Molecular & Biochemical Parasitology 135 (2004) 165–169

Short communication

Regulatory pathways in ion homeostasis involving calcineurinand a calcium transporting ATPase are different between

yeast and schistosomes

Alessandro Rossia,∗, Michel Ghislainb, Mo-Quen Klinkerta,1a Department of Parasitology, Institute for Tropical Medicine, University of Tübingen, Wilhelmstrasse 27, 72074 Tübingen, Germany

b Department of Physiological Biochemistry, University of Louvain-la-Neuve, Louvain-la-Neuve, Belgium

Received 21 November 2003; received in revised form 16 January 2004; accepted 21 January 2004

Keywords: Schistosoma mansoni; Calcineurin; SERCA ATPase; Ion homeostasis; Heterologous expression

Calcineurin (CN) is a calcium and calmodulin-dependentphosphatase, which is involved in a variety of signal trans-duction pathways (reviewed in[1]). The protein is com-posed of two subunits. The catalytic subunit A (CNA) isorganized into four conserved domains. The N-terminalcatalytic domain shows extensive homology to other knownphosphatases and is followed by two amphipathic heliceswhich, respectively, bind the regulatory subunit of cal-cineurin (CNB) and calmodulin (CaM). The fourth domainis known as the autoinhibitory domain (AID) as it interactswith the catalytic pocket to maintain the enzyme in an inac-tive conformation. The CNB subunit is a calcium-bindingprotein and is structurally similar to CaM. Upon CaM bind-ing, the C-terminal AID is displaced from the catalytic siteof CNA, which results in a 20-fold stimulation of the basalphosphatase activity (reviewed in[1]).

The genes coding for the calcineurin subunits from theblood flukeSchistosoma mansoni (SmCN) have been cloned[2,3]. Indirect immunofluorescence as well as SmCN pro-moter analysis, using the green fluorescence protein as areporter gene, indicate that the protein phosphatase is ex-pressed in the excretory system of adult worms[2,3]. The lo-calization of SmCN in the proximal part of the schistosome’sexcretory system is analogous to the situation of the ver-tebrate kidney, where high CN activity is detected in the

∗ Corresponding author. Present address: Department of Neurobiologyand Anatomy, University of Utah School of Medicine, 401 MedicalResearch and Education Building, 20 North 1900 East, Salt Lake City,UT 84132, USA. Tel.:+1-801-5853674; fax:+1-801-5855171.

E-mail address: rossi@neuro.utah.edu (A. Rossi).1 Present address: Bernhard Nocht Institute for Tropical Medicine,

Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.

proximal tubules (reviewed in[4]). It has been shown thatCN stimulates activity of Na+, K+-ATPase in renal tubulecells [5]. The molecular mechanisms underlying this regu-lation remain to be clarified. However, the finding that CNis implicated in the regulation of ion homeostasis in a largenumber of eukaryotic cells, including yeast, neurons or plantcells [6–8], opens the possibility that SmCN may also ex-ert an important function in the control of ion fluxes in theparasite excretory system.

In the yeastSaccharomyces cerevisiae CN up-regulatesthe TCN1/CRZ1 transcription factor[9,10], which inducesthe expression of genes indispensable for cell viability un-der salt stress. Amongst them are genes encoding transporterproteins, P-type ATPases and proteins involved in cell wallsynthesis/maintenance[11]. Mutations in theCNB1, CNA1,andCNA2 genes or in theTCN1/CRZ1 gene cause a similardecreased tolerance to high concentrations of Na+, Li+ andMn2+ [12]. It has also been shown that CN controls cal-cium homeostasis through regulation of Ca2+ channel andCa2+ pump [13–15]. The vacuolar Ca2+-ATPase, PMC1,provides the largest contribution in the depletion of Ca2+ions from the cytosol, while the Golgi Ca2+-ATPase, PMR1,and the Ca2+-exchanger, VCX1, contribute to a lesser ex-tent [14]. Mutants in thePMC1 gene fail to grow in amedium containing 200 mM CaCl2. Deletion of bothPMC1andPMR1 leads to cell death under normal growth condi-tion [15]. Interestingly, the growth defects of thepmc1 mu-tants are suppressed by the disruption of CN function[15].Under these conditions, down regulation of VCX1 activityby CN is relieved and the activated exchanger is capableof substituting for the vacuolar Ca2+ pump, thereby pre-venting the lethal accumulation of Ca2+ ions in the cytosol[14].

