + and Li + can be the result of increased uptake, decreased efflux, or inability to sequester these toxic cations into intracellular compartments, such as the vacuole. To get insight into a possible cause for sensitivity in the ref2 mutant, the intracellular concentration of lithium in cells grown in the presence of 25 or 50 mM LiCl was tested. As observed in Figure 4A, ref2 mutants accumulate 60 to 70% more lithium than the wild type strain. This suggested that lack of Ref2 might favour lithium entry or make detoxification of the cation more difficult by interfering with the efflux systems. To this end, we tested the ability of ref2 cells to extrude lithium. As shown in Figure 4B, lithium efflux was markedly impaired in cells lacking Ref2, suggesting that the absence of this Glc7 regulatory subunit interferes with the normal efflux mechanisms.

Mutation of Ref2 affects expression of the ENA1 ATPase gene under saline and alkaline pH stresses.

The Na + -ATPase Ena1 is a major determinant of sodium and lithium tolerance. The expression of the gene is dramatically increased by exposure to stressing concentrations of Li + or Na + or alkalinization of the medium, and many mutations impairing ENA1 induction upon stress have been shown to lead to hypersensitivity to these cations. Therefore, we considered it necessary to monitor the expression of ENA1 in ref2 cells under cation stress conditions. To this end, cells were transformed with plasmid pKC201, which carries the entire ENA1 promoter fused to the lacZ reporter gene. As shown in Figure 5A, expression driven from the ENA1 promoter in cells exposed to Na + or Li + cations, as well as to alkaline pH, is drastically reduced in ref2 cells. Therefore, the increased sensitivity of the ref2 mutant to these stresses could be attributable, at least in part, to a defect in ENA1 induction.

The expression of ENA1 under saline or alkaline stress is controlled by a variety of signalling pathways, including activation of calcineurin, which is triggered by a burst of cytosolic calcium. As observed in Figure 5A, the effect of the ref2 mutation on ENA1 expression is even more dramatic than that of mutation of CNB1, encoding the regulatory subunit of calcineurin. Furthermore, the cnb1 mutation seems additive to ref2 both with respect to ENA1 expression (Figure 5A) or Li + , Na + and alkaline pH tolerance (Figure 5B). Therefore, ref2 phenotypes cannot be exclusively explained by a hypothetical impairment of calcineurin signalling under stress. Most regulatory inputs acting on the ENA1 promoter target two relatively small regions named ARR1 and ARR2 (see [START_REF] Ruiz | Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system[END_REF] for a recent review). To gain insight into the effect of the ref2 mutation, cells were transformed with plasmids pMRK212 and pMRK213. The former contains a region which mostly integrates calcium/calcineurin inputs, whereas the latter is regulated by calcineurin-independent stimuli. As shown in Figure 5A, deletion of REF2 decreases expression driven from both promoter regions, suggesting a complex and diverse effect of the mutation. Analysis of the expression from these constructs further supports an additive effect of the cnb1 and ref2 mutations on cation homeostasis.

ref2 cells display a hyperactivated calcium/calcineurin pathway in the absence of stress and show altered vacuole morphology

A striking phenotype that can be observed in Figure 1 is that lack of Ref2 results in a substantial sensitivity to calcium cations. It has been reported that hyperactivation of calcineurin results in a calcium-sensitive phenotype, whereas deletion of calcineurinencoding genes yields calcium hypertolerance. We then hypothesized that the calciumsensitive phenotype of ref2 mutants could be attributed to a higher-than-normal basal calcineurin activity (that is, in the absence of saline or alkaline pH stress). To test this possibility, we evaluated calcium tolerance in cells lacking both Ref2 and Cnb1, in comparison with the single ref2 and cnb1 mutants. As shown in Figure 6A, the calcium sensitivity conferred by the ref2 mutation is largely abolished in the absence of Cnb1. This suggests that the ref2 growth defect in the presence of calcium is caused by hyperactivation of calcineurin and leads to the possibility that calcium levels might be higher-than-normal in ref2 cells. This was directly tested by introducing the calcium-reporter plasmid pEV11/AEQ in wild type and ref2 mutant cells. Our measurements indicated that the luminescence observed in ref2 cells was 141.9 ± 3.7 RLU/10 6 cells, whereas this parameter was 64.8 ± 3.5 in the wild type strain, thus confirming the existence of higher free cytosolic calcium levels in the mutant. On the basis of this result, one would expect that expression from a specific calcium/calcineurin-sensitive promoter would be increased in ref2 cells grown under basal (non-stressing) conditions. To test this conjecture, wild type and ref2 strains (as well as their cnb1 derivatives) were transformed with plasmid pAMS366, which carries a tandem of four copies of the CDRE from the promoter of the FKS2 gene. As shown in Figure 6B, under basal growth conditions, expression from this synthetic promoter is 4 to 5-fold higher in ref2 cells than in the wild type strain. As expected, this effect is abolished in the absence of calcineurin activity (cnb1 ref2 strain). Remarkably, while in the wild type strain addition of calcium ions to the medium triggers a dramatic, calcineurin-mediated increase in expression, this effect is much less prominent in cells lacking Ref2. This could be explained if the ref2 mutation somehow interferes with stress-triggered effects mediated by activation of calcineurin. Increased sensitivity to alkaline pH and calcium is a characteristic phenotype of yeast strains with deficient vacuolar function. Therefore, the possibility that ref2 mutants may display some alteration in this subcellular compartment was considered. As shown in Figure 6C, incubation of wild type and ref2 cells with the lypophilic fluorescent dye FM4-64, which stains the vacuole membrane, reveals that vacuolar structure is dramatically altered in the ref2 mutant, with a punctuated fluorescent pattern and lack of discernible vacuolar structure. Because of the similarity between the saline phenotypes of the ref2 deletion and the glc7-109 allele, we considered it interesting to evaluate vacuolar morphology in the latter. As observed in Figure 6C, FM4-64 staining of strain YJFD17, which carries a centromeric plasmid-borne glc7-109 allele as only source of GLC7 function reveals that this strain displays vacuoles with essentially wild-type morphology (similarly to the original cation-sensitive strain KT1935, not shown). Interestingly, combination of the ref2 deletion and the glc7-109 mutation results in cells with ref2 phenotype, that is, altered vacuoles. The disruption of REF2 in the KT1935 background was repeatedly attempted without success. This is likely due to the close genetic relationship of this strain with the W303 genetic background (K. Tatchell, personal communication), in which the REF2 deletion was reported to be lethal [START_REF] Dheur | Pti1p and Ref2p found in association with the mRNA 3' end formation complex direct snoRNA maturation[END_REF].

Mutants in components of the APT complex do not share the cation-related phenotypes of the ref2 strain.

