ACCELERATED PUBLICATION
A New Pathway for Heavy Metal Detoxification in Animals

PHYTOCHELATIN SYNTHASE IS REQUIRED FOR CADMIUM TOLERANCE IN CAENORHABDITIS ELEGANS*

Olena K. VatamaniukDagger§, Elizabeth A. BucherDagger, James T. Ward, and Philip A. Rea§||

From the § Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 and the  Department of Cell and Developmental Biology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058

Received for publication, March 27, 2001, and in revised form, April 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma -Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

Plants and some fungi post-translationally synthesize novel peptides termed phytochelatins (PCs)1 when exposed to heavy metals. Fabricated from the ubiquitous thiol tripeptide GSH and related thiols in a novel transpeptidation reaction catalyzed by PC synthases (gamma -glutamylcysteinyltransferase; EC 2.3.2.15), PCs have the general structure (gamma -Glu-Cys)n-Xaa, contain 2-11 gamma -Glu-Cys repeats, chelate heavy metals at high affinity, and facilitate the vacuolar sequestration of heavy metals, most notably Cd2+ (1-3). Although it is more than a decade since the first report of the partial purification of a heavy metal-, primarily Cd2+-activated PC synthase from plant extracts (1), it is only recently that the small family of genes encoding these enzymes has been identified in plants and the fission yeast Schizosaccharomyces pombe (4-6). As exemplified by the clone from Arabidopsis thaliana (AtPCS1), these genes encode 45-55-kDa proteins that are sufficient for heavy metal-activated PC synthesis from GSH both in vivo and in vitro (6, 7).

An unexpected outcome of the cloning of AtPCS1 and its equivalents from other plants and S. pombe was the identification of a single-copy gene homolog (accession number Z66513) in the nematode worm Caenorhabditis elegans (4-6). Designated ce-pcs-1, this gene encodes a hypothetical 40.8-kDa protein (CePCS1) bearing 32% identity (45% similarity) to AtPCS1 in an overlap of 367 amino acid residues (6). Disclosure of a PCS1 homolog in the genome of C. elegans was surprising in that it raised for the first time the possibility that not only GSH and metallothioneins (8) but also PCs might participate in metal homeostasis in at least some animals.

In the report that follows we demonstrate unequivocally that ce-pcs-1 encodes a bona fide PC synthase whose activity is necessary for the detoxification of heavy metals in the intact organism. Discovery of the PC synthase-dependent pathway in the model organism C. elegans establishes a firm basis for determining the ubiquity of this pathway in other animals and for elucidation of the identity and organization of the cellular machinery likely involved in the eventual elimination, sequestration, and/or metabolism of heavy metal·PC complexes in animals.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

Isolation and Heterologous Expression of ce-pcs-1-- ce-pcs-1 was isolated from C. elegans N2 total RNA by reverse transcription polymerase chain reaction (9), and the resulting cDNA (accession numbers AF299332 and AF299333) was subcloned into yeast-Escherichia coli shuttle vector pYES3 (10) to place ce-pcs-1 under control of the constitutive yeast PGK gene promoter. For immunodetection of the translation product, vector pYES3-CePCS1::FLAG, containing the ce-pcs-1 insert engineered to code CePCS1 C-terminally fused with a FLAG (DYDDDDK) epitope tag, was constructed. Both constructs or empty vector pYES3 were used to transform (11) Saccharomyces cerevisiae ycf1Delta strain DTY167 (MATalpha ura3-52 leu2-3,-112 his-Delta 200 trp1-Delta 901 lys2-801 suc2-Delta 9 ycf1::hisG), a Cd2+-hypersensitive mutant deficient in the YCF1-mediated vacuolar sequestration of Cd·glutathione complexes (12, 13). The pYES3-CePCS1, pYES3-CePCS1::FLAG, and empty vector transformants were selected for uracil prototrophy by plating on AHC medium supplemented with glucose (2% w/v) and tryptophan (50 µg/ml). pYES3-AtPCS1::FLAG-transformed DTY167 cells were generated as described (6).

