Research Article

Functional analysis of the Neurospora crassa PZL-1protein phosphatase by expression in budding and®ssion yeast

Emese Vissi1, Josep Clotet2, Eulalia de Nadal2, Anna BarceloÂ2, EÂva BakoÂ1, PaÂl Gergely1, Viktor DombraÂdi1

and JoaquõÂn ArinÄo2*1 Department of Medical Chemistry, Faculty of Medicine, Medical and Health Science Centre, University of Debrecen, H-4026 Debrecen,Hungary

2 Departament de BioquõÂmica i Biologia Molecular, Universitat AutoÁnoma de Barcelona, Bellaterra 08193 Barcelona, Spain

*Correspondence to:J. ArinÄo, Dept. BioquõÂmica iBiologia Molecular, Facultat deVeterinaÁria, Ed. V, UniversitatAutoÁnoma de Barcelona,Bellaterra 08193, Barcelona,Spain.E-mail: J.Arino@cc.uab.es

Received: 9 June 2000

Accepted: 2 August 2000

Abstract

The gene pzl-1 from the ®lamentous fungus Neurospora crassa encodes a putative Ser/Thr

protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from

Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-

1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia colias active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed

in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype

of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to

that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth.

Expression of PZL-1 in ®ssion yeast from a weak version of the nmt1 promoter fully

rescued the growth defect of a pzh1D strain in high potassium, but only partially

complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassaphosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears

to ful®l every known function carried out by its S. cerevisiae counterpart, despite the

marked divergence in sequence within their NH2-terminal moieties. Copyright # 2000

John Wiley & Sons, Ltd.

Keywords: Ppz phosphatases; functional expression; yeast; salt tolerance

Introduction

The PPZ enzymes de®ne a novel type of Ser/Thrprotein phosphatases that have been identi®ed inbudding yeast (Saccharomyces cerevisiae; Posaset al., 1992) and ®ssion yeast (Schizosaccharomycespombe; Balcells et al., 1997). These phosphatasesdisplay a COOH-terminal catalytic domain ofabout 300 residues, which is about 60% identicalto the catalytic subunit of protein phosphatase-1,and a NH2-terminal moiety that is unrelated toother known phosphatases. Budding yeast containstwo genes for PPZ, encoding the Ppz1 (692residues) and Ppz2 (710 residues) proteins (Posaset al., 1992; Lee et al., 1993, Hughes et al., 1993).

They are about 93% identical within their COOH-terminal portion, whereas their NH2-terminalregions are less related (43%). Strains lackingPPZ1 are prone to cell lysis under some stresssituations, such as exposure to low concentrationsof caffeine (Posas et al., 1993), and the deletion ofPPZ1 is synthetically lethal in combination with theabsence of the Slt2/Mpk1 MAP kinase (Lee et al.,1993). In addition, lack of PPZ1 results in increasedtolerance to sodium and lithium ions and this effecthas been attributed to an increased expression ofthe Na+-ATPase encoded by the gene ENA1 (Posaset al., 1995a). Deletion of PPZ2 in a ppz1 back-ground intensi®es these effects, although it does notresult in a detectable phenotype in an otherwise

YeastYeast 2001; 18: 115±124.

Copyright # 2000 John Wiley & Sons, Ltd.

wild-type cell. Recent evidences suggest that Ppz1

might play a role in the regulation of the cell cycle,

as we have observed that the deletion of PPZ1

improves the defective growth of mutants in the

SIT4 gene (Clotet et al., 1999). SIT4 encodes a Ser/

Thr protein phosphatase involved in the G1±S

transition of the cell cycle (Arndt et al., 1989;

Sutton et al., 1991; FernaÂndez-Sarabia et al., 1992).

