FEBS Letters 584 (2010) 2415–2420

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Regulation of Trk-dependent potassium transport by the calcineurin pathwayinvolves the Hal5 kinase

Carlos Casado a, Lynne Yenush b, Carmen Melero c, María del Carmen Ruiz c, Raquel Serrano a,Jorge Pérez-Valle b, Joaquín Ariño a,*, José Ramos c

a Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autónoma de Barcelona, Barcelona, Spainb Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Valencia, Spainc Departamento de Microbiología, Universidad de Córdoba, Córdoba, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 July 2009Revised 14 April 2010Accepted 16 April 2010Available online 20 April 2010

Edited by Ivan Sadowski

Keywords:High-affinity potassium transportTrk1Gene expressionSaccharomyces cerevisiae

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.04.042

Abbreviations: CDRE, Calcineurin Dependent Redithioerythritol

* Corresponding author. Address: Departament deular, Ed. V, Campus de Bellaterra, Universitat Autónode Vallès 08193, Barcelona, Spain. Fax: +34 93 58120

E-mail address: Joaquin.Arino@uab.es (J. Ariño).

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake inSaccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is neces-sary for high-affinity potassium uptake even in the absence of Na+ stress. HAL5 expression is inducedin response to stress in a calcineurin–dependent manner through a newly identified functionalCDRE (nt �195/�189). Lack of calcineurin decreases Hal5 protein levels, although with little effecton Trk1 amounts. However, the growth defect of cnb1 cells at K+-limiting conditions can be rescuedin part by overexpression of HAL5, and this mutation further aggravates the potassium require-ments of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expressionmay be biologically relevant for Trk1 regulation.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

The regulation of the intracellular concentrations of the majormonovalent cations (H+, K+ and Na+) is crucial to ensure properfunction of many cellular systems. In most plants and fungi, K+ isthe major intracellular cation and it is largely responsible for themaintenance of appropriate cellular volume, electrical membranepotential and ionic strength. In contrast, most cells strive to main-tain a relatively low concentration of sodium cations, particularlywhen K+ supply is sufficient [1,2]. The concentration of K+ in mostnatural environments is much lower than the intracellular concen-tration of this cation. Therefore, many organisms have developedhigh-affinity K+ uptake systems able to accumulate potassium [1] .

In the model yeast Saccharomyces cerevisiae, high-affinity potas-sium transport is mediated by the partially redundant Trk1 andTrk2 plasma membrane proteins [3,4]. Trk1, by far the most activetransporter, can work in a dual mode, showing low or high-affinityfor the cation [1]. Work in the last few years has identified the

chemical Societies. Published by E

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Bioquímica i Biologia Molec-ma de Barcelona, Cerdanyola06.

existence of diverse regulatory factors for high-affinity potassiumtransport, such as the Hal3/Ppz system [5,6], the SR protein kinaseSky1 [7] or the osmo-induced protein Hal1 [8], although theprecise regulatory mechanisms remain to be elucidated. A pair ofpartially redundant protein kinases, Sat4/Hal4 and Hal5, positivelyregulate Trk1 transporter function and confer salt tolerance whenoverexpressed [9]. Very recently, it has been shown that they arerequired for maintenance of Trk1 at the plasma membrane, partic-ularly when K+ levels in the medium are low [10]. Finally, an earlyreport identified the calcium-activated protein phosphatase calci-neurin as a necessary element for the transition of the transportersystem to the high-affinity potassium state induced by Na+ stress,which helps to discriminate K+ over Na+ [11]. However, in spite ofthis information, the molecular basis of the regulation of Trk1 func-tion and the possible interconnection of the regulatory processesdescribed above remain largely unexplored.

Calcineurin plays an important role in the response to differentforms of stress, including cation stress [12]. In S. cerevisiae, the en-zyme is a heterodimer composed of one catalytic subunit (encodedby CNA1 or CNA2) and one regulatory subunit (encoded by CNB1).Its activation under stress conditions is triggered by a transientincrease in intracellular calcium and results in changes in geneexpression, essentially mediated through the Crz1/Tcn1/Hal8 tran-scription factor [13–16]. Upon dephosphorylation by calcineurin,Crz1 enters the nucleus [17] and binds to specific CDRE sequences

lsevier B.V. All rights reserved.