0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.molbiopara.2004.01.014

166 A. Rossi et al. / Molecular & Biochemical Parasitology 135 (2004) 165–169

The S. mansoni sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), SMA2, has been shown to restore via-bility of pmc1 pmr1 yeast double mutants and to function inCN-mediated Ca2+ tolerance[16]. Strikingly, SMA2 is ableto restore calcium tolerance inpmc1 vcx1 double mutantsonly upon CNB1 deletion or incubation with cyclosporinA, a CN inhibitor. These results therefore indicate that theSMA2 pumping activity is negatively regulated by yeastCN.

Since stable transformation procedures in schistosomeshave not yet been established nor are schistosome celllines available for functional studies, we chose the yeastexpression system to assess whether SMA2 is a potentialtarget for dephosphorylation by SmCN. This further inves-tigation into CN-mediated regulation of Ca2+ pumps at apost-translational level can help us to gain an insight intoparasite physiology. Moreover, the yeast expression systemis a generally useful approach for the analysis of molecularpathways in the schistosome parasite.

We first sought to functionally express SmCNA inS. cere-visiae and test whether this schistosomal phosphatase can re-store salt tolerance incna1 cna2 double mutants. Full lengthSmCNA cDNA was cloned in the yeast expression vectorpRS426(PPMA1), a URA3-based, high copy number plas-mid which contains the strong and constitutive promoter ofthe plasma membrane H+-ATPase (PMA1) gene[16]. Theresulting construct, p426SmCNA, was transformed into aCN-deficient yeast strain, K571. As a consequence of thecna1 cna2 double mutation, K571 is unable to grow in thepresence of elevated Li+ concentrations[6]. K571 and theisogenic wild-type, K601, were also transformed with anempty pRS426 plasmid, as a negative control.

Using a spotting assay, salt sensitivity of the transformedcells was determined. The K601 strain containing pRS426grows in the absence or presence of 400 mM LiCl at compa-rable rates (Fig. 1A, line 1). As expected, the mutant straincontaining the empty vector failed to form colonies in thepresence of elevated Li+ concentrations (line 2). Upon Sm-CNA expression, however, the growth defects of thecna1cna2 mutant were partially suppressed (line 3). To betterquantify the level of salt tolerance in mutant cells expressingSmCNA, we performed the assay in liquid medium usingLiCl concentrations ranging from 0 to 600 mM (Fig. 1B).The wild-type strain shows an IC50 (defined as the salt con-centration causing a 50% decrease in cell growth relative tothe maximal growth) greater than 600 mM. In comparison,the mutant strain transformed with pRS426 shows a reducedIC50 of about 300 mM. Upon SmCNA expression, however,LiCl tolerance of K571 increases to about 400 mM. The re-sults of the phenotypic analysis therefore indicate that Sm-CNA is functional in yeast and can partially substitute forthe yeast counterpart.

Thecna1 cna2 mutant strain expressing SmCNA shows alower level of salt tolerance when compared to the wild-typestrain. The difference could be explained if SmCNA overex-pression has a detrimental effect on cell growth, through per-

turbed regulation of essential genes. To reduce the levels ofSmCNA expression, we used aTRP1-based low-copy num-ber (Cen) plasmid, pRS314(PGAL1) which, instead of theconstitutivePMA1 promoter, contains a galactose inducibleGAL1 promoter. pRS314(PGAL1) was created by ligating the3-kb BglI fragment of pRS314[17] with the 2.3-kbBglIfragment of p424GAL1[18]. The growth defects of the CNmutant K571 were partially suppressed by the expressionof SmCNA from pRS314(PGAL1) (data not shown), arguingagainst the deleterious effects of SmCNA overexpression.