It has been proposed that Ppz1 might integrate

diverse stimuli relevant for cell cycle progression

(Clotet et al., 1999).The Sz. pombe gene pzh1+ encodes a homologue

of the PPZ phosphatases (Balcells et al., 1997). Pzh1

is a 515-residue protein, whose COOH-terminal

segment is 78% identical to Ppz1 and Ppz2, whereas

its NH2-terminal domain is shorter and, with the

exception of the ®rst 40 amino acids, shows a low

level of similarity to other proteins (Figure 1). Pzh1

is also involved in the regulation of salt tolerance,

although its role in Sz. pombe might differ substan-

tially from that of the PPZ phosphatases in S.

cerevisiae (Balcells et al., 1997, 1998, 1999). In fact,

in our hands the Pzh1 protein has been unable to

restore the wild-type phenotype of S. cerevisiae ppz1

strain, or to substitute for Ppz1 under different

conditions (Balcells et al., 1997; Clotet J, ArinÄo J,

unpublished results).Recently, a phosphatase gene and cDNA encod-

ing a PPZ-like protein (pzl-1) have been identi®ed

and cloned in Neurospora crassa (SzoÈoÄr et al.,

1998). The predicted open reading frame is similar

in size to ®ssion yeast Pzh1 (531 residues) and can

be also divided in two segments. The COOH-terminal portion, spanning about 300 residues, ishighly similar to the budding and ®ssion yeast PPZphosphatases (Figure 1). The NH2-terminal moietyof PZL-1 is rich in Ser and Pro and, althoughoverall it is quite unrelated to Ppz1/Ppz2 or Pzh1, itretains a signi®cant level of identity over the ®rst 50residues. This includes a NH2-terminal myristoyla-tion consensus sequence, which appears as acommon trait for all Ppz phosphatases, and isimportant for the proper function of Ppz1 (Clotetet al., 1996).

The identi®cation of a PPZ-like enzyme in the®lamentous fungus N. crassa raised the question ofits functional role. We have addressed this questionby expressing this protein in bacteria, to character-ize its catalytic activity, and in budding and ®ssionyeast, to evaluate whether or not the PZL-1 proteincan ful®l the biological functions of Ppz1 and/orPzh1.

Materials and methods

Growth of Escherichia coli and yeast strains

E. coli cells (strain DH5a) were grown at 37uC inLB medium (containing 50 mg/ml ampicillin forplasmid selection), unless otherwise stated. Sz.pombe cells were grown at 28uC in YES (0.5%yeast extract, 3% glucose, plus 225 mg/ml of ade-nine, uracil and leucine) or essential minimalmedium (EMM) supplemented with the necessaryrequirements (Moreno et al., 1991). The pH of themedium was buffered at 5.2 with 20 mM MES. S.cerevisiae cells were grown at 28uC in YPD mediumor, when indicated, in CM synthetic medium (Sher-man et al., 1986). The relevant genotypes of thedifferent S. cerevisiae strains used in this work areshown in Table 1. The Sz. pombe strain tested forexpression of PZL-1 was strain LB2 (pzh1::ura4+),described earlier (Balcells et al., 1997).

Recombinant DNA techniques

E. coli cells were transformed using the standardcalcium chloride method (Sambrook et al., 1989). S.cerevisiae cells were transformed by a modi®cationof the lithium acetate method (Schiestl andGietz, 1989), that includes treatment with dimethylsulphoxide. Sz. pombe cells were transformed bya modi®cation of the lithium acetate method

Figure 1. Schematic alignment of the S. cerevisiae Ppz1/Ppz2,Sz. pombe Pzh1 and N. crassa PZL-1 proteins. The dottedareas represent the conserved NH2-terminal element. Theasterisk denotes the conserved myristoylation consensussequence surrounding Gly-2. Numbers indicate the percen-tage of amino acid identities within the highly conservedphosphatase carboxyl-terminal domains, represented byblack boxes

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(Norbury and Moreno, 1997). Restriction diges-tions, DNA ligations and other standard recombi-nant DNA techniques were performed essentially asdescribed (Sambrook et al., 1989).

Expression of PZL-1 in bacterial cells

The entire open reading frame of N. crassa pzl-1was ampli®ed by PCR using a forward oligo-nucleotide 5k-CGGAATTCATGGGCAACTCATCCTC-3k and the reverse oligonucleotide 5k-GATGCGGCCGCTCAGACACTCTGGGGG-3k (Eco-RI and NotI sites, respectively, which are under-lined, were added to facilitate cloning). Theampli®ed 1.6 kbp fragment was cloned into theplasmid pGEX 4T1 (Pharmacia), previouslydigested with EcoRI and NotI, to yield plasmidpGEX±Pzl1. The carboxy-terminal moiety of PZL-1was ampli®ed with the forward primer 5k-CGGAATTCGGGTGTTGCCATGAG-3k (addedEcoRI site underlined) and the reverse primerdescribed above. The ampli®ed fragment (about1.0 kbp) was cloned into EcoRI and NotI digestedpGEX 4T3 vector (Pharmacia), to generatepGEX±Pzl1/CT.