Given the previously described function of Ref2 as a component of the APT complex, it was reasonable to consider that the striking cation-related phenotypes of the ref2 strain could be attributed to the role of the protein in this complex. We reasoned that, if so, mutations in components of the complex should yield phenotypes reminiscent of those of the ref2 strain. Since many members of the APT complex are essential (including Pta1), we had to resort in most cases to temperature sensitive (ts) mutants cultured at sub-lethal temperatures. Figure 7A shows that deletion of SYC1, encoding a non-essential member of the complex, does not alter cell growth under conditions that clearly affect proliferation of ref2 mutants. The yth1-1 mutant displayed a slight sensitivity to LiCl, but was indistinguishable from the wild type in its sensitivity to NaCl and alkaline pH. The swd2-3 and ssu72-2 strains did not show any sensitivity to LiCl or NaCl and only a marginal sensitivity to high pH. None of these strains displayed sensitivity to high calcium levels in the medium. Similarly, a ts strain lacking the N-terminal region of Pta1 (strain pta1 1-75) exhibited a near wild type tolerance to cations or alkaline pH even at 37 ºC. We confirm here the phenotype of sensitivity to formamide attributed to the yth1-1 strain when grown at 30 ºC [START_REF] Barabino | The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins[END_REF]. Interestingly, we observe a similar behaviour for the swd2-3 mutant at 30 ºC or the ref2 and pta1 1-75 strains at 37 ºC, clearly showing that under these conditions the function of the APT complex is compromised. The almost complete lack of overlap between specific ref2 phenotypes and those of other mutants composing the APT complex is exemplified in the phenotypic array shown in Figure 7B.

We also examined the basal expression level of ENA1 in these strains and the ability of the ENA1 promoter to be activated by salt stress. As shown in Figure 8, changes in expression levels induced by exposure to LiCl or NaCl were smaller in the swd2-3 strain. However, the expression observed in syc1, ssu72-2 or pta1 1-75 derivatives was virtually identical to that of their respective wild type strains. Taken together, these results demonstrate that the mutation of diverse components of the APT complex does not mimic the cation-related phenotypes of the ref2 strain.

Discussion

In this work we present a screen for defects in cation tolerance of eighteen strains lacking specific regulatory subunits of the yeast type 1 protein phosphatase Glc7. Among them, only the ref2 mutant presented phenotypes undoubtedly associated to altered cation homeostasis, such as decreased tolerance to lithium and sodium, sensitivity to alkaline pH and decreased growth at limiting external potassium. We also observe that ref2 cells are sensitive to diverse toxic organic cations which interfere with very different cellular functions. It is generally accepted that these compounds enter the cell driven by the electrochemical gradient generated by the Pma1 plasma membrane proton ATPase and it has been observed that many mutations that affect Na + and/or K + homeostasis result in altered electrochemical gradient (i.e. trk1, ppz1, hal4,5, …). Therefore, the observation that ref2 cells are sensitive to hygromycin B, spermine and TMA is consistent with the notion that Ref2 is a relevant component of cation homeostasis. Our data suggest that the decreased tolerance to alkaline cations of the ref2 mutant could be due, at least in part, to the inability to fully induce expression of the ENA1 Na + -ATPase gene, which is a major determinant in saline tolerance. Since lack of Ref2 blocks the response of ENA1 to diverse stresses that are mediated through a variety of signalling pathways [12;48], the effect of Ref2 on ENA1 expression is possibly pleiotropic and may involve multiple targets.

Ref2 and Glc7 are components of the APT complex and they are required for efficient 3' processing of mRNAs and transcription termination of certain snoRNA genes ([25;29;30;47]. Therefore, it would be reasonable to speculate that the cation-related phenotypes of the ref2 mutant could be caused by malfunction of this complex, which is itself a component of the holo-CPF. However, our results show that deletion or ts mutations of diverse components of the APT complex, tested under conditions that impair their function in the complex [START_REF] Dichtl | Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation[END_REF][START_REF] Dichtl | A role for SSU72 in balancing RNA polymerase II transcription elongation and termination[END_REF][START_REF] Ghazy | The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing[END_REF], do not mimic consistently the growth defects or impaired ENA1 expression of the ref2 strain (Figure 7B and8). The same situation is observed for the conditional mutation of YTH1, whose product participates in polyadenylation factor I, a different subcomplex of the CPF [START_REF] Barabino | The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins[END_REF]. Furthermore, we also observe that while the ref2 strain fails to grow on non-fermentable carbon sources and grows poorly on low glucose, none of the APT-related mutations tested display such phenotypes (Figure 7B and Supplementary Figure 2), thus providing an additional example of disparate phenotypes. On the other hand, Ref2 has been related to the COMPASS/Set1 complex through the Swd2 protein [START_REF] Nedea | Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'ends[END_REF]. The function of Swd2 in this complex has been shown to be independent from its role in APT complex [START_REF] Dichtl | Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation[END_REF]. The COMPASS/Set1 complex is required for Histone H3 methylation at K4, which is a fingerprint of actively transcribed genes [START_REF] Shilatifard | Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation[END_REF]. Therefore, defective COMPASS function could be invoked to explain the effect of the ref2 mutation on ENA1 expression. However, this seems to be unlikely since: 1) a survey of the literature (and our own results, not shown) indicates that mutants in the non-essential components of the complex do not display salt-related phenotypes nor are they sensitive to organic toxic cations; 2) no evidence for a role of Glc7 on COMPASS function has been reported so far, and 3) deletion of REF2 does not perturb Histone H3 methylation at K4 [START_REF] Cheng | The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3[END_REF].

Our observation that ref2 cells are calcium-sensitive is consistent with the identification of REF2 in a screen for mutations sharing multiple pmr1 phenotypes [START_REF] Yadav | A phenomics approach in yeast links proton and calcium pump function in the Golgi[END_REF]. PMR1 encodes an ATPase that sequesters Ca +2 and Mn +2 into the Golgi/secretory pathway that is pivotal to maintain proper cytosolic concentrations of these ions. In addition, similarly to vma and pmr1 mutants [START_REF] Yadav | A phenomics approach in yeast links proton and calcium pump function in the Golgi[END_REF], the ref2 mutant displays strong sensitivity to the cell-wall perturbing agent calcofluor white (data not shown). Remarkably, we observe in ref2 cells grown in the absence of any source of stress increased cytosolic calcium levels and higherthan-normal expression from a synthetic calcium/calcineurin-regulated promoter (Figure 6B). This suggests that basal calcineurin activity is abnormally high in ref2 mutants. Further activation of calcineurin caused by exposure to high concentrations of extracellular calcium would explain the hypersensitive phenotype of ref2 cells to this cation, since it is known that excessive calcineurin activity is detrimental for the cell [52;53]. The observation that deletion of CNB1, which blocks calcineurin signalling, relieves calcium toxicity provides further support to this hypothesis. We show here that ref2 cells display altered vacuolar morphology, which is reminiscent of the class B/C vps mutants [45;54;55]. Since the vacuole is the most important calcium store in yeast [START_REF] Halachmi | Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1[END_REF], it is reasonable to assume that the calciumrelated phenotypes displayed by ref2 cells could be derived from their incapacity for normal vacuole assembly. It must be noted, however, that whereas lack of Ref2 increases basal calcineurin activity, this mutation also interferes with stress signals that transiently activate the phosphatase, as deduced from the lower-than-normal short-term response of the calcineurin-regulatable promoter (Figure 6B).