To compare the expression levels of SmCNA in the dif-ferent yeast strains, whole-cell extracts were analyzed byimmunoblotting with a polyclonal anti-SmCNA antibody.However, no band with a molecular size corresponding tothat of SmCNA could be detected (data not shown). Thus,a most likely explanation for the partial suppression of thegrowth defects of the CN mutant could be the low expres-sion level of SmCNA in yeast cells. However, we cannotrule out the possibility that SmCNA and the yeast regulatoryB subunit are only weakly interacting, thereby affecting thereconstitution of a fully active holoenzyme.

An alternative explanation for the low activity of Sm-CNA in yeast cells is based on the presence of a longC-terminal tail downstream from the AID, which may haveinhibitory effects on the activity of the protein in yeast,either simply by steric hindrance or by an unknown reg-ulated mechanism. The presence of such long C-terminaltails is observed in other CNA subunits, such asDrosophilamelanogaster (accession number M97012),Cryptococ-cus neoformans (AF042082) andDictyostelium discodeum(X97280). The function of the additional sequences thatare rich in polar amino acids (S. mansoni 72.3%; D.melanogaster 75.4%;C. neoformans 49.6%;D. discodeum67.9%) and repetitive stretches (such as the poly-Ser6/Asn6stretch in theDrosophila sequence; the poly-Gln11 stretchin the Dyctiostelium sequence or the four-fold repeatedmotif RXNSX(G/A)(E/D)LX in the same protein) in theseorganisms is not known. To determine whether SmCNA ac-tivity in vivo is inhibited by the C-terminal tail, we analyzedthe effect of a chimeric schistosome-yeast CNA cassettein transformed yeast cells. In the SmCNA�C construct,the last 170 amino acids of SmCNA were replaced by the27 terminal residues of the yeast CNA2 protein (Fig. 1C).As shown inFig. 1A, cna1 cna2 mutant cells transformedeither with p426SmCNA or p426SmCNA�C display nodifference in their LiCl sensitivities (lines 3 and 4), therebydisproving the possibility that the 170 amino acids couldregulate or inhibit SmCN activity. The same transformedstrains displayed no significant differences in their IC50values when tested in liquid cultures (data not shown).

SmCNA functionality inS. cerevisiae was further charac-terized in a growth assay, based on CN-mediated repressionof the VCX1 exchanger. The deletion of theCNA1 andCNA2genes in apmc1 mutant (strain K559) leads to high Ca2+tolerance, as a result of relieved CN-mediated repression ofVCX1. Reintroduction of the yeastCNA genes in the K559

A. Rossi et al. / Molecular & Biochemical Parasitology 135 (2004) 165–169 167

Fig. 1. Functional expression of SmCNA in yeast strains lacking calcineurin. TheS. cerevisiae strains K601 (Mata ade2-1 can1-100 his3-11,15 leu2-3,112trp1-1 ura3-1), K571 (Mata ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 cna1::hisG cna2::HIS3), K559 (Mata ade2-1 can1-100 his3-11,15leu2-3,112 trp1-1 ura3-1 pmc1::LEU2 cna1::hisG cna2::HIS3) have been described[14]. (A) The wild-type K601 (line1) and mutant K571 (lines 2–4)strains containing pRS426 or p426SmCNAFL and p426SmCNA�C were grown overnight in SD medium (0.7% yeast nitrogen base, 2% glucose) lackinguracil. Serial dilutions of the saturated cultures were spotted onto a solid medium containing or lacking 400 mM LiCl. The plates were photographedafter a 2- or 3-day incubation at 30◦C. (B) Precultures from the same strains as in panel A (except for p426SmCNA�C) were diluted to an OD600 nm