For bacterial expression, DH5a E. coli cells weretransformed with plasmids and grown up to OD600

of 1.0 in 2rYT-G media (16 g/l tryptone, 10 g/lyeast extract, 5 g/l NaCl and 20 g/l glucose) contain-ing 1 mM MnCl2. Cells were induced by adding1 mM IPTG (0.2 mM for cells bearing pGEX-Pzl1/CT) and growth was resumed for 2 h at 28uC. Cellswere collected and disrupted with an Ultra Thuraxhomogenizer (4r1 min at 20 000 rpm) in thepresence of a buffer containing 50 mM Tris±HCl,pH 8.0, 1 mM MnCl2, 1% Triton X-100, 10mM

DTT, 0.2 mM phenylmethylsulphonyl ¯uoride,5 mM benzamidine and 1 mM o-phenantroline at0uC. Extracts were centrifuged at 3000rg for15 min at 4uC and the glutathione-S-transferase(GST) fusion proteins were puri®ed from the

supernatants by incubation with glutathione-Sepharose beads (Pharmacia) for 1 h at 4uC. Thebeads were washed three times with 50 mM Tris,pH 8.0, 0.1% b-mercaptoethanol, and the fusionproteins were eluted with the same buffer contain-ing 20 mM reduced glutathione.

Expression of N. crassa PZL-1 in S. cerevisiaeand Sz. pombe

For expression of PZL-1 in S. cerevisiae, thepromoter region (±525/±10) of S. cerevisiae PPZ1was ampli®ed by PCR using oligonucleotides A1 5k-GCG AGC TCG TCC TCC AAT TCA AC-3k andA2 5k-CGT CTA GAA GGA AAG ATA AGCAGA G-3k (added SacI and XbaI sites are under-lined) and Expand High Fidelity DNA polymerase(Roche). The 0.6 kbp fragment was cloned into theSacI and XbaI sites of plasmids YCplac111 andYEplac181 (which contain a LEU2 selectionmarker). The complete open reading frame of N.crassa pzl-1 was ampli®ed from the cloned cDNA(SzoÈoÄr et al., 1998) by PCR using oligonucleotidesA3 5k-CGT CTAGA A ATC ATG GGC AACTCA TC-3k and A4 5k-CAG AAG CTT TGG CTAGCG TGC AG-3k (added XbaI and HindIII sitesunderlined). This fragment was cloned into theabove-mentioned constructs to yield YCplac111±Pzl1 and YEplac181±Pzl1. The hybrid fragment(PPZ1 S. cerevisiae promoter plus N. crassa pzl-1open reading frame) was released by digestion withSacI±HindIII and used for cloning in the alternativeyeast vector YEplac195, bearing a URA3 selectionmarker, to generate YEplac195±Pzl1.

For expression of PZL-1 in ®ssion yeast, theentire pzl-1 open reading frame was ampli®ed witholigonucleotides A5 5k-CGC TGA TCA TGG GCAACT CAT CCT C-3k (added BclI site underlined)and A4 (see above). The fragment was digestedwith BclI, to create a BamHI-compatible end,and ligated into vectors pREP3x, pREP41x and

Table 1. S. cerevisiae strains used in this work

Strains Relevant genotype Reference

JA-100* MATa PPZ1 PPZ2 SIT4 MPK1 De Nadal et al., 1998

JA-101 MATa ppz1::URA3 PPZ2 SIT4 MPK1 De Nadal et al., 1998JC-10 MATa PPZ1 PPZ2 mpk1::LEU2 SIT4 This work

JA-110 MATa PPZ1 PPZ2 MPK1 sit4::TRP1 Clotet et al., 1999

JA-112 MATa ppz1::URA3 PPZ2 MPK1 sit4::TRP1 Clotet et al., 1999

*JA-100 is a haploid derivative from strain DL790 (Lee et al., 1993) and is ura3-52 leu2-3,112 trp1-1 his4 can-1r.

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pREP81x (Forsburg, 1993), previously digestedwith BamHI and SmaI, to yield pREP3x±Pzl1,pREP41x±Pzl1 and pREP81x±Pzl1. The insertswere sequenced using an automated DNA sequen-cer to verify the absence of unwanted mutations.These constructs were used to transform wild-typeand LB2 (pzh1) Sz. pombe cells. Positive clones weremaintained in medium containing 4 mm thiamine,to avoid undesired expression from the nmt1promoter, unless otherwise stated.