2416 C. Casado et al. / FEBS Letters 584 (2010) 2415–2420

(Calcineurin Dependent Response Elements), present in the pro-moter region of target genes.

Calcineurin exerts a complex influence on cation homeostasis.First, activation of the phosphatase upon sodium stress or alkalinepH increases the expression of the ENA1 gene [11,18,19], which en-codes a Na+-ATPase required for sodium detoxification, by directbinding of Crz1 to the ENA1 promoter [20–22]. Second, as men-tioned above, in the presence of sodium stress, calcineurin affectspotassium transport, presumably through regulation of the Trk1transporter [11]. However, neither the possibility that calcineurinmay play a role in the regulation of potassium transporters inthe absence of stress nor the possible interaction between calci-neurin and other pathways regulating Trk1 function have beeninvestigated. In this work, we demonstrate that disruption of calci-neurin signalling affects potassium uptake and increases potas-sium requirements even under standard growth conditions. Inaddition, we show that expression of the HAL5 protein kinase geneis under the control of calcineurin and Crz1, thus establishing afunctional link between two important regulators of Trk1 activityand potassium homeostasis.

2. Materials and methods

2.1. Yeast strains and growth conditions

Yeast cells were grown at 28 �C in YPD medium (10 g/L yeastextract, 20 g/L peptone and 20 g/L dextrose) or, when carryingplasmids, in synthetic complete medium [23] containing 2% glu-cose and lacking the appropriate selection requirements. Strainsused in this work are listed in Table 1. Strain ESV212 was describedin Ref. [24]. Strain MAR89 was constructed by transforming strainEDN92 with a previously described cnb1::TRP1 disruption cassette[25]. Strain MAR91 was made by transforming MAR89 with a 2.44Kbp trk2::HIS3 cassette obtained from plasmid pRH-3 [26] byEcoRV/BamHI digestion. This strain was transformed with a 4.5Kbp trk1::LEU2 fragment, derived from plasmid pRH2.23 [26] di-gested with SacI, to yield strain MAR108. Construction of strainsCCV31 and CCV32 is described in Supplementary data. PlasmidYEp-HAL5 contains the HAL5 gene cloned into the multicopy vectorYEp352, and includes 517 bp of 50 and 201 bp of 30 untranslated re-gions [10].

Potassium requirements on solid media was tested by spottingcells (5 lL) at initial OD600 of 0.05 on ammonium phosphate (AmP)medium agar plates containing different KCl concentrations. Dou-bling time was calculated as in [27]. In some cases (i.e. Fig. 4Band C), growth under limiting external potassium concentrationswas carried out using a newly developed YNB-based liquid med-ium (Translucent K-free medium, [28]) whose final potassium con-tent is negligible (�15 lM).

Table 1Yeast strains used in this work.

Strain Genotype Source/Reference

DBY746 MAT a, trp1, leu2-3, ura3, his3 Botstein et al.MAR15 DBY746 cnb1::KAN [19]EDN92 DBY746 crz1::KAN [19]ESV212 DBY746 trk1::LEU2 trk2::HIS3 [24]MAR89 DBY746 crz1::KAN cnb1::TRP1 This workMAR91 DBY746 crz1::KAN cnb1::TRP1 trk2::HIS3 This workMAR 108 DBY746 crz1::KAN cnb1::TRP1 trk1::LEU2 trk2::HIS3 This workCCV31 DBY746 hal4::HIS3 This workCCV32 DBY746 cnb1::KAN hal4::HIS3 This work

2.2. K+ cellular content and Rb+ transport experiments

The K+ content of cells growing in AmP medium was deter-mined essentially as in Ref. [29]. The kinetic constants for rubidiumtransport were determined in K+-starved cells as described inSupplemental data.