We show here that a version of Ref2 that cannot interact with Glc7 [START_REF] Nedea | The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes[END_REF] is unable to rescue the saline phenotypes of the ref2 strain. Therefore, it is reasonable to consider that the perturbed cation homoeostasis observed in a ref2 strain is due to a deregulation of Glc7 activity. Altered cation homeostasis has been described in the past for specific mutations of Glc7, such as the glc7-109 allele [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF], which carries an Arg-260 to Ala mutation near the hydrophobic channel that is likely to be responsible for interaction with the binding motif present in Ref2. Remarkably, the glc7-109 mutant displays a subset of phenotypes that resembles very much those reported here for the ref2 disruption, such as enhanced sensitivity to sodium, lithium, alkaline pH and organic toxic cations [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF]. We have also observed that deletion of REF2 in a strain carrying the glc7-109 allele (YJFD18) does not result in enhanced sensitivity to NaCl or alkaline pH (not shown). In addition, the saline phenotypes of the glc7-109 allele are additive to those caused by disruption of calcineurin activity, a circumstance also observed for the ref2 strain (Figure 5B). All this evidence suggests that the cation-related phenotypes of the glc7-109 allele could be caused, at least in part, by its incapacity to interact with Ref2. It must be stressed, however, that ref2 and glc7-109 phenotypes are not always consistent. For instance, the glc7-109 strain hyperaccumulates glycogen [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF], whereas the ref2 mutant does not (data not shown). More importantly, the glc7-109 strain does not display altered calcium levels and increased calcium sensitivity [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF], nor altered vacuolar morphology (Figure 6C). Therefore, it could be hypothesized that the R260A mutation in glc7-109 may affect interaction of the phosphatase catalytic subunit with diverse regulatory subunits, one of these being Ref2. The relevance of the role of Ref2 is highlighted by the observation that the salt-related defects of ref2 cells cannot be overridden by simply overexpressing Glc7.

In conclusion, our data demonstrates that the role of Ref2 on cation tolerance is not attributable to the function of this protein in regulating Glc7 in the CPF complex but, instead, it suggests that Ref2 must direct Glc7 to alternative target(s). This implies that Ref2 may have multiple cellular functions. While remarkable, this is not fully surprising, since a similar scenario has been reported for the Gac1 regulatory subunit, which functions as a molecular scaffold to tether Glc7 to Gsy2 (encoding glycogen synthase), thus controlling glycogen accumulation [START_REF] Williams-Hart | Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae[END_REF], and also binds the transcription factor Hsf1, thus controlling the transcription of certain heat-shock responsive genes, such as CUP1 [START_REF] Lin | Glycogen synthase phosphatase interacts with heat shock factor to activate CUP1 gene transcription in Saccharomyces cerevisiae[END_REF]. Therefore, Ref2 would represent a novel example of multifunctional Glc7 regulatory subunit. A. Cultures (~3x10 3 and 3x10 2 cells) of strains described on the left were spotted on plates and grown at the indicated temperatures (right) for three days. YPDF, YPD plus 3% formamide, LiCl was at 100 mM (30 ºC) or 150 mM (37 ºC). Conditions for NaCl (800 mM), pH (8.2) or CaCl 2 (200 mM) treatments were identical for both temperatures. B. Phenotypic array comparing multiple ref2 phenotypes with those of other APT-related mutants. Growth of each mutant was compared with that of the corresponding wild type strain grown under the same set of conditions. Filled squares denote strong sensitivity; grey squares indicate a mild phenotype and empty squares correspond to wild type behavior. Conditions tested to construct the array were: LiCl (50-200 mM); NaCl (0.4-1.2 M); High pH (8.0-8.3); Ca +2 , (100-300 mM CaCl 2 ), YPGly, YP plus 2% glycerol; YP-EtOH, YP plus 2% Ethanol; YP-LowGlu, YP plus 0.05% Glucose; Hyg B, Hygromycin B (20-60 g/ml); TMA, Tetramethyl ammonium (0.2-0.6 M). The indicated mutants and their corresponding wild type strains were transformed with plasmid pKC201. Stress conditions were as in Figure 5, except that growth temperature was 30 ºC for all strains but PTA1 and pta1( 1-75), which were grown at 37 ºC. Data are mean ± SEM from 12 independent transformants. Bars at the bottom connect isogenic strains.

Biochemical

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Biochemical

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  Biochemical Journal Immediate Publication. Published on 22 Dec 2009 as manuscript BJ20091909 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091909 A c c e p t e d M a n u s c r i p t

Biochemical

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Biochemical

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Biochemical

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Biochemical

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LEGENDSFigure 1 .

 1 Figure 1. Effect of the mutation of several Glc7-interacting proteins on cation-related phenotypes. Two dilutions (~3x10 3 and 3x10 2 cells) from cultures of wild-type strain BY4741 and the indicated isogenic derivatives were grown on YPD plates in the presence of 0.6 M NaCl, 50 mM LiCl, 0.2 M CaCl 2 or alkaline pH for 2 days.

Figure 2 .

 2 Figure 2. Mutation of REF2 increases potassium requirements for growth. Cultures of wild type strain BY4741 (open bars) and its ref2 derivative (closed bars) were inoculated at A 600 of 0.004 in Translucent K-free medium supplemented with the indicated amounts of KCl and grown for 16 h. Results are represented as percentage of growth compared with cells incubated with 50 mM KCl and are mean ± SEM from 3 independent cultures.

Figure 3 .

 3 Figure 3. Mutation of Phe-374 within the Glc7-binding consensus sequence abolishes the function of Ref2 in cation tolerance. A.Wild type BY4741 (+) and ref2 strains (-) were transformed with the empty plasmid pRS415 or the same plasmid expressing the wild type Ref2 protein (pJFD1) or the Ref2 F374A mutated version (pJFD2). Transformants were spotted on plates adjusted to pH 8.1 or containing LiCl (150 mM), NaCl (1 M), TMA (0.3 M), Spermine (0.6 mM) or Hygromycin B (20 g/ml) and growth was monitored after 3 days, except for LiCl and NaCl plates (6 days). B. Wild type and ref2 strains were transformed with the indicated plasmids and growth was tested at limiting concentration of external potassium (1 mM). Experimental conditions were as described in the legend of Figure2.

Figure 4 .

 4 Figure 4. Effects of the ref2 mutation on intracellular lithium accumulation and efflux. A. Wild-type BY4741 cells (open bars) and the ref2 derivative (closed bars) were incubated with 25 or 50 mM LiCl as described in Experimental procedures, and the intracellular content of lithium ions measured. Data are mean ± SD from at least six independent determinations. B. Strains BY4741 (--) and ref2 (--) were loaded with 75 mM LiCl, samples taken at different times and the efflux of the cation determined as described. Results are expressed as percentage of the intracellular Li + content for each strain at time zero and are mean ± SD from at least six independent assays.

Figure 5 .

 5 Figure 5. Effects of the ref2 mutation in wild type and cnb1 strains on the ENA1 promoter activity. A. The indicated strains were transformed with plasmids pKC201, carrying the entire ENA1 promoter, or plasmids pMRK212 or pMRK213, which contain specific regions of the ATPase gene promoter. See cartoon for details and relevant regulatory elements (MIG, Mig1/2 binding sequence; CRE, cAMP Regulatory Element). Cells received no treatment (NI), or either were shifted to pH 8.2 (pH) or received 0.4 M NaCl (Na) or 0.2 M LiCl (Li) for 1 h. -galactosidase activity was measured in permeabilized cells as described in Experimental. Data are mean ± SEM from 8-9 independent transformants. B. Three dilutions of the indicated cultures were spotted on plates at the conditions shown and growth recorded after 72 h (LiCl and NaCl) or 48 h (alkaline pH).