of 0.05 and grown overnight in a liquid medium supplemented with different LiCl concentrations. K601 transformants in line 1(�), K571 transformantsin line 2 (�), and K571 transformants in line 3 (�). The values presented are the average of duplicate cultures. (C) Schematic representation of thechimeric construct SmCNA�C. The tail of SmCNAFL was replaced by the last 27 amino acids downstream Asp577 of the yeast CNA2 protein. Thechimeric CNA cassette was generated by PCR, recombining the first 1422 bp of SmCNA coding region with a 177 bp stretch of the yeastCNA2 gene(including the last 81 bp of the coding region and 96 bp of the 3′ UTR). This latter sequence was PCR amplified using genomic DNA from the K601strain as template. For PCR amplifications, proofreading polymerase (Expand, Roche) was used and the absence of mutations and correct ligation wereconfirmed by DNA sequencing.

strain restores Ca2+ sensitivity[15]. To test the interchange-ability of the CN catalytic subunits betweenS. mansoni andS. cerevisiae in the regulation of Ca2+ homeostasis, we in-troduced the SmCNA expression plasmid (p426SmCNA)in K559 cells and analyzed the sensitivity of the trans-formed cells to increasing Ca2+ concentrations. No pheno-typic change was detected (data not shown), suggesting thatthe putative yeast effector molecule that acts by repressingVCX1 is not recognized by SmCNA as target substrate. Thisresult raises the possibility that both yeast and schistosomal

CNs have different substrate specificities and that criticalamino acid differences in the SmCNA catalytic domain maybe responsible for the poor recognition and activation of theTCN1/CRZ1 transcription factor and, hence, to an incom-plete restoration of salt tolerance.

We next analyzed the effect of SmCNA expression on thefunction of a schistosomal SERCA isoform, SMA2[16]. TheYAR1 strain contains multiple mutations inPMC1, CNA1,CNA2, and VCX1. The growth of the mutant cells in thepresence of 100 mM CaCl2 is supported by the heterologous

168 A. Rossi et al. / Molecular & Biochemical Parasitology 135 (2004) 165–169

Fig. 2. Growth assay showing the lack of interaction between SMA2 and SmCNA2 when expressed in yeast. The YAR1 strain is avcx1::KanR derivative ofK559. TheVCX1 gene in K559 was replaced by thevcx1::KanR null allele after yeast transformation and homologous gene recombination, as previouslydescribed[16]. The p426SMA2 plasmid expressing SMA2 from thePMA1 promoter (PPMA1) was constructed by Talla et al.[16]. (A) The pmc1 cna1cna2 vcx1 quadruple mutant (YAR1) containing empty pRS426 (line 1) or the SMA2 expression plasmid (line 2) were tested for calcium sensitivity byspotting serial dilution of saturated cultures on SD (yeast nitrogen base concentration was lowered to 0.17% in order to prevent precipitation) supplementedwith 100 mM CaCl2. (B) Relative growth rates of YAR1 cells containing pRS426 and pRS314 (�), pRS426+ p314(GAL1)SmCNA (�); p426SMA2+ pRS314 (�); and p426SMA2+ p314(GAL1)SmCNA (�). The different strains were grown overnight in a minimal medium containing 2% raffinoseand lacking uracil and tryptophan. The cultures were then diluted to an OD600 nm of 0.05 and incubated for 24 h in minimal medium supplemented with4% galactose to induce SmCNA expression and the indicated CaCl2 concentrations. The values presented are the means of duplicate cultures.

expression of SMA2 from the p426SMA2 plasmid (Fig. 2A,line 2). In order to assess whether SmCNA can antagonizethe calcium tolerance induced by the activity of SMA2, thep314(GAL1)SmCNA plasmid was introduced in YAR1 cellsexpressing SMA2. Transformations with the empty pRS426and pRS314 plasmids in all possible combinations were alsodone (Fig. 2B). The YAR1 cells containing the different plas-mids were cultured in a raffinose-containing medium beforethey were shifted to galactose to induce the expression ofSmCNA under high CaCl2 concentrations. These cells con-taining the pRS426 and pRS314 plasmids show the samelevel of calcium sensitivity as the cells expressing SmCNAalone (IC50 of about 100 mM CaCl2) (Fig. 2B). Expressionof SMA2 in the YAR1 strain results in increased Ca2+ tol-erance, with an IC50 of about 300 mM CaCl2. Simultaneousexpression of SmCNA, however, seems to have no effect onthe levels of Ca2+ tolerance as the IC50 value is comparableto that obtained when SMA2 alone is expressed. There aretwo possible explanations for this finding. It can be arguedthat SmCNA does not play a direct role in SMA2 regulationand consequently does not directly inhibit the Ca2+ pump-