2.3. LacZ reporters and b-galactosidase assays

Translational fusions of the HAL4 and HAL5 promoters to theLacZ gene were generated as follows. The regions �936/+45 ofHAL4 and �615/+33 of HAL5 were amplified from genomic DNAusing oligonucleotides pairs 50-promHAL4/30-promHAL4 and 50-promHAL5/30-promHAL5, respectively (see Supplementary data).Artificial EcoRI and XbaI ends were added to facilitate cloning intothese same sites of plasmid YEp357, yielding pHAL4 and pHAL5.Mutagenesis of putative CDREs in the HAL5 promoter was per-formed by two-step PCR as described in Supplementary data toyield constructs pHAL5-PCDRE1 and pHAL5-PCDRE2, respectively. As-say of constructs was performed essentially as described [21].Alkaline stress was carried out by resuspending cells in YPD buf-fered at pH 8.2 with 50 mM TAPS. For calcium stress, CaCl2 wasadded to reach a final concentration of 0.2 M. In both cases cellswere collected after 1 h.

2.4. Protein extractions and immunoblot analysis

For NaCl and CaCl2 treatments, the indicated yeast strains weregrown to an OD660 of 0.4 (5 � 106 cells/mL) in YPD media. Cellswere treated with 0.8 M NaCl or 0.1 M CaCl2 for 30 min, harvestedby centrifugation and frozen at �70 �C. For K+ starvation experi-ments, cells were grown to an OD660 of 0.4 in AmP media supple-mented with 10 mM KCl. Cells were washed extensively in waterand resuspended at the same density in ammonium phosphatemedia with or without K+ supplementation (10 mM) and incubatedfor 2 h, harvested by centrifugation and frozen at �70 �C. For pro-tein extraction, cells were resuspended in homogenization buffer(50 mM Tris pH 8.0, 0.1 M KCl, 5 mM EDTA, 5 mM 1,4-dithioeryth-ritol (DTE), 20% sucrose (w/v), plus protease inhibitor cocktail(Roche)) and lysed by vortexing with glass beads. The lysate wascollected after centrifugation for 5 min at 500�g. The crude extractwas separated into soluble and particulate fractions by centrifuga-tion for 30 min at 16 000�g at 4 �C. The particulate fraction wasresuspended directly in Laemmli sample buffer, electrophoresedand immunoblots probed for Trk1 and Hal5 as described [30].The relative intensity of each band was determined by quantifyingdigital images of the band corresponding to Hal5 (or Trk1) and arepresentative band in the same lane of the PonS-stained filter(loading control) using the Image Gauge V4.0 software (FUJIFILM).The local background was subtracted in each case. The value of thefirst control lane (defined as Hal5 intensity/loading control inten-sity) was set at 1.0 and the rest of the results are expressed as rel-ative intensities in arbitrary units. Similar results were observed intwo different experiments in all cases.

3. Results and discussion

Almost 15 years ago, a functional interaction between the Trk1high-affinity potassium transporter and the calcineurin phospha-tase was proposed [11]. The authors reported that the presence offunctional calcineurin was important for proper Na+/K+ discrimina-tion by the Trk1 transporter under conditions of salt stress. Toinvestigate the mechanism of this regulation we performed, as a firststep, a detailed characterization of the potassium requirements ofmutant strains lacking the regulatory subunit of calcineurin, CNB1,

C. Casado et al. / FEBS Letters 584 (2010) 2415–2420 2417

the calcineurin-dependent transcription factor, CRZ1, as well as inthe cnb1 crz1 double mutant. As observed in Fig. 1A, both the cnb1and the cnb1 crz1 mutant display growth defects when grown on so-lid AmP medium containing limiting concentrations of potassium.Quantification of the growth defect on liquid AmP medium(Fig. 1B) showed that, whereas the duplication time when the med-ium was supplemented with 0.7 mM KCl was 5 h for the wild typestrain, this parameter increased slightly for the crz1 strain (5.25 h)and markedly for the cnb1 (7.75 h) and cnb1 crz1 (11 h) strains.Increasing the external KCl concentration up to 3 mM or above elim-inated the growth defect of cnb1 and crz1 strains, although in allcases the cnb1 crz1 strain showed a slower growth rate (around6.5 h vs. 5 h in the wild type).These results suggest that the calcineu-rin/Crz1 signalling pathway plays a role in high-affinity potassiumuptake also in the absence of sodium stress.