Other techniques

Sensitivity of S. cerevisiae strains to caffeine, NaCland LiCl was tested as previously reported (Clotetet al., 1996). Recovery from a-factor arrest wasmonitored as in Clotet et al. (1999). Sensitivity ofSz. pombe cells to ions was tested as describedearlier (Balcells et al., 1997). Growth rate determi-nations were performed essentially as in Clotet et al.(1996). Measurements of protein phosphatase activ-ity were carried out essentially as described pre-viously (Posas et al., 1995b), except that theincubation assay contained no MnCl2. One unit ofphosphatase is de®ned as the amount of enzymewhich releases one mmol phosphate/min from 32P-labelled myelin basic protein.

Results and discussion

Characterization of bacterially expressedPZL-1

The entire open reading frame encoding the PZL-1protein, as well as the region from residue 196 tothe stop codon (comprising the presumed phospha-tase catalytic domain), were successfully expressedas a soluble GST fusion protein in E. coli. The yieldwas relatively low (about 1.5±2 mg/l of culture). Thesoluble protein was puri®ed through glutathione-Sepharose columns, yielding essentially homoge-neous proteins (not shown). The estimated molecu-lar mass was 84 kDa for the full-length GST±Pzl1construct, and 61 kDa for the GST±catalyticdomain fusion protein, in good agreement with thecalculated values. Activity measurements demon-strated that the recombinant proteins were able todephosphorylate 32P-labelled myelin basic protein,as previously shown for Ppz1 and Pzh1 (Posas et al.,1995b; Balcells et al., 1997). The speci®c activity ofthe catalytic moiety was higher than that of the

entire protein (3.55 vs. 0.22 mU/mg protein), sug-gesting an inhibitory function for the NH2-terminalsegment. Extracts of bacteria harbouring no phos-phatase-containing plasmids showed a negligiblephosphatase activity in this assay (0.002 mU/mg).As indicated in Table 2, the phosphatase activity ofPZL-1 was inhibited by ATP and ¯uoride, similarlyto its homologues, but was less sensitive to micro-cystin LR and okadaic acid than bacteriallyexpressed Ppz1 or Pzh1 (Posas et al., 1995b; Balcellset al., 1997).

Functional analysis of PZL-1 in budding yeast

To test the possibility that N. crassa PZL-1phosphatase might functionally replace Ppz1, weexpressed the pzl-1 open reading frame from thePPZ1 promoter in ppz1 S. cerevisiae mutants.Budding yeast strains lacking PPZ1 display aphenotype of increased tolerance to sodium andlithium (Posas et al., 1995a). We ®rst tested whetherPZL-1 was able to rescue the lithium-tolerantphenotype of ppz1 cells. As can be observed inFigure 2, expression of pzl-1 from a low-copy,centromeric plasmid resulted in a reduction in thelithium tolerance of a ppz1 strain, similarly to theexpression of budding yeast PPZ1. The effect ofhigh-copy number expression of pzl-1 on salttolerance was dif®cult to assess because it resultedin a reduced cell growth rate, even in the absence ofsalt stress (see Figure 5).

In addition to the salt tolerance phenotype, inbudding yeast the absence of Ppz1 results inhypersensitivity to caffeine, as it has been reportedthat exposure to low concentrations of this drugcauses cell lysis (Posas et al., 1993). We show inFigure 3 that expression of N. crassa PZL-1 from

Table 2. Enzymological characteristics of bacteriallyexpressed PZL-1

Inhibitor*

Expressed protein

Pzl1 Pzl1/CT

ATP (mM) 23t2 40t4

NaF (mM) 25t2 20t2

Microcystin (nM) 66t3 66t5

*Data represent the IC50 values for each compound and are

meantSD from three to seven determinations. 2 mm okadaic acidhas no signi®cant effect on the phosphatase activity. Pzl1/CT refers to

the C-terminal catalytic fragment of Pzl1.