ing activity of SMA2. As mentioned above, however, it isalso likely that the expression level of SmCNA is too lowto have any effect on SMA2 activity.

The mechanism with which yeast CN can inhibit SMA2expression is still unknown and it is conceivable that the reg-ulation is mediated through a not yet identified effector pro-tein. Based on differences in substrate specificity betweenthe schistosomal and the yeast proteins, we favor the hy-pothesis that SmCNA is unable to dephosphorylate the yeasteffector protein and consequently fails to regulate SMA2.However, such an effector protein homolog, capable of rec-ognizing and interacting with SmCN to regulate the calciumpump has not yet been reported to be present in schisto-somes.

A major argument for an indirect mechanism responsiblefor the CN-mediated repression of SERCA pumps stemsfrom the differences obtained with a rabbit SERCA isoformbetween the effects on Ca2+ homeostasis and ATPase ac-tivity measured in vitro. Similarly to SMA2, SERCA1a in-creases its contribution to Ca2+ tolerance upon impairmentof CN function. However, mutation ofCNB1 or inhibition

A. Rossi et al. / Molecular & Biochemical Parasitology 135 (2004) 165–169 169

by cyclosporin leads to decreased ATPase activity in yeastSERCA1a-enriched membrane fractions[19]. This discrep-ancy may suggest that the control of SERCA1a functionrequires a CN-dependent regulator that is lost during prepa-ration of the SERCA1a fractions from yeast.

A further step in obtaining a full picture of the phys-iological relevance of the CN-mediated regulation ofSERCA pumps would be to use conventional biochemicalapproaches to purify yeast or schistosomal proteins asso-ciating with SMA2. In this respect, we have been able toexpress inS. cerevisiae and purify by affinity chromatog-raphy a His6-FLAG-tagged version of SMA2 (unpublishedresults). The eluted protein was purified to 70% homo-geneity and found to exhibit a Ca2+-dependent ATPaseactivity of 1.6�mol of Pi min−1 mg−1, comparable to thespecific activity of a similarly purified yeast-expressedrabbit SERCA1a[20]. The identification of protein com-ponents involved in the CN-mediated repression of SMA2via SMA2-based affinity chromatography could form thebasis of future experiments to help us understand bettercalcium homeostasis control. Elucidation of the mechanismbehind such regulatory events will have a general impacton eukaryotic cell biology.

Acknowledgements

We are greatly indebted to Dr. K.W. Cunningham (JohnsHopkins University) for providing the yeast strains K601,K571, and K559. Our special thanks also go to Joseph Naderand Catherine Tillieux for skillful technical help. This workwas supported by a grant from the Deutsche Forshungs-gemeinschaft (KL 587/6-1) and by a short-term fellowshipfrom EMBO (ASFT 9665).

References

[1] Klee CB, Ren H, Wang X. Regulation of the calmodulin-stimulatedprotein phosphatase, calcineurin. J Biol Chem 1998;273:13367–70.

[2] Mecozzi B, Rossi A, Lazzaretti P, et al. Molecular cloning ofSchis-tosoma mansoni calcineurin subunits and immunolocalization to theexcretory system. Mol Biochem Parasitol 2000;110:333–43.

[3] Rossi A, Wippersteg V, Klinkert MQ, Grevelding CG. Cloning of5′and 3′ flanking regions of theSchistosoma mansoni calcineurin Agene and their characterization in transiently transformed parasites.Mol Biochem Parasitol 2003;130:133–8.