Figure 6 .

 6 Figure 6. Lack of REF2 results in altered calcium homeostasis and vacuolar morphology. A. The indicated strains were grown in the presence or absence of calcium cations (300 mM CaCl 2 ) and growth was monitored after 72 h. B. Yeast strains were transformed with plasmid pAMS366, which contains a tandem of four CDREs, and incubated for 1 h in the absence (empty bars) or presence (closed bars) of 0.2 M CaCl 2 . -

Figure 7 .

 7 Figure 7.-Comparison of ref2 phenotypes with those of diverse APT-related mutants.A. Cultures (~3x10 3 and 3x10 2 cells) of strains described on the left were spotted on plates and grown at the indicated temperatures (right) for three days. YPDF, YPD plus 3% formamide, LiCl was at 100 mM (30 ºC) or 150 mM (37 ºC). Conditions for NaCl (800 mM), pH (8.2) or CaCl 2 (200 mM) treatments were identical for both temperatures. B. Phenotypic array comparing multiple ref2 phenotypes with those of other APT-related mutants. Growth of each mutant was compared with that of the corresponding wild type strain grown under the same set of conditions. Filled squares denote strong sensitivity; grey squares indicate a mild phenotype and empty squares correspond to wild type behavior. Conditions tested to construct the array were: LiCl (50-200 mM); NaCl (0.4-1.2 M); High pH (8.0-8.3); Ca +2 , (100-300 mM CaCl 2 ), YPGly, YP plus 2% glycerol; YP-EtOH, YP plus 2% Ethanol; YP-LowGlu, YP plus 0.05% Glucose; Hyg B, Hygromycin B (20-60 g/ml); TMA, Tetramethyl ammonium (0.2-0.6 M).

Figure 8 .

 8 Figure 8.-Expression of ENA1 under saline stress in diverse APT-related mutants.The indicated mutants and their corresponding wild type strains were transformed with plasmid pKC201. Stress conditions were as in Figure5, except that growth temperature was 30 ºC for all strains but PTA1 and pta1( 1-75), which were grown at 37 ºC. Data are mean ± SEM from 12 independent transformants. Bars at the bottom connect isogenic strains.

Fig. 2

 2 Fig. 2 Ferrer-Dalmau et al.

A c c eFig. 6 Fig. 8

 68 Fig. 6 Ferrer-Dalmau et al.

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

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Acknowledgements

The authors acknowledge the help of H. Sychrovà and G. Wiesenberger in developing and testing the YNB-based, K-free medium. Thanks are given to C. Moore, B. Ditchtl, K. Tatchell and M. Marquina for diverse strains and plasmids. The excellent technical assistance of Anna Vilalta and Montserrat Robledo is acknowledged. Work supported by grants BFU2008-04188-C03-01 and GEN2006-27748-C2-1-E/SYS (SysMO ERA-NET) to JA; BFU2007-60342 to AC and BFU2008-04188-C03-03 and GEN2006-27748-C2-2-E/SYS (SysMO ERA-NET) to JR (Ministry of Science and Innovation, Spain). J.A. is recipient of an "Ajut de Suport a les Activitats dels Grups de Recerca" (2009SGR-1091, Generalitat de Catalunya) Biochemical Journal Immediate Publication. Published on 22 Dec 2009 as manuscript BJ20091909 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091909

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.


Mimi Høier-Madsen

Jan Holm

Steen Ingemann Hansen

Ingemann Steen

Hansen

ALPHA -ISOFORMS OF SOLUBLE AND MEMBRANE-LINKED FOLATE BINDING PROTEIN IN HUMAN BLOOD by

Keywords:

The high affinity folate binding protein/ folate receptor (FBP/FR) is expressed in three isoforms. FR alpha and beta are attached to cell membranes by hydrophobic glycosylphosphatidyl inositol (GPI) anchors, while FBP gamma is a secretory protein. Mature neutrophil granulocytes contain a nonfunctional FR beta on the surface , and in addition nanomolar concentrations of a secretory functional FBP (29 kDa) can be present in the secondary granules. A statistically significant correlation between the concentrations of functional FBP, probably a gamma isoform, in granulocytes and serum supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were for the first time established in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes, and in granulocytes and serum only constituted an almost undetectable fraction of the functional FBP. The alpha FBP in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of alpha FBP (> 120 kDa) which could represent receptor fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, while FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors have also been used for therapeutic targeting in malignancy.

INTRODUCTION

A high-affinity folate binding protein (FBP) was first identified in cow's milk [START_REF] Antony | The biological chemistry of folate receptors[END_REF][START_REF] Antony | Folate receptors[END_REF][START_REF] Ghitis | The folate binding in milk[END_REF]. The protein has been purified and sequenced; it consists of 222 amino acids with a molecular weight of 30 kDa based on amino acid composition and carbohydrate content [START_REF] Svendsen | The complete amino acid sequence of folate binding protein from cow´s milk[END_REF]. FBP purified from human milk has a similar molecular weight as estimated from its amino acid composition, carbohydrate content and sequence homology with FBP from cow's milk [START_REF] Svendsen | Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk[END_REF]. The purified human milk FBP exists in two forms: one is water-soluble and elutes at 27 kDa on gel filtration and one is equipped with a hydrophobic glycosyl phosphatidyl inositol (GPI) residue and forms micellar aggregates with Triton X-100 eluting at 100 kDa [START_REF] Svendsen | Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk[END_REF][START_REF] Hansen | Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C[END_REF]. The micellar FBP is transformed to the water-soluble FBP after enzymatic cleavage of the GPI tail by phosphatidylinositol-specific phospholipase C, PI-PLC [START_REF] Hansen | Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C[END_REF].

The ubiquitous existence of FBPs in mammalian cells, tissues and body fluids is now well-established [START_REF] Antony | The biological chemistry of folate receptors[END_REF][START_REF] Antony | Folate receptors[END_REF]. Three isoforms of FBP , alpha, beta and gamma, are expressed on chromosome 11q13 in humans [START_REF] Ross | Differential regulation of folate receptor isoforms in normal and malignant tissues and in established cell lines. Physiological and clinical implications[END_REF][START_REF] Shen | Identification of a novel folate receptor , a truncated receptor, and receptor type beta in hematopoietic cells : cDNA cloning, expression, immunoreactivity, and tissue specificity[END_REF]. Only the former two isoforms can be equipped with a GPI tail [START_REF] Lacey | Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidyl inositol[END_REF], which enables them to be anchored to plasma membranes as membrane-bound folate receptors (FRs) susceptible to enzymatic cleavage by PI-PLC or proteases. FR alpha is normally expressed in secretory epithelial cells and overexpressed in certain epithelial tumor cells, e.g. ovarian carcinoma [START_REF] Weitman | Cellular localization of the folate receptor : potential role in drug toxicity and folate homeostasis[END_REF] ; its amino acid sequence is homologous to that of human milk FBP which represents the soluble form of FR alpha [START_REF] Svendsen | Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk[END_REF][START_REF] Coney | Cloning of a tumor-associated antigen : MOv18 and Mov19 antibodies recognize a folate binding protein[END_REF].