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Figure 2. Expression of N. crassa PZL-1 rescues the lithium hypertolerant phenotype of a ppz1 S. cerevisiae strain. StrainsJA-100 (PPZ1) or JA-101 (ppz1) were transformed with plasmids YEplac181 (YEp), YCplac111±PPZ1 (YCp±PPZ1),YEplac181±PPZ1 (YEp±PPZ1), YCplac111±Pzl1 (YCp±Pzl1), or YEplac181±Pzl1 (YEp±Pzl1) and plated (initial A660 of 0.05) onCM plates (lacking leucine) containing LiCl at the indicated concentrations. Plates were incubated at 30uC and growth wasscored after 2 days (except for cells grown in the absence of LiCl, which were scored for growth after 32 h to show theeffect of high-level expression of pzl-1 in the absence of salt)

Figure 3. Expression of N. crassa PZL-1 rescues the caffeine-sensitive phenotype of a budding yeast ppz1 strain. StrainsJA-100 (PPZ1) or JA-101 (ppz1) were transformed with plasmids YEplac181 (YEp), YCplac111±PPZ1 (YCp±PPZ1),YCplac111±Pzl1 (YCp±Pzl1), or YEplac181±Pzl1 (YEp±Pzl1) and plated on CM plates (lacking leucine) containing caffeine atthe indicated concentrations. Plates were incubated at 30uC and growth was scored after 2 days

Expression of the N. crassa PZL-1 phosphatase 119

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the S. cerevisiae PPZ1 promoter results in a

complete rescue of the caffeine-sensitive phenotype

of a ppz1 strain. It is remarkable that the presence

of PZL-1 virtually mimics the expression of bona

®de Ppz1, and that rescue is achieved even when

PZL-1 is expressed from a low-copy, centromeric

plasmid. By contrast, rescue of the caffeine-sensitive

phenotype in budding yeast is achieved by ®ssion

yeast Pzh1 only when expressed from a high copy

number plasmid (Balcells et al., 1997). It must be

emphasized that the Pzh1 phosphatase was also

expressed from the PPZ1 promoter in budding

yeast.The caffeine-sensitive phenotype of ppz1 mutants

has been related to the role of this phosphatase in

the maintenance of cell integrity (Posas et al., 1993).

A functional relationship between Ppz1 and the

PKC1-regulated Slt2±Mpk1 MAP kinase pathway

has been established (Lee et al., 1993). mpk1

mutants, like ppz1 mutants, are caffeine-sensitive.

In addition, mpk1 cells are temperature-sensitive

and cannot grow at 37uC unless osmotically

stabilized. High copy number expression of PPZ1

allows mpk1 to grow in the absence of osmotic

stabilizers (Lee et al., 1993). Figure 4 shows that

high copy number expression of PZL-1 is also able

to allow growth of a mpk1 mutant under non-

permissive conditions in the absence of 1 M sorbitol.

This observation suggests that the N. crassa PZL-1

phosphatase is able to perform certain function(s)

related to the maintenance of cell integrity in

budding yeast, similarly to endogenous Ppz1. It is

remarkable that, by contrast, even high copy

number expression of ®ssion yeast pzh1+ is unable

to rescue the temperature-sensitive phenotype of

mpk1 cells (J. Clotet & J. ArinÄo, unpublished).We have recently reported that, in budding yeast,

Ppz1 is negatively regulated by the Hal3 protein

(De Nadal et al., 1998). The gene HAL3/SIS2 was

isolated as a determinant of salt tolerance (Fer-

rando et al., 1995). On the basis that high copy

number expression of HAL3/SIS2 was able to

rescue the growth defect of sit4 mutants, it was

also identi®ed as a component of the machinery

that regulates the progression of the cell cycle

through the G1±S transition (Di Como et al.,

1995). This evidence led us to postulate that the

effect of Hal3 on the cell cycle might be mediated

by Ppz1 and that, in consequence, Ppz1 might be a

regulatory component of the cell cycle. This

hypothesis is in agreement with the fact that high

levels of Ppz1 are detrimental for growth (Clotet

Figure 4. High-copy number expression of N. crassa PZL-1 rescues the temperature-sensitive lytic phenotype of a MAPkinase-de®cient mpk1 S. cerevisiae strain. Wild-type strain JA-100 (wt) was transformed with plasmid YEplac195 (YEp),whereas the isogenic strain JC10 (mpk1::LEU2) was transformed with the same plasmid or with plasmid YEplac195±Pzl1(YEp±Pzl1). Cells were plated on CM plates lacking uracil, with or without 1 M sorbitol, and were grown at 37uC for 2 days