To confirm this hypothesis and to gain insight into the mecha-nisms involved, cells were grown for 24 h in AmP medium contain-ing 50 mM KCl and then were switched to the same mediumwithout added potassium. As shown in Fig. 1C, an increase in boththe rate and total amount of K+ loss in the cnb1 crz1 mutant wasobserved, suggesting a defect in the retention and/or recapture of

Fig. 1. Disruption of the calcineurin pathway increases potassium requirements in the a(MAR15), crz1 (EDN92) and cnb1 crz1 (MAR89) derivatives were spotted on AmP platmonitored after 4 days. (B) The indicated strains were inoculated at OD600 of 0.04 on AmP(C) Strains DBY746 and MAR89 were grown for 24 h on liquid AmP medium containing 5taken at the indicated times and the intracellular concentration of potassium measured.difference (P < 0.01) between the wild type and the cnb1 crz1 strains. (D) Strains ESV212containing the indicated amounts of KCl. Dilutions were spotted in the indicated plates

K+ from the external medium. Measurement of Rb+ uptake inpotassium-starved cells demonstrated that disruption of the calci-neurin pathway markedly decreased the high-affinity Rb+ uptake.The Km and Vmax of Rb+ uptake in the same set of strains was deter-mined (Table 2). In agreement with early results [11] we did notobserve significant changes in the relative affinity for rubidiumin any of the strains tested (Table 2 and Ref. [11]). However, wedid observe a significant decrease in the Vmax for rubidium uptakein all three mutant strains, being most pronounced in the cnb1 crz1mutant. Finally, we investigated if disruption of calcineurin signal-ling would have any effect in the absence of the high-affinitypotassium Trk uptake system. As shown in Fig. 1D, lack of cnb1crz1 did not aggravate the phenotype of the trk1 trk2 strain at lim-iting potassium concentrations. Taken together, these results indi-cate that the defects in potassium homeostasis observed in thecnb1 crz1 mutants are mediated by the high-affinity potassiumtransporters Trk1 and Trk2.

We next studied the mechanism by which the calcineurin/Crz1pathway may be regulating the high-affinity potassium transport-ers. We noticed that: (1) analysis of transcriptomic data in the lit-erature revealed that HAL5 was induced upon salt treatment in a

bsence of sodium stress. (A) Two dilutions of wild type strain DBY746 and its cnb1es (pH 5.8) supplemented with the indicated concentrations of KCl. Growth wasliquid medium containing 0.7 mM KCl and grown for the indicated number of hours.0 mM KCl. Cells were then resuspended in AP medium lacking potassium, samples

Data are mean ± S.E.M. from four determinations. The asterisk denotes a significant(trk1 trk2) and MAR108 (cnb1 crz1 trk1 trk2) were inoculated into AmP agar platesand growth recorded after 4 days.

Table 2Kinetic parameters of strains deficient in the calcineurin pathway. Cells werepotassium-starved for 5 h in ammonium phosphate medium. Km and Vmax values forRb+ uptake were obtained from the double reciprocal plot of the experimental data.See Supplementary data for details.

Strains Km (mM) Vmax (nmol mg�1 min�1)

DBY746 (CNB1 CRZ1) 0.13 ± 0.01 30 ± 2MAR15 (cnb1) 0.14 ± 0.01 22 ± 1EDN92 (crz1) 0.14 ± 0.01 24 ± 1MAR89 (cnb1 crz1) 0.15 ± 0.01 19 ± 1

Fig. 2. HAL5 expression is regulated by the calcineurin pathway. DBY746 cells andthe indicated mutant strains were transformed with plasmids pHAL4 and pHAL5.Cells were incubated at pH 8.2 (closed bars) or with 0.1 M CaCl2 (stripped bars) for1 h. Open bars denotes standard growth conditions. Cells were collected and b-galactosidase activity measured. Data are means ± S.E.M. from six independentexperiments.

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calcineurin/Crz1-dependent manner [16]. In addition, our owndata on the transcriptional response to alkaline pH, a situation thatactivates calcineurin, also revealed induction of the HAL5 gene[31]; (2) we recently showed that Hal4 and Hal5 are required forthe maintenance of Trk1 at the plasma membrane [10]; and (3)the present study revealed a Trk1/Trk2-dependent defect in potas-sium uptake in the cnb1 crz1 mutant. Based on these observations,we considered that the effect of calcineurin on Trk1 could be med-iated, at least in part, through the regulation of HAL5 expression.We first sought to confirm and characterize the presumed role ofcalcineurin on HAL4/HAL5 expression. To this end, the promoterregions of HAL4 or HAL5 were fused to LacZ and the expression ofb-galactosidase activity driven by these promoters in response toalkaline pH or CaCl2, two conditions known to trigger a calcineu-rin/Crz1-dependent transcriptional response [12,19,21,31], wastested. As shown in Fig. 2, basal HAL4-driven expression was rela-tively higher than that of HAL5. However, the HAL4 promoter didno respond to the stress conditions tested and was only marginallyaffected by the cnb1 mutation. In contrast, we observed a clearinduction of HAL5-mediated expression in response to alkalinepH and CaCl2 treatments. Moreover, this induction was completely