Preparation of S. cerevisiae Cell Extracts-- For investigations of their PC content and PC synthase activities, aliquots of stationary phase cultures of CePCS1::FLAG-, AtPCS1::FLAG-, or empty vector-transformed DTY167 cells were inoculated into 40-ml volumes of AHC medium with or without added CdCl2 (25 µM), grown for 16-18 h at 30 °C to an A600 nm of 0.8-1.0, and harvested by centrifugation. After resuspension and disruption of the cells by the glass bead method (14) in homogenization buffer (10 mM Tris-HCl, 10% (w/v) glycerol, 10 mM 2-mercaptoethanol, pH 7.6) containing 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml each of leupeptin, pepstatin, and aprotinin, the homogenates were cleared by centrifugation at 10,000 × g for 20 min and assayed immediately for PCs and PC synthase activity.

Measurement of PCs and PC Synthase Activity-- The cellular PC content and PC synthase activities of the cell-free extracts from yeast CePCS1::FLAG-, AtPCS1::FLAG-, and empty vector-transformants were estimated as described (7) by a combination of reverse-phase HPLC and thiol quantitation with Ellman's reagent. Individual PC fractions were identified by estimating their Glu/Gly ratios (ratio = n = number of Glu-Cys repeats per Gly) after acid hydrolysis and amino acid analysis of the appropriate HPLC fractions and/or on the basis of their comigration with PC standards synthesized in vitro by purified AtPCS1-FLAG (7).

SDS-PAGE and Immunoblot Analyses-- Protein samples were prepared for and subjected to SDS-PAGE for immunoblot analysis against anti-FLAG M2 antibody (Sigma) as described (6).

dsRNA Synthesis-- For in vitro ce-pcs-1 RNA synthesis, the cDNA insert of pYES3-CePCS1 was subcloned into pBluescript, and RNA was transcribed from the T3 and T7 promoters using an in vitro RNA transcription kit (Stratagene). After DNase digestion of the template, the sense and antisense preparations were gel-purified, mixed in an equimolar ratio in 1 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, heated for 1 min in boiling water, and annealed at room temperature (15). The single electrophoretic species corresponding in size to that expected of dsce-pcs-1 RNA was purified by phenol/chloroform extraction, dissolved in water, diluted to a concentration of 500 ng/µl with injection buffer (16), and stored at -80 °C.

dsRNA Microinjection and Analysis of Phenotypes-- The ce-pcs-1 RNA interference experiments were performed on wild type (N2) worms. The body cavities of adult hermaphrodites were injected as described (16). Injected worms were cultured for 12-18 h on standard nematode growth medium (NGM) plates seeded with E. coli OP50 before transfer onto plates supplemented with CdCl2. Two treatment regimes were used to examine the effects of Cd2+. In the first regime, individual worms were transferred sequentially onto NGM plates supplemented with 0, 25, 50, and 100 µM CdCl2. After transfer onto each Cd2+ concentration, egg laying was allowed to proceed for 6 h, after which time the worms were transferred to the next higher Cd2+ concentration for another round of egg laying. In this way, it was determined that 25 µM CdCl2 was sufficient to cause severe morphological and developmental changes in the CePCS1-deficient worms but not the controls. In the second regime, individual injected worms were transferred to NGM plates supplemented with 0, 5, 10, 25, 50, or 100 µM CdCl2 and allowed to lay eggs for 6 h, after which time they were transferred for another 6 h onto plates containing 25 µM CdCl2. Only the progeny of worms that exhibited the CePCS1-deficient phenotype upon transfer to plates containing 25 µM Cd2+ (65-75% of the progeny of the total worms injected) were scored at the other concentrations. Control worms were treated in an identical manner except that they were injected with injection buffer alone. The phenotypes of the worms were examined 4-6 days after hatching. Features scored included gross morphology, developmental stage, and fertility. The specificity of the effects exerted by the injection of dsce-pcs-1 RNA and the efficacy of the injection protocol employed were assessed by the injection of wild type N2 worms and strain EE86 worms (genotype upIs1(mup-4::gfp(CeMup-4.99.1617);rol-6(pRF4))) with dsgfp RNA by the same procedure. In both cases the worms were screened for Cd2+ tolerance; in the latter case the worms were also screened for diminished hypodermal cell MUP-4::GFP expression (17). GFP levels were assessed under a Leica stereomicroscope equipped with UV illumination.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

CePCS1 Catalyzes PC Synthesis-- The functional equivalence of CePCS1 with its plant and S. pombe homologs was determined by heterologous expression in S. cerevisiae, which otherwise lacks PC synthase (4-6). For this purpose and to render its translation product immunodetectable, ce-pcs-1::FLAG fusions were cloned into expression vector pYES3, and these (pYES3-CePCS1::FLAG), the untagged construct (pYES3-CePCS1), and empty vector were transformed into Cd2+-hypersensitive S. cerevisiae strain DTY167 (see "Materials and Methods").