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et al., 1996), and it has recently received furthersupport from a number of observations, includingthe evidence that deletion of Ppz1 results in therescue of the sit4 growth defect similarly to the

overexpression of HAL3 (Clotet et al., 1999). Wereport here that high copy expression of PZL-1 alsoresults in a decrease in cell growth rate in a wild-type background (Figure 5, upper panel). In addi-tion, we have expressed PZL-1 from a low copy,centromeric plasmid in a sit4 ppz1 strain andcompared the growth of this strain with sit4 andsit4 ppz1 cells. As shown in Figure 5 (lower panel),the improvement in growth caused by the lack ofPpz1 in a sit4 background is eliminated by expres-sion of PZL-1, in a similar way to what is observedupon expression of budding yeast Ppz1. We haveshown (Clotet et al., 1999) that high levels of Ppz1result in a delayed entry into cell cycle after a-factorarrest. We demonstrate here (Figure 6) that asimilar effect is obtained by high copy expressionof N. crassa PZL-1, indicating that the growthdefect observed upon overexpression of PZL-1 is

Figure 5. Effects on growth of the expression of N. crassaPZL-1 in wild-type and sit4 ppz1 S. cerevisiae strains. Upperpanel: S. cerevisiae wild-type JA-100 cells were transformedwith plasmid YEplac181 ($) or plasmid YEplac181±Pzl1 (#).Cells were grown overnight on CM medium lacking leucine,re-inoculated on fresh medium at an initial OD660 of 0.1, andgrowth was monitored as indicated. Lower panel: strain JA-110 (sit4) was transformed with plasmid YCplac111 (().Strain JA-112 (sit4 ppz1) was transformed with plasmidsYCplac111 ($), YCplac111±Pzl1 (#), and YCplac111±PPZ1((). Growth conditions were as indicated above. Data aremeantSEM of three independent experiments

Figure 6. Overexpression of N. crassa PZL-1 in S. cerevisiaeresults in delayed recovery from a-factor G1 arrest. Wild-type JA-100 cells were transformed with plasmid YEplac195($), YEp195±PPZ1 (() or YEp195±Pzl1 (&). Cells werearrested by exposure of a-factor (20 mg/ml) for 2 h. Aftercentrifugation, cells were resuspended in fresh medium,washed, and growth was resumed. Samples were taken atthe indicated times and the percentage of budded cellsmonitored under the microscope. In each experiment, atleast 300 cells were monitored per time point. A represen-tative experiment is shown

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the result of a delayed G1±S transition. Therefore,all of the experimental evidences strongly suggestthat the heterologous phosphatase is functionallyreplacing Ppz1.

Functional analysis of PZL-1 in ®ssion yeast

We proceeded to express pzl-1 in the Sz. pombestrain LB2 (pzh1D ) by using three different versionsof the nmt1+ promoter that differ in their strength(Forsburg, 1993). We observed that, while cellsgrowing in the presence of 4 mm thiamine (whichrepresses expression form the promoter) did notshown any apparent defect in growth, in theabsence of this compound, cells harbouringpREP3x±Pzl1 or pREP41x±Pzl1 constructs didshow a reduced growth rate (Figure 7A). In fact,expression of PZL-1 from the strongest promoter(wild-type nmt1, present in plasmid pREP3x±Pzl1)almost abolished cell growth. Microscopic examina-tion indicated that many of these cells exhibited an

abnormal morphology, a situation that was repro-duced up to some extent by the expression from theless powerful version of the nmt promoter presentin plasmid pREp41x (Figure 7 B). Expression ofPZL-1 from the weakest promoter system, thepREP81x±Pzl1 construct, did not affect either cellgrowth rate or morphology, indicating that theobserved effect was dose-dependent. This ®nding isin agreement with previous reports indicating thatstrong overexpression of type 1 protein phospha-tases in yeast results in aberrant morphologicalchanges (Cannon et al., 1995).