The presence of soluble and membrane-linked FBPs in human blood cells and plasma have been reported. The outer membrane surface of erythrocytes [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF][START_REF] Antony | Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro[END_REF] and neutrophil granulocytes contain FRs [START_REF] Ross | Folate receptor type β is a neutrophil lineage marker and is differentially expressed in myeloid leukaemia[END_REF][START_REF] Wang | Differentiation-independent retinoid induction of folate receptor type β, a potential tumor target in myeloid leukaemia[END_REF][START_REF] Reddy | Expression and functional characterization of the β-isoform of the folate receptor on CD 34+ cells[END_REF] ; in addition the secondary cytoplasmic granules of neutrophils contain a high-affinity FBP [START_REF] Colman | Folate-binding proteins[END_REF][START_REF] Costa | Appearance of a folate binder in leukocytes and serum of women who are pregnant or taking oral contraceptives[END_REF][START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF]. The membrane receptor of erythrocytes immunoreacts with polyclonal antibodies against human placenta FR [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF], which is a mixture of alpha and beta isoforms [START_REF] Ratnam | Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA[END_REF], while that of neutrophil granulocytes is a beta isoform [START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF]. The water-soluble FBP in neutrophil granules is supposed to be a gamma isoform without GPI residue and most likely a source of origin of plasma/serum FBP [START_REF] Shen | Folate receptor type γ is primarily a secretory protein due to lack of an efficient signal for glycosylphosphatidyl inositol modification : protein characterization and cell type specificity[END_REF][START_REF] Kane | Biology of disease: Role of folate binding proteins in folate metabolism[END_REF].

As yet, no alpha isoforms of FBP/FR have been identified in human blood. A major issue of this report has therefore been to establish whether these isoforms are present in human blood. For this purpose we reviewed earlier data on FR/FBP in human blood , and employed a combination of radioligand (tritiated folate) binding and immunotechniques involving a polyclonal antibody against purified alpha FBP from human milk [START_REF] Høier-Madsen | Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological bsr20080031.doc 13 17-04-2008 characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)[END_REF]. The latter antibody exhibits an immunoreactive profile similar to that of the monoclonal antibody, MOv18 . Like MOv18 it only immunoreacts , as evidenced by immuno precipitation, immunoblotting , ELISA and immunohistochemistry , with FR alpha, e.g. in serous bsr20080031.doc ovarian adenocarcinoma (7,10,11,26 ,27) ; normal ovarian tissue where FR beta prevails [START_REF] Ross | Differential regulation of folate receptor isoforms in normal and malignant tissues and in established cell lines. Physiological and clinical implications[END_REF] shows no immunoreactive response [START_REF] Holm | High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites : Radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity[END_REF].

MATERIALS and METHODS

Proteins and antibodies: FBPs were purified from human and bovine milk as previously described [START_REF] Svendsen | The complete amino acid sequence of folate binding protein from cow´s milk[END_REF][START_REF] Svendsen | Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk[END_REF]. Polyclonal antibodies against human and bovine milk FBPs were raised in rabbits [START_REF] Høier-Madsen | Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological bsr20080031.doc 13 17-04-2008 characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)[END_REF][START_REF] Høier-Madsen | Rabbit antibodies against the folate binding protein from cow's milk. Production,characterization and use for development of an enzyme-linked immunosorbent assay (ELISA)[END_REF].

Rabbit antibodies against human lactoferrin (A 0186) and FITC labelled Ig fraction of swine antirabbit serum (F 0205) were from DAKO. Rabbit serum was from State Serum Institute.

Radiochemicals: [ 3 H ] Folate with a specific activity of 26-45 kCi/mol was supplied by Amersham International Ltd. Amersham, U.K.

Preparation of hemolysate from erythrocytes:

Hemolysates were prepared as previously reported in a study dealing with binding of tritiated folate to hemoglobin [START_REF] Hansen | Evidence that the low-affinity folate-binding protein in erythrocyte hemolysate is identical to hemoglobin[END_REF]. Briefly, specimens of EDTA stabilized venous blood were drawn from healthy volunteers with normal hematological parameters ( hemoglobin, RBC count, MCV, WBC count, and differential count). The plasma and buffy coat layer was discarded after gentle centrifugation (1500 x g ) for 15 min, and erythrocytes were washed several times in a volume of isotonic NaCl equal to the plasma volume to remove contaminating plasma . The washed erythrocytes were finally diluted with a volume of distilled water equal to the plasma volume and subjected to repeated freeze-thaw procedures. Aliquots of hemolysates were dialysed overnight against 0.17 M Tris-HCl buffer of pH 7.4 before analysis to remove endogenous folate .

Preparation of leukocyte lysates :Specimens of EDTA stabilized venous blood were drawn from healthy volunteers with normal haematological parameters (cf above), leukocytes harvested , washed several times in isotonic NaCl to remove contaminating plasma and then subjected to repeated freeze-thaw procedures [START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF]. The cells were homogenized and solubilized in 1 g/l Triton X-100,dialysed against 0.17 M Tris HCl of pH 7.4 for 24 h to remove endogenous folate, and finally centrifuged at 2000 x g for 15 min [START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF]. Aliquots of the supernatants were used for analysis.

Preparation of pooled sera :Pooled sera were prepared as previously reported [START_REF] Holm | High and low affinity binding of folate to proteins in serum of pregnant women[END_REF]. Briefly, sera were pooled, dialysed overnight against 0.05 M imidazole buffer of pH 6. Radioligand binding:Aliquots of supernatants were dialysed to equilibrium against [ 3 H ] Folate in 0.17 M Tris-HCl buffer of pH 7.4 for 24 h at 37 o C with Triton X-100 1g/l added to both sides of the dialysis membrane [START_REF] Holm | High and low affinity binding of folate to proteins in serum of pregnant women[END_REF]. Radioactivity was determined as previously described [START_REF] Holm | High and low affinity binding of folate to proteins in serum of pregnant women[END_REF].

Enzyme linked immunosorbent assay (ELISA):

A previously described ELISA employing an anti-human milk FBP antibody [START_REF] Høier-Madsen | Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological bsr20080031.doc 13 17-04-2008 characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)[END_REF] which specifically immunoreacts with FBP/FR alpha was used [START_REF] Holm | High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites : Radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity[END_REF] . ELISA with antibodies against FBP purified from cow's milk was also conducted for further testing of the specificity of the immunoreactive response in ELISA [START_REF] Høier-Madsen | Rabbit antibodies against the folate binding protein from cow's milk. Production,characterization and use for development of an enzyme-linked immunosorbent assay (ELISA)[END_REF]. FBPs purified from human and bovine milk were analyzed in ELISA to control the analytical performance of the assays [START_REF] Høier-Madsen | Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological bsr20080031.doc 13 17-04-2008 characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)[END_REF][START_REF] Høier-Madsen | Rabbit antibodies against the folate binding protein from cow's milk. Production,characterization and use for development of an enzyme-linked immunosorbent assay (ELISA)[END_REF].