The involvement of CePCS1 in heavy metal tolerance was examined at three levels, by determining its capacity for alleviating Cd2+ hypersensitivity, by defining its facility for promoting the Cd2+-dependent intracellular accumulation of PCs, and by determining its ability for de novo Cd2+-activated synthesis of PCs from GSH in vitro.

Regardless of the level at which it was examined, CePCS1 had the properties expected of a PC synthase. Plasmid-borne ce-pcs-1::FLAG suppressed the Cd2+-hypersensitivity of yeast strain DTY167, as did untagged ce-pcs1, at efficacies comparable with those of AtPCS1::FLAG (and AtPCS1; data not shown). CePCS1::FLAG and CePCS1 increased the concentration of CdCl2 in the growth medium required for 50% inhibition of growth by 12.0- and 12.4-fold and 1.5- and 1.4-fold versus empty vector-transformed controls or AtPCS1::FLAG-transformed cells (Fig. 1). As shown previously for AtPCS1 (6), the tolerance conferred by CePCS1::FLAG (and CePCS1) was not restricted to Cd2+ but extended to other soft metals and metalloids, including mercury (as HgCl2) and arsenic (as AsO43- or AsO2-) (data not shown).


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Fig. 1.   Suppression of Cd2+ hypersensitivity of S. cerevisiae ycf1Delta mutant strain DTY167 by plasmid-borne ce-pcs-1. Yeast ycf1Delta strain DTY167 was transformed with pYES3-CePCS1 (open circle ), pYES3-CePCS1::FLAG (), pYES3-AtPCS1::FLAG (), or empty pYES3 vector (black-square). Cells were grown at 30 °C to an A600 nm of ~1.8 in AHC medium supplemented with glucose and tryptophan before inoculating aliquots into 2-ml volumes of the same medium containing the indicated concentrations of CdCl2. A600 nm was measured after growth for 36 h.

Whenever enhanced Cd2+ tolerance was observed, it was associated with Cd2+-dependent intracellular PC accumulation. Reverse-phase HPLC analysis of nonprotein thiols in the soluble fraction from ce-pcs-1-transformed DTY167 cells after growth in media containing CdCl2 revealed prominent peaks eluting after the GSH/2-mercaptoethanol injection peak whose chromatographic properties were indistinguishable from those of PCs (PC2, PC3, PC4, and PC5) synthesized in vitro by purified AtPCS1::FLAG (7) (Fig. 2A). The aggregate thiol content of these nonprotein thiol peptides was 178 nmol/mg soluble protein. The corresponding fractions from CdCl2-grown empty vector control cells and from ce-pcs-1-transformed cells after growth in medium lacking Cd2+ were devoid of PC-like nonprotein thiols (Fig. 2A), indicating that both CePCS1 and exposure to heavy metal were essential prerequisites for net PC synthesis in vivo.


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Fig. 2.   Cadmium-activated CePCS1-FLAG-dependent PC synthesis in vivo and in vitro. A, reverse-phase HPLC analysis of nonprotein thiols in the soluble fractions extracted from CePCS1::FLAG-transformed DTY167 cells after growth in liquid medium containing CdCl2 (25 µM). B, reverse-phase HPLC analysis of the nonprotein thiols formed after incubation of whole cell extracts prepared from CePCS1::FLAG-transformed DTY167 cells (180 µg protein/ml) with GSH (3.3 mM) and CdCl2 (25 µM). The peaks designated PC2 and PC3 were identified on the basis of their Glu/Gly ratios (2.1 ± 0.1 and 3.0 ± 0.2, respectively) and comigration with PC2 and PC3 standards synthesized in vitro using purified AtPCS1-FLAG (7). Inset, immunoblot analysis of CePCS1-FLAG and AtPCS1-FLAG in whole cell extracts from CePCS1::FLAG-, AtPCS1::FLAG-, and empty vector-transformed DTY167 cells. The Mr 46,000 and 58,000 species were the only anti-FLAG antibody-reactive polypeptides detected in the extracts.