The negative effect of strong overexpression ofPZL-1 on cell growth forced us to performcomplementation experiments using the constructpREP81x±Pzl1. It has been previously reported thatSz. pombe cells lacking the pzh1+ phosphatase geneare hypertolerant to sodium and lithium ions and,in addition, they display a decreased tolerance forpotassium ions. We show in Figure 8 that expres-sion of PZL-1 from the pREP81x±Pzl1 plasmid is

Figure 7. Overexpression of the PZL-1 phosphatase in Sz. pombe severely affects cells growth and cell morphology. (A) Sz.pombe LB2 cells (pzh1D) were transformed with plasmids pREP3x ($), pREP3x±Pzl1 (#), pREP41x±Pzl1 (,), pREP81x±Pzl1(() and grown on EMM medium until saturation in the presence of 4 mm thiamine. Cell were centrifuged, washed,resuspended in fresh EMM medium lacking thiamine to an A595 of 0.1, and growth was resumed. Samples were taken at theindicated times and growth of the culture was monitored. Data are meantSEM of three independent experiments.(B) Samples of LB2 cultures (24 h) carrying pREP3x or pREP41x±Pzl1 were ®xed with formaldehyde (7%) and micrographswere taken with Nomarsky optics (r1000)

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able to restore sensitivity to potassium ions to wild-type levels, indicating that N. crassa was able toperform the function of Pzh1. However, onlypartial complementation was observed when cellswere challenged with different concentrations ofNaCl (Figure 8).

Conclusions

The results presented in this paper demonstrate thatthe product of the N. crassa pzl-1 gene encodes aSer/Thr protein phosphatase whose catalytic site iscon®ned to its COOH-terminal domain. We char-acterized its physiological role by expressing theprotein in budding and ®ssion yeast cells. It isremarkable that low-copy expression of PZL-1 in S.cerevisiae ppz1 mutants, from the PPZ1 promoter,essentially restores wild-type phenotypes, suggestingthat PZL-1 is behaving as a bona ®de Ppz1 enzyme.In addition, overexpression of the N. crassaphosphatase mimics the effects of high levels of

the budding yeast enzyme. Therefore, our resultssuggest that, despite the marked differences in theirNH2-terminal regions, the important structuraldeterminants of these proteins, as well as theircellular targets must have been conserved in bothorganisms. Because the Ena1 sodium ATPase, themajor determinant for sodium ef¯ux in buddingyeast, is a known cellular target for Ppz1, we couldspeculate on the existence of a similar ef¯ux systemin N. crassa. In the case of ®ssion yeast, the failureof PZL-1 to fully complement the sodium-hypertolerant phenotype of a pzh1 mutant mightre¯ect the differences in sodium ef¯ux systemsbetween budding and ®ssion yeast.

Acknowledgements

The skilful technical help of Anna Vilalta, Mireia Zaguirre

and Margit BõÂro is acknowledged. This work was supported

by grants PB98-0565-C04-02 (DireccioÂn General de Investi-

gacioÂn Cientõ®ca y TeÂcnica, Spain), and 1999SGR97-00100

(Generalitat de Catalunya), to J.A., by grant MKM FPFP

Figure 8. Effect of mild expression of PZL-1 on the altered potassium and sodium tolerances of a S. pombe pzh1D strain. Sz.pombe wild-type cells were transformed with plasmid pREP3x (empty bars) and, in parallel, strain LB2 (pzh1D) wastransformed with plasmid pREP3x (dashed bars) or pREP81x±Pzl1 (crossed bars). Cells were grown as in Figure 7, washedand diluted 10-fold in fresh medium lacking thiamine. To monitor tolerance to potassium (A), growth was resumed for 14 hand then cultures were diluted with EMM medium until A595 0.01. Cultures were mixed with EMM medium containing KCl toachieve the indicated concentrations of salt. For sodium tolerance measurement (B), cells were diluted with YES medium andthen mixed with YES medium containing the appropriate amounts of NaCl. ($), Sz. pombe wild-type cells containing plasmidpREP3x. (#), strain LB2 (pzh1D) plus plasmid pREP3x or (,), plasmid pREP81x±Pzl1. Data represent the percentage ofgrowth with respect to cells grown without added salts, and are meantSEM of ®ve to eight independent experiments

Expression of the N. crassa PZL-1 phosphatase 123

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2001; 18: 115±124.

0784/1997 (Ministry of Education) to G.P, and by grant

OTKA 22675 (Hungarian Research Foundation) to V.D. E.

de N. was recipient of a Fellowship from the `Ministerio de

EducacioÂn y Cultura', Spain. E.V. was recipient of a

fellowship within the TEMPUS-PHARE and ERASMUS

frameworks.

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