Gel filtration:

Gel filtration experiments on an Ultrogel AcA 44 column (LKB) were performed as previously described in detail [START_REF] Holm | High and low affinity binding of folate to proteins in serum of pregnant women[END_REF]. Briefly, the column ( 5.3 cm 2 x 96 cm ) was eluted at 5 0 C with Tris-HCl buffer of pH 7.4 containing 1 g/l Triton X-100. The column was calibrated as previously reported [START_REF] Holm | High and low affinity binding of folate to proteins in serum of pregnant women[END_REF] .

Indirect immunofluorescence:To demonstrate human alpha FBP in neutrophil granulocytes we used ethanol fixed smears of normal peripheral blood leukocytes as substrate [START_REF] Wiik | Methods to detect autoantibodies to neutrophil granulocytes[END_REF]. The smears were incubated with 1.5 mg protein /ml solutions of rabbit anti human alpha FBP, anti human lactoferrin, and rabbit serum (control) for 30 min in a humid chamber. Each smear was individually washed with excess of PBS and all smears are submersed in the chamber in PBS for 10 min to remove nonspecifically attached serum. After removal of all PBS from the slides, the submersion of slides in PBS was repeated for another 10 min. Excess PBS was removed around the circle of each slide, but a minute amount of PBS was left in the circle to avoid drying of the sample. Fluorescein isothiocyanate-labelled Ig fraction of swine anti-rabbit serum (DAKO F0205) diluted 1:25 in PBS was added as a drop onto the encircled cell smear of each slide. The slides were again incubated for 30 min at room temperature in a humid chamber. The washing procedures were repeated. A drop of a 2:1 glycerol/PBS mixture was then applied to each smear and a cover glass was applied. The smears were read directly in a fluorescence microscope.

To show the specificity we absorbed the rabbit anti-human FBP antibodies with excess of human FBP and repeated the immunofluorescence procedures. The lightening of the cytoplasm in the neutrophil granulocytes disappeared.

The specificity was further tested by replacing rabbit anti human FBP antibodies by rabbit anti bovine FBP antibodies or rabbit serum.

RESULTS

alpha FBP/FR in erythrocytes

Antibodies against alpha FBP from human milk immunoreacted with serial dilutions of hemolyzed erythrocytes (Fig1). To test the specificity of response we performed a parallel analysis in ELISA with antibodies against bovine milk FBP ( Fig1 inset). The analytical performance of the two ELISA methods were controlled by running serial dilutions of purified human and bovine milk FBP

( Fig 1, Fig 1 inset).
Immunoreactive FBP/FR in hemolyzed erythrocytes did not bind [ 3 H] folate in equilibrium dialysis experiments.

FBP in leukocytes

The concentration of functional FBP, in terms of maximum binding of [ 3 H] folate in equilibrium dialysis experiments, was determined in leukocyte lysates obtained from three healthy subjects. The FBP concentration in leukocyte lysate showed a great interindividual variation: one was < 0.01, one 3.8 and one 5.0 nmol/l. Gel filtration of Triton X-100 solubilized leukocyte lysates containing 3.8 and < 0.01 nmol/l FBP were performed after preincubation with [ 3 H]folate. The former sample exhibited a single peak of radioactivity at 29 kDa, whereas no peak was observed in the latter sample (Fig 2). No micellar aggregates of GPI-tailed FR appeared at 100 kDa.

alpha FBP/FR in neutrophil granulocytes

ELISA with antibodies against human milk alpha FBP was performed to identify alpha FR/FBP in neutrophil granulocytes. The specificity of the present anti-alpha FBP antibody was documented as follows.

Serial dilution curves (two-fold initial dilution) of the three leukocyte lysates in

Immunostaining disappeared when the indirect immunofluorescence procedure was repeated after absorption of the antibody with excess of human milk FBP (data not shown).

The typical immunostaining pattern disappeared when antibodies against bovine milk FBP were used (data not shown).

No immunostaining of beta FR on the surface of neutrophil granulocytes (14 -16, 22) .

Correlation between functional FBP in serum and leukocyte lysate

Matched data for serum and leukocyte concentrations of functional FBP from previous studies [START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF] were re-analysed in order to test a hypothesized relationship between the FBPs in granulocytes and plasma.

A weak positive correlation (statistically significant) between the concentrations of functional FBP in serum and leukocyte lysate was found in 15 healthy subjects with > 0.5 nmol FBP / 10 9 leukocytes (Fig 4).

Functional FBP and alpha FBP in serum

The concentration of [ 3 H] folate bound FBP (functional FBP) was determined by equilibrium dialysis in pooled sera from pregnant women ( FBP, 0.73 nmol/l ) , blood donors ( FBP, 1.10 nmol/l ) , and umbilical cord blood ( FBP, 0.71 nmol/l). The pooled sera were analysed in ELISA with antibodies against human milk alpha FBP to screen for the presence of alpha FBP (Fig5).

Serial dilution curves of the three types of pooled sera showed weak positive responses in ELISA suggesting the presence of alpha FBP in serum. No response was seen when a parallel ELISA with antibodies against bovine milk FBP was performed to test the specificity (data not shown). The analytical performance of the ELISA was controlled by running serial dilutions of purified human milk FBP (Fig 5). The elution profile of alpha FBP determined in ELISA with antibodies against human milk alpha FBP contained a small peak at 43 kDa continuing into a broad peak with a maximum height (OD 0.6) at >120 kDa (void volume). No alpha FBP was found at a position of 29 kDa where the major peak of functional FBP had its maximum height (Fig 6).

The gel filtration experiments in

DISCUSSION

The most important finding herein was the identification of alpha FBP in human blood cells and plasma/serum.

A high-affinity FBP ( K= 10 -11 M -1 ) anchored to surface membranes of mature human erythrocytes by a hydrophobic residue was first identified by Antony et al [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF][START_REF] Antony | Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro[END_REF]. Incubation of erythrocytes with trypsin resulted in the release of soluble hydrophilic FBP to the external medium [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF][START_REF] Antony | Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro[END_REF]. Indirect evidence suggested that erythrocyte FR is an alpha isoform: It immunoreacts with antibodies against human placental FR (12), which represents a mixture of alpha and beta isoforms [START_REF] Ratnam | Homologous membrane folate binding proteins in human placenta: cloning and sequence of a cDNA[END_REF], and erythrocyte membranes do not show immunostaining with antibodies against FR beta [START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF]. Our ELISA data in Fig1 obtained with antibodies specifically directed against alpha FBP [START_REF] Campbell | Folate -binding protein is a marker for ovarian cancer[END_REF][START_REF] Holm | High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites : Radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity[END_REF] provide direct evidence for the presence of FR alpha in mature erythrocytes.