The CePCS1- and Cd2+-dependent synthesis of PCs measured in vivo was precisely replicated in vitro. Analyses of the capacity of CePCS1::FLAG yeast cell-free extracts for the Cd2+-dependent incorporation of GSH into PCs demonstrated net synthesis of PC2 and PC3 at an aggregate rate of 28.5 ± 4.3 nmol/mg/min (Fig. 2B). No peaks other than those corresponding to GSH were detected when the same extracts were incubated in the media lacking CdCl2. The corresponding fraction from DTY167 cells transformed with AtPCS1::FLAG catalyzed PC2 and PC3 synthesis at an aggregate rate of 107.0 ± 36.0 nmol/mg/min (Fig. 2B). By contrast, net PC synthesis by the cell-free extracts from vector controls was undetectable (<0.01 nmol/mg/min) (data not shown). Because approximate proportionality between net PC synthetic activity in vitro and the amount of recoverable fusion protein in the extracts was demonstrable immunologically (Fig. 2, inset), CePCS1::FLAG and AtPCS1::FLAG were inferred to have similar intrinsic catalytic capacities.

CePCS1 Is Required for Cadmium Tolerance in the Intact Organism-- The discovery of ce-pcs-1 in C. elegans and the demonstrated facility of its translation product for Cd2+-dependent PC synthesis was completely unexpected. PCs had never before been considered to be involved in metal homeostasis in animals (8). Hence, to determine whether PC synthase-mediated PC synthesis might indeed contribute to metal detoxification in C. elegans, the in vivo function of ce-pcs-1 was examined at the level of the whole organism. Toward this end, the double-stranded RNA interference technique (18) was employed. This technique for the targeted suppression of specific genes was thought to be particularly applicable to the study of ce-pcs-1, a single-copy gene. Wild type (N2) or a GFP-expressing line (EE86) of C. elegans were injected with injection buffer alone, injection buffer containing dsgfp RNA, or with injection buffer containing dsce-pcs-1 RNA and cultured on standard NGM or on NGM containing different concentrations of CdCl2. 4 days later, the progeny of these worms were scored for gross morphology, development, and fertility.

The progeny of control worms injected with either injection buffer or dsgfp RNA were indistinguishable from wild type (see Fig. 3 and Fig. 4A), although all of the progeny of the dsgfp RNA-injected GS86 worms exhibited markedly diminished or no GFP expression as determined by fluorescence microscopy. At low (5, 10, and 25 µM) concentrations of Cd2+, the wild type control progeny developed into normal-sized, gravid adults, comparable with those grown in the media devoid of heavy metal (see Fig. 3 and Fig 4B). At higher concentrations (50 and 100 µM), the controls grew more slowly but nevertheless reached adulthood and laid eggs after about 6 days (data not shown). In contrast, marked differences were evident in the progeny of worms injected with dsce-pcs-1 RNA, even at the lowest concentrations of Cd2+. When raised on 5 and 10 µM Cd2+, the CePCS1-deficient animals reached adulthood (Fig. 3); however, at 5 µM most individuals retained their eggs and at 10 µM showed severe necrosis (Fig. 4B), producing fewer eggs. A fraction (21%) of the worms reached adulthood at 25 µM CdCl2 (Fig. 3), but they were small, necrotic, and sterile and eventually died. At 50 and 100 µM Cd2+, CePCS1-deficient worms arrested at the L2-L4 larval stage, were extensively necrotic and had died by day 6 (see Fig. 3 and Fig. 4C). The differential effects seen were conditional on exposure to Cd2+, because CePCS1-deficient worms were identical to wild type when grown on standard NGM plates (see Fig. 3 and Fig. 4A). As would be expected if the effects of Cd2+ on the CePCS1-deficient worms were irreversible, transfer of arrested larvae to standard NGM plates did not restore growth (data not shown).