Fig. 3. Mutagenesis of the HAL5 promoter reveals a functional CDRE. (A) The 615 nt doCDRE1 and CDRE2 denote the sequences identified by computer analysis as putative CDRtransformed with the reporter plasmid pHAL5 or with versions carrying the indicated m

absent in mutant strains lacking cnb1 and/or crz1. Direct measure-ment of mRNAs by quantitative RT-PCR using HAL5-specific prim-ers showed an increase of 3 to 3.5-fold in HAL5 mRNA in cellstreated for 10 min with 0.2 M CaCl2 or stressed at pH 8.1 for thesame period of time. These increases were again fully dependent

wnstream the initiating Met codon (underlined) of the HAL5 gene are represented.E and subjected to mutagenesis (to yield a TCTAGA XbaI site). (B) DBY746 cells wereutations in CDRE1 or CDRE2 and treated as indicated in the legend of Fig. 2.

C. Casado et al. / FEBS Letters 584 (2010) 2415–2420 2419

on the presence of a functional calcineurin pathway (data notshown).

Based on these results, we searched the HAL5 promoter regionfor CDREs by using an alignment matrix based in the G(A/T)GGC(C/T)(C/G) sequence and the Regulatory Sequence AnalysisTools WEB interface [32]. Two possible sites were identified at�195/�189 (ln(P) = �8.27) and �160/�154 bp (ln(P) = �8.56) up-stream of the ATG, subsequently referred to as CDRE1 and CDRE2,respectively (Fig. 3A). To establish the functionality of these pro-posed sites, a PCR-based approach was used to mutate these se-quences in the context of a LacZ reporter construct. As shown inFig. 3B, mutation of the CDRE1 site resulted in a substantial lossof b-galactosidase activity in response to alkaline pH and, espe-cially, CaCl2 treatment, whereas mutation of CDRE2 led to a verymodest reduction of the Ca2+-dependent induction. Mutation ofboth sites added only marginally to the effect of mutation of CDRE1(not shown). These results demonstrate that transcription of theHAL5 gene is induced in response to several stress conditions in aCrz1-dependent manner and define the specific CDRE elementprincipally responsible for this regulation.

To confirm the physiological relevance of this transcriptionalstress response, we analyzed the amount of Hal5 present aftersimilar stress treatments in both wild type and cnb1 crz1 mutantstrains. As observed in Fig. 4A (upper panel), the Hal5 proteinaccumulates in wild type strains after treatment with CaCl2 andNaCl. In some experiments, we also observed a change in theelectrophoretic mobility of the protein under stress conditions

Fig. 4. Influence of the calcineurin pathway on Hal5 and Trk1 protein expression, and inMAR89 (cnb1 crz1) strains were grown YPD media and subjected to 0.8 M NaCl or 0.1 Mpanel) Cells were grown to mid-log phase in AmP medium (10 mM KCl) and transferredresumed for 2 h and protein extracts probed with anti-HA (for HA-Trk1 detection) or anrelative intensity of the Hal5 or Trk1 signal, determined as described in Section 2.4, anindicated strains were inoculated at OD660 of 0.005 on K-free Translucent medium containas percentage of growth respective to equivalent cultures grown on 50 mM KCl and arestrains were transformed with plasmid YEp-HAL5 or an empty plasmid (YEpØ), and cellwith 1 mM KCl. Data is expressed as in panel B and was obtained from six independent exstrain with or without plasmid-borne HAL5.

(i.e. after treatment with NaCl). This shift is often attributed tochanges in the phosphorylation state of the protein. WhetherHal5 undergoes autophosphorylation or is regulated by upstreamkinases is currently under investigation. In the cnb1 crz1 mutant,we observed a modest reduction in the amount of Hal5 detectedunder normal growth conditions and less accumulation afterstress treatments.