[4] Tumlin JA. Expression and function of calcineurin in the mammaliannephron: physiological roles, receptor signaling, and ion transport.Am J Kidney Dis 1997;30:884–95.

[5] Aperia A, Ibarra F, Svensson LB, Klee C, Greengard P. Calcineurinmediates alpha-adrenergic stimulation of Na+, K(+)-ATPase activityin renal tubule cells. Proc Natl Acad Sci USA 1992;89:7394–7.

[6] Mendoza I, Rubio F, Rodriguez-Navarro A, Pardo JM. The proteinphosphatase calcineurin is essential for NaCl tolerance ofSaccha-romyces cerevisiae. J Biol Chem 1994;269:8792–6.

[7] Norris CM, Blalock EM, Chen KC, Porter NM, Landfield PW.Calcineurin enhances L-type Ca(2+) channel activity in hippocam-pal neurons: increased effect with age in culture. Neuroscience2002;110:213–25.

[8] Allen GJ, Sanders D. Calcineurin, a type 2B protein phosphatase,modulates the Ca2+-permeable slow vacuolar ion channel of stomatalguard cells. Plant Cell 1995;7:1473–83.

[9] Stathopoulos AM, Cyert MS. Calcineurin acts through theCRZ1/TCN1-encoded transcription factor to regulate gene expres-sion in yeast. Genes Dev 1997;11:3432–44.

[10] Boustany LM, Cyert MS. Calcineurin-dependent regulation of Crz1pnuclear export requires Msn5p and a conserved calcineurin dockingsite. Genes Dev 2002;16:608–19.

[11] Yoshimoto H, Saltsman K, Gasch AP, et al. Genome-wide analysis ofgene expression regulated by the calcineurin/Crz1p signaling pathwayin Saccharomyces cerevisiae. J Biol Chem 2002;277:31079–88.

[12] Matheos DP, Kingsbury TJ, Ahsan US, Cunningham KW. Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentiallyregulates gene expression inSaccharomyces cerevisiae. Genes Dev1997;11:3445–58.

[13] Muller EM, Locke EG, Cunningham KW. Differential regulation oftwo Ca(2+) influx systems by pheromone signaling inSaccharomycescerevisiae. Genetics 2001;159:1527–38.

[14] Cunningham KW, Fink GR. Calcineurin inhibits VCX1-dependentH+/Ca2+ exchange and induces Ca2+ ATPases inSaccharomycescerevisiae. Mol Cell Biol 1996;16:2226–37.

[15] Cunningham KW, Fink GR. Calcineurin-dependent growth controlin Saccharomyces cerevisiae mutants lacking PMC1, a homolog ofplasma membrane Ca2+ ATPases. J Cell Biol 1994;124:351–63.

[16] Talla E, de Mendonca RL, Degand I, Goffeau A, Ghislain M.Schisto-soma mansoni Ca2+-ATPase SMA2 restores viability to yeast Ca2+-ATPase-deficient strains and functions in calcineurin-mediated Ca2+tolerance. J Biol Chem 1998;273:27831–40.

[17] Sikorski RS, Hieter P. A system of shuttle vectors and yeast hoststrains designed for efficient manipulation of DNA inSaccharomycescerevisiae. Genetics 1989;122:19–27.

[18] Mumberg D, Muller R, Funk M. Regulatable promoters ofSaccha-romyces cerevisiae: comparison of transcriptional activity and theiruse for heterologous expression. Nucleic Acids Res 1994;22:5767–8.

[19] Degand I, Catty P, Talla E, et al. Rabbit sarcoplasmic reticulumCa2+-ATPase replaces yeast PMC1 and PMR1 Ca2+-ATPases for cellviability and calcineurin-dependent regulation of calcium tolerance.Mol Microbiol 1999;31:545–56.

[20] Lenoir G, Menguy T, Corre F, et al. Overproduction in yeast andrapid and efficient purification of the rabbit SERCA1a Ca2+-ATPase.Biochim Biophys Acta 2002;1560:67–83.