In spite of removal of endogenous folate , free as well as receptor bound, by repeated washes at low pH, Antony et al. [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF] who used a radiofolate preparation with a high specific activity only succeeded in identifying two molecules of functional FBP/cell in reticulocyte-rich young cells. A progressive up to seven-fold loss of functional FBP was observed as erythrocytes aged [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF]. No folate was released after boiling of the washed erythrocyte membranes indicating a complete removal of endogenous folate [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF]. Hence, the extremely small number of functional receptors explains why we failed to detect any binding of our low-specific activity [ 3 H] folate to hemolysates despite repeated washes and prolonged dialysis to remove endogenous folate.. The functional role of erythrocyte FR is not clear, but the addition of anti-placental FR antiserum induced a decrease of intracellular folate and megaloblastic changes during in vitro erythropoiesis [START_REF] Antony | Effect of perturbation of specific folate receptors during in vitro erythropoiesis[END_REF]. Mature erythrocytes contain polyglutamated forms of folate suggesting that folate is taken up [START_REF] Antony | Identification of high affinity folate binding proteins in human erythrocyte membranes[END_REF]. Human hemoglobin has been identified as a low-affinity/high capacity binding protein for folate [START_REF] Hansen | Evidence that the low-affinity folate-binding protein in erythrocyte hemolysate is identical to hemoglobin[END_REF], and binding of folate polyglutamates to hemoglobin has also been reported [START_REF] Benesch | The binding of folyl-and antifolylpolyglutamates to hemoglobin[END_REF].

Several studies have focussed on the presence of different isoforms of FBP in mature neutrophil granulocytes.

A FR beta has thus been identified on the surface of mature neutrophils and subpopulations of CD 34 positive cells in the myelomonocytic lineage by use of immunostaining and flow cytometry with anti-human FR beta antibodies [START_REF] Ross | Folate receptor type β is a neutrophil lineage marker and is differentially expressed in myeloid leukaemia[END_REF][START_REF] Wang | Differentiation-independent retinoid induction of folate receptor type β, a potential tumor target in myeloid leukaemia[END_REF][START_REF] Reddy | Expression and functional characterization of the β-isoform of the folate receptor on CD 34+ cells[END_REF][START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF]. No FR alpha is present on the surface membrane as evidenced by flow cytometry with monoclonal antibodies against FR alpha [START_REF] Ross | Folate receptor type β is a neutrophil lineage marker and is differentially expressed in myeloid leukaemia[END_REF] as well as by our indirect immunofluorescence data (Fig 3). The FR beta had a variant GPI membrane anchor insensitive to PI-PLC but cleaved by nitrous acid [START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF]. The FR beta in mature normal granulocytes remained non-functional in spite of acid washing to remove endogenous folate , whereas it was active in acute myeloid leukaemia cells with 0.72 pmol folate bound per 10 6 cells [START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF]. The physiological role of FR beta expressed during neutrophil maturation is obscure, but in CD 34 positive cells it may serve a function that is distinct from its role in folate uptake,i.e. signal transduction and cell adhesion [START_REF] Reddy | Expression and functional characterization of the β-isoform of the folate receptor on CD 34+ cells[END_REF]. The FR beta in myeloid leukemia cells is up-regulated 20-fold at the level of mRNA by all-trans retinoic acid [START_REF] Wang | Differentiation-independent retinoid induction of folate receptor type β, a potential tumor target in myeloid leukaemia[END_REF] meaning that the receptor could be a promising target for therapeutic intervention, e. g.with folate conjugated cytotoxins, due to its selective upregulation in leukemic cells [START_REF] Ross | Folate receptor type β is a neutrophil lineage marker and is differentially expressed in myeloid leukaemia[END_REF][START_REF] Pan | Strategy for the treatment of acute myelogenous leukemia based on folate receptor β-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid[END_REF].

Besides the FR beta on the surface, neutrophil granulocytes contain a cytoplasmic functional highaffinity ( K d = < 1 nM ) FBP only present in detectable amounts in a minority of healthy subjects [START_REF] Colman | Folate-binding proteins[END_REF][START_REF] Costa | Appearance of a folate binder in leukocytes and serum of women who are pregnant or taking oral contraceptives[END_REF][START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF]. It was originally found in women who were pregnant or taking oral contraceptives [START_REF] Costa | Appearance of a folate binder in leukocytes and serum of women who are pregnant or taking oral contraceptives[END_REF], but the present data (Fig 2 ) as well as other studies reveal great inter-individual variations in concentration of functional FBP in both males and females [START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF], and clustering of persons with high concentration levels in certain families [START_REF] Holm | Inheritance of high leukocyte folate binding activity. Relationship between folate binding in leukocytes and serum[END_REF]. The protein seems to be located in the matrix of secondary neutrophil granules, and just like lactoferrin released therefrom during degranulation induced by high lithium concentrations [START_REF] Bloomfield | Influence of lithium and fluoride on degranulation from human neutrophils in vitro[END_REF][START_REF] Herbert | Release of vitamin binding proteins from granulocytes by lithium: vitamin B 12 and folate binding proteins[END_REF][START_REF] Holm | Li+ -induced release of the folate binding protein from human granulocytes[END_REF][START_REF] Holm | Lithium-induced release of lactoferrin from human granulocytes in vitro[END_REF]. It is supposed to be identical to the secretory FBP gamma isoform which is expressed in hematopoietic tissue and lacks a hydrophobic GPI residue [START_REF] Shen | Folate receptor type γ is primarily a secretory protein due to lack of an efficient signal for glycosylphosphatidyl inositol modification : protein characterization and cell type specificity[END_REF][START_REF] Kane | Biology of disease: Role of folate binding proteins in folate metabolism[END_REF]. The appearance of a single peak of radioactivity at 29 kDa in the gel filtration profile of leukocyte lysate (Fig 2) is consistent with a gamma isoform possessing no GPI-tail. is located to a cytoplasmic structure in neutrophils. The exact nature of this structure is difficult to establish since the ethanol fixation procedure disrupts cytoplasmic membranes. A putative locus could be secretory vesicles since their membranes contain several GPI-linked proteins [START_REF] Sengeløv | Secretory vesicles of human neutrophils[END_REF][START_REF] Borregaard | Granules of the human neutrophilic polymorphonuclear leukocyte[END_REF].

One may argue that subpopulations of hematopoietic stem and progenitor cells do not contain FR alpha mRNA [START_REF] Ross | Folate receptor type β is a neutrophil lineage marker and is differentially expressed in myeloid leukaemia[END_REF]. This finding, however, does not exclude the presence of quiescent FR alpha mRNA having been or becoming active at other stages of differentiation.

At least two isoforms of FBP seem to be present in human serum.

A functional high-affinity FBP with a molecular size of 29 kDa similar to that of functional FBP in leukocytes (Fig 2 and6 ). Data in Fig 4 strongly suggest that this FBP mainly derives from neutrophil granules [START_REF] Colman | Folate-binding proteins[END_REF][START_REF] Costa | Appearance of a folate binder in leukocytes and serum of women who are pregnant or taking oral contraceptives[END_REF][START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF].

An alpha isoform of FR/FBP that contributes very little to the total concentration of functional FBP in serum ( Fig 5 and6). It appears as a minor peak at 43 kDa continuing into a broad immunoreactive peak reaching its maximum in void volume (> 120 kDa) . This isoform is present in serum at a low concentration as judged from its weak immuno-response, and could represent fragments of FR alpha from decomposed erythrocytes and neutrophils.

To summarize, the present report and other studies have revealed a rather complex expression pattern of FBP/FR isoforms in human blood cells. FR alpha on reticulocyte/erythrocyte membranes seems to mediate folate uptake and to be of great importance for the erythropoiesis. The functional FR beta on membranes of leukemic granulocytes has been utilized for therapeutic targeting while its non-functional counterpart on membranes of normal neutrophil granulocytes could be a putative signal protein. At this point it is difficult to assign a biological/functional role to the cytoplasmic FR/FBP alpha identified in neutrophil granulocytes.

