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
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ABSTRACT |
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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,
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
( 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.
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
ycf1 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 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.
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
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.
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).
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.
-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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
CONCLUSIONS
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-glutamylcysteinyltransferase; EC 2.3.2.15), PCs have the
general structure (
-Glu-Cys)n-Xaa, contain 2-11
-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).
MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
CONCLUSIONS
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strain DTY167 (MAT
ura3-52
leu2-3,-112 his-
200 trp1-
901 lys2-801
suc2-
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).
80 °C.
RESULTS
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MATERIALS AND METHODS
RESULTS
CONCLUSIONS
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or
AsO2
) (data not shown).
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Fig. 1.
Suppression of Cd2+
hypersensitivity of S. cerevisiae ycf1 mutant
strain DTY167 by plasmid-borne ce-pcs-1. Yeast ycf1
strain DTY167 was transformed with pYES3-CePCS1 (
),
pYES3-CePCS1::FLAG (
), pYES3-AtPCS1::FLAG (
),
or empty pYES3 vector (
). 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.
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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.
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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
(
) 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.
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RESULTS
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Emerson and D. Standiford for the use of microscope facilities.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are: PC(s), phytochelatin(s); NGM, nematode growth medium; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein.
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