Importantly, as shown in Fig. 4A, the amount of Hal5 proteindetected in the cnb1 crz1 mutant is markedly reduced when thecells are grown in minimal AmP media. This decrease in theamount of Hal5 protein may explain the reduced capacity of thecnb1 crz1 mutant to accumulate potassium. We did not observe,however, significant differences in the amount of Trk1 present inthe cnb1 crz1 mutant after up to 4 h of potassium starvation(Fig. 4A and data not shown). Although this may seem surprising,in comparison with our previous studies with hal4 hal5 mutantstrains in which, upon potassium starvation, the amount of Trk1present at the plasma membrane is markedly reduced [10], it mustbe considered that the defect in potassium uptake is much moredrastic in the hal4 hal5 mutant than in cnb1 crz1 strains, wheresome Hal5 protein is still present and HAL4 expression is notaffected (Fig. 2). Moreover, it is likely that Trk1 activity is regulatedby more than one mechanism, i.e. protein trafficking/turnover andalteration of the affinity state. The present results are consistentwith the observed weak effect on Trk1 activity caused by blockageof calcineurin signalling and support the original hypothesisinvolving regulation of Trk1 affinity. Since Hal4 and Hal5 have

cell growth under limiting potassium concentrations. (A) Upper panel. DBY746 andCaCl2 for 30 min (+). Immunoblots were probed with anti-Hal5 antibodies. (Lowerto the same medium in the presence (+) or absence (�) of 10 mM KCl. Growth wasti-Hal5 antibodies. PonS, Ponceau S staining. Numbers below each blot denote thed similar results were obtained in at least two independent experiments. (B) Theing the indicated amounts of KCl. Growth was resumed for 16 h. Data are expressedmean ± S.E.M. from 12 independent experiments. (C) Wild type and MAR15 (cnb1)

s were grown as indicated in panel B on K-free Translucent medium supplementedperiments. Asterisk denotes a significant difference (P < 0.005) in growth of the cnb1

2420 C. Casado et al. / FEBS Letters 584 (2010) 2415–2420

been postulated to be functionally redundant, we hypothesisedthat deletion of HAL4 may enhance the functional effects derivedfrom calcineurin regulation of HAL5 expression. As shown inFig. 4B, when limiting amounts of extracellular potassium are pres-ent, growth of the cnb1 hal4 strain is decreased when comparedwith the cnb1 strain (in which Hal4 is active). In addition(Fig. 4C), under conditions of potassium limitation, expression ofHAL5 from a high-copy plasmid is able to significantly increasegrowth of the cnb1 strain supporting the notion that, at least inpart, the increased potassium requirements of the cnb1 strainsare caused by a defect in Hal5 expression.

These results suggest that the calcineurin-mediated regulationof HAL5 expression constitutes a biologically significant mecha-nism for controlling high-affinity potassium uptake through theTrk1 transporter. Of course, our data do not rule out additionalHal5-independent, calcineurin-mediated mechanisms for Trk1 reg-ulation. However, our results suggest that this calcineurin-depen-dent mechanism of Trk1 regulation is relevant not only in thepresence of sodium, but also in the absence of stress conditionsand suggest that Hal4 and Hal5 may have a dual role in Trk1 reg-ulation, affecting both its trafficking and affinity state.

Acknowledgements

We thank A. Rodriguez-Navarro for plasmids and Amparo Ruizfor support. The skillful technical help of Montse Robredo, AnnaVilalta and Jessica Rothe is acknowledged. Supported by grantsBFU2008-04188-C03-01 and GEN2006-27748-C2-1-E/SYS (SysMO)to JA; BFU2008-04188-C03-02 to L.Y., and BFU2008-04188-C03-03and GEN2006-27748-C2-2-E/SYS (SysMO) to J.R. (Ministry of Sci-ence and Innovation, Spain and FEDER). J.A. was recipient of an‘‘Ajut 2009SGR-1091” (Generalitat de Catalunya). J. Pérez-Vallewas supported by a predoctoral fellowship from the Spanish Min-istry of Science and Technology.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2010.04.042.

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