The biological role of the high-affinity FBP in serum is still unresolved. Like other soluble nutrient and vitamin binding proteins in serum , e.g. lactoferrin and B 12 vitamin binding protein which are released during degranulation of neutrophil granulocytes [START_REF] Bloomfield | Influence of lithium and fluoride on degranulation from human neutrophils in vitro[END_REF][START_REF] Herbert | Release of vitamin binding proteins from granulocytes by lithium: vitamin B 12 and folate binding proteins[END_REF][START_REF] Holm | Li+ -induced release of the folate binding protein from human granulocytes[END_REF][START_REF] Holm | Lithium-induced release of lactoferrin from human granulocytes in vitro[END_REF] , FBP may exert bacteriostatic effects by depriving bacteria of endogenous folates [START_REF] Kane | Biology of disease: Role of folate binding proteins in folate metabolism[END_REF] . Binding to FBP protected tetrahydrofolate, the endogenous form of folate in pig serum, from biological degradation, whereas Correlation coefficient 0.644 ; p< 0.01 ( Spearman Rank Correlation). Data taken from references [START_REF] Holm | Great interindividual variations in maximum folate binding of leukocyte lysates from normal persons[END_REF][START_REF] Holm | Maximum folate binding in leukocyte lysates and sera from blood donors[END_REF]. A parallel ELISA of serum was performed with antibodies against bovine milk FBP to test the specificity of antibodies against human milk FBP.

3 17 -

 17 04-2008 Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T

  3 /30 mM NaCl to remove endogenous folate and then centrifuged at 1000 x g for 15 min . The supernatant was used for analysis. bsr20080031.doc 4 17-04-2008 Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T

  Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T

  Fig 2 showed a weak positive response in ELISA. The response was rather similar in the three samples despite widely different concentrations of functional FBP suggesting that alphaFR/FBP contributed very little -or most likely not at all to the total amount of functional FBP. The analytical performance of the ELISA was tested by running serial dilutions of purified human milk FBP (Fig 2). Indirect immunofluorescence with antibodies against human milk alpha FBP identified the alpha isoform of FR/FBP in neutrophil granulocytes subjected to ethanol fixation (31). The positive immunostaining in Fig 3a exhibited a characteristic pattern involving the nuclei or perinuclear zone bsr20080031.doc 6 17-04-2008 Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T of neutrophils. This type of pattern is supposed to be due to redistribution of cytoplasmic components to anionic components of the nucleus upon removal of lipids from granule membranes by ethanol fixation (31). Indirect immunofluorescence with antibodies against another cytoplasmic protein in neutrophils, lactoferrin, showed a very similar pattern of immunostaining (Fig 3b). No immunostaining was seen in Fig 3c with rabbit serum (control).

  Fig 6 were performed to estimate the molecular size of functional FBP and alpha FBP in serum. A Triton X-100 solubilized pool of sera from blood donors was bsr20080031.doc 7 17-04-2008 Bioscience Reports Immediate Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T preincubated with [ 3 H] folate to identify functional FBP. The elution profile revealed a major peak (400 cpm) at a position of 29 kDa similar to that in leukocyte lysates as well as a small (60 -80 cpm) broad and shoulder-like peak from 70-120 kDa.

  Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T at a more immature stage of erythrocyte development since the enzyme polyglutamate synthetase is only present in immature cells

  Publication. Published on 30 Jun 2008 as manuscript BSR20070033 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20070033 S t a g e 2 ( a ) P O S T -P R I N T The ELISA data in Fig 2 showed virtually similar concentrations of FBP alpha in three samples of leukocyte lysate despite widely different concentrations of functional FBP. Hence, FBP/FR alpha seems to contribute very little, most likely not at all, to the total content of functional FBP in the granulocyte. Indirect immunofluorescence data (Fig 3) revealed that FR alpha just like lactoferrin

LEGENDSFig 1

 1 Fig 1 The FBP alpha isoform in haemolysates identified by ELISA (OD, optical density) with antibodies directed against FBP alpha from human milk. Erythrocyte samples from five healthy subjects were analyzed in serial dilutions (each symbol represents haemolysate from a single individual). Analytical performance of the ELISA methods controlled by running serial dilutions of purified human milk FBP,1.0 nM (filled circles).Inset: A parallel ELISA of haemolysate performed with antibodies against bovine milk FBP to test the specificity of anti human milk FBP antibodies. Analytical performance of the ELISA controlled by running serial dilutions of purified bovine milk FBP,1.0 nM (filled circles).

Fig 2

 2 Fig 2 The FBP alpha isoform in leukocyte lysate. Leukocyte lysates containing 3.8 nmol (circles), 5.0 nmol (squares), and <0.01 nmol (diamonds) [ 3 H] folate bound (functional) FBP /10 9 cells were sampled from three healthy persons, and analyzed serially diluted ( two-fold initial dilution) in ELISA with antibodies against human milk alpha FBP. Abscissa, concentration of [ 3 H]folate bound FBP. Ordinate, OD in ELISA. Analytical performance of the ELISA controlled by running serial dilutions of purified human milk FBP,1.0 nM (filled circles). Inset: Gel filtration profile of functional FBP in leukocyte lysates. Leukocyte samples containing 3.8 nmol (circles) and <0.01 nmol (triangles down) [ 3 H] folate bound FBP / 10 9 cells (cf above) were preincubated with 10 nmol/l [ 3 H]folate in Tris HCl buffer of pH 7.4 containing Triton X-100 1 g/l before column application. Ordinate, cpm. Abscissa, elution volume.

Fig 3 Fig 4

 34 Fig 3 Indirect immunofluorescence for identification of cytoplasmic alpha FBP in neutrophil granulocytes. Typical immunostaining patterns with primary antibodies against human milk alpha FBP (a), and another cytoplasmic protein, lactoferrin (b) Rabbit serum as control (c). All solutions containing 1.5 mg protein/ml.Immunofluorescence disappeared when anti human milk FBP antibodies were preabsorbed with excess of human milk FBP. The immunostaining pattern disappeared when antibodies against human milk FBP were replaced with antibodies against bovine milk FBP (data not shown).

Fig 5

 5 Fig 5 The FBP alpha isoform in serum. Concentration of [ 3 H] folate bound FBP determined in pooled sera from pregnant women (triangles up), umbilical cord blood (triangles down) , and blood donors (diamonds) by equilibrium dialysis. Serial dilutions of the pooled sera analyzed in ELISA with antibodies against human milk alpha FBP. Abscissa, concentration of [ 3 H]folate bound FBP. Ordinate, OD in ELISA. Analytical performance of the ELISA controlled by running serial dilutions of purified human milk FBP (filled circles).

Fig 6

 6 Fig 6 Gel filtration profile of FBP isoforms in pooled sera from blood donors. Pooled sera were preincubated with 10 nmol/l [ 3 H] folate prior to column application. Left ordinate, cpm in effluent fractions (filled circles). Right ordinate, OD measurements in ELISA on effluent fractions (open circles).

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S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society