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Fig. 3.   Increased sensitivity of CePCS1-deficient worms to Cd2+. Shown are the percentages of the progeny of dsce-pcs-1 RNA-injected (), dsgfp RNA-injected (numbers), and injection buffer-injected worms (open circle ) that had reached adulthood 4 days after hatching on NGM plates supplemented with the indicated concentrations of CdCl2. Because the two treatment regimes described under "Materials and Methods" yielded identical results, the data presented incorporate both sets of results. The number of worms injected and the number of progeny scored at each concentration of Cd2+ were as follows. For injection buffer control: 0 µM Cd2+, 8 injections and 268 progeny; 5 µM Cd2+, 6 injections and 144 progeny; 10 µM Cd2+, 5 injections and 191 progeny; 25 µM Cd2+, 10 injections and 206 progeny; 50 µM Cd2+, 10 injections and 295 progeny; 100 µM Cd2+, 9 injections and 178 progeny. For dsgfp RNA injection control: 0 µM Cd2+, 4 injections and 74 progeny; 5 µM Cd2+, 4 injections and 76 progeny; 10 µM Cd2+, 3 injections and 140 progeny; 25 µM Cd2+, 3 injections and 62 progeny; 50 µM Cd2+, 3 injections and 65 progeny; 100 µM Cd2+, 3 injections and 156 progeny. For dsce-pcs-1 RNA injection: 0 µM Cd2+, 17 injections and 248 progeny; 5 µM Cd2+, 12 injections and 278 progeny; 10 µM Cd2+, 10 injections and 234 progeny; 25 µM Cd2+, 19 injections and 300 progeny; 50 µM Cd2+, 16 injections and 264 progeny; 100 µM Cd2+, 18 injections and 397 progeny.


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Fig. 4.   Morphology of control worms (Control) and CePCS1-deficient worms (CePCS1) after growth on NGM plates containing (A) 0 µM CdCl2, (B) 10 µM CdCl2, or (C) 50 µM CdCl2. 5-day-old worms were immobilized in buffer containing 5 mM sodium azide and photographed within 5 min. Nomarski images were collected using a Leica DAS microscope equipped with a DMR camera.


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

The findings reported here represent the first demonstration of the PC synthase-mediated detoxification of Cd2+ and probably other heavy metals in an animal. Moreover, although other gene products have been inferred to contribute to Cd2+ tolerance in C. elegans, CePCS1 is the first for which a firm biochemical basis has been established for the effects seen at the whole organism level. Elegant studies of C. elegans mutants for a multidrug resistance-associated protein subclass ATP binding cassette transporter gene (19) and for a mitogen-activated kinase kinase (MEK1) gene (20) have revealed an increase in sensitivity to Cd2+ versus wild type, but the effects seen were less acute and/or less specific than those reported here for CePCS1-deficient worms and in neither case could be attributed to a specific biochemical process or event.

The ubiquity of the PC synthase-dependent pathway in other animals and in groups of organisms other than plants remains to be delineated, but the fact that many nematode species are pathogenic and that the expressed sequence tag data bases for the parasitic protists Eimeria, Leishmania, and Plasmodium contain cDNAs whose predicted proteins are also PC synthase homologs markedly extends the significance of the findings reported here. On one hand, its discovery in C. elegans, in conjunction with the prominence of heavy metals as environmental toxins (21) implicated in many disease states including cancers in humans (22), may mean that PC synthase-dependent metal detoxification in animals will prove to be of environmental toxicological significance. On the other hand, the likely operation of equivalent metal detoxification pathways in pathogenic nematodes and protists, organisms responsible for untold human suffering and agronomic losses (23, 24), may, in view of the conditional lethality of the ce-pcs-1 RNA interference phenotype, spawn new chemotherapeutic and agrochemical approaches for combating the many infections caused by these pathogens.

    ACKNOWLEDGEMENTS

We thank Drs. C. Emerson and D. Standiford for the use of microscope facilities.

    FOOTNOTES

* This work was supported in part by National Science Foundation Grant MCB-0077838 (to P. A. R.) and National Institutes of Health Grant RO1-HL 59680-0 (to E. A. B.). O. K. V. was sponsored by PlantGenix, Inc., Philadelphia, PA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF29932 and AF29933.

Dagger Contributed equally to this work.

|| To whom correspondence should be addressed. Tel.: 215-898-0807; Fax: 215-898-8780; E-mail: parea@sas.upenn.edu.

Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.C100152200

    ABBREVIATIONS

The abbreviations used are: PC(s), phytochelatin(s); NGM, nematode growth medium; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
REFERENCES

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