1. No category

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 8, pp. 4936 –4943, February 20, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
A Common Highly Conserved Cadmium Detoxification
Mechanism from Bacteria to Humans
HEAVY METAL TOLERANCE CONFERRED BY THE ATP-BINDING CASSETTE (ABC)
TRANSPORTER SpHMT1 REQUIRES GLUTATHIONE BUT NOT METAL-CHELATING
PHYTOCHELATIN PEPTIDES *□
S
Received for publication, October 23, 2008, and in revised form, November 24, 2008 Published, JBC Papers in Press, December 2, 2008, DOI 10.1074/jbc.M808130200
Cadmium poses a significant threat to human health due to its
toxicity. In mammals and in bakers’ yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to
glutathione. In fission yeast, phytochelatins constitute the cosubstrate with cadmium for the transporter SpHMT1. In plants,
a detoxification mechanism similar to the one in fission yeast is
supposed, but the molecular nature of the transporter is still
lacking. To investigate further the relationship between
SpHMT1 and its co-substrate, we overexpressed the transporter
in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression
of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO3, As(III), As(V), CuSO4, or
HgCl2. Abolishment of the catalytic activity by expression of
SpHMT1K623M mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but
not that of AtHMA4, a P-type ATPase, indicating that GSH is
necessary for the SpHMT1-mediated cadmium resistance. In
E. coli, SpHMT1 was targeted to the periplasmic membrane and
led to an increased amount of cadmium in the periplasm. These
results demonstrate that SpHMT1 confers cadmium tolerance
in the absence of phytochelatins but depending on the presence
* This work was supported by a grant from the “Commissariat a` l’Energie
Atomique” (“Toxicologie Nucle´aire Environnementale” program), a grant
from the European Commission Marie Curie Research Training Network,
and a grant from the “Agence Nationale pour la Recherche” (ANR 2007SEST-023-01). The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
a supplemental figure.
1
These authors contributed equally to this work.
2
Present address: CNRS, UMR 5534, Centre de Ge´ne´tique Mole´culaire et Cellulaire, Universite´ de Lyon 1, 69622 Villeurbanne, France.
3
To whom correspondence should be addressed: CEA Cadarache, DSV, IBEB,
LEMS, UMR 6191 CNRS-CEA-Aix-Marseille II, F-13108 St. Paul lez Durance,
France, Tel.: 33-4-4225-3048; Fax: 33-4-4225-2364; E-mail: cforestier@
cea.fr.
4936 JOURNAL OF BIOLOGICAL CHEMISTRY
of GSH and ATP. Our results challenge the dogma of the two
separate cadmium detoxification pathways and demonstrate
that a common highly conserved mechanism has been selected
during the evolution from bacteria to humans.
Cadmium is a trace element, the presence of which in the
environment is essentially due to human activities. It is a highly
toxic non-biological heavy metal able to enter living cells via
transporters usually used for the uptake of essential cations
such as calcium, iron, zinc, and so forth (1). The reactivity of
cadmium with thiol groups and its ability to displace essential
biological metals result in oxidative stress and eventually cell
death (2). To cope with cadmium toxicity, living organisms
have developed different strategies.
In animals, as in the bakers’ yeast cytoplasmic cadmium is
complexed with the thiol tripeptide glutathione, a general
redox regulator (3, 4). Bis(glutathionato)-cadmium complexes
(Cd-GS2)4 are then driven from the cytoplasm to lesser sensitive cellular compartments by dedicated transporters. The prototypical transporter of Cd-GS2 is the GS-X pump, ScYCF1, in
Saccharomyces cerevisiae (5) and, even if still controversial, to a
lesser extent HsMRP1 in humans (6). HsMRP1 probably acts as
an efflux pump at the plasma membrane, delivering cadmium
in the extracellular medium, whereas ScYCF1 allows sequestration of cadmium into the vacuole (5). A study of a deficient
Scycf1 strain has shown that it was extremely cadmium-sensitive, pointing to a major role of ScYCF1 in cadmium tolerance
and detoxification (5). Additionally, ScYCF1 was also found
4
The abbreviations used are: Cd-GS2, bis(glutathionato)-cadmium complexes; ABC transporter, ATP-binding cassette transporter; HMT1, heavy
metal tolerance factor 1; SpHMT1, S. pombe heavy metal tolerance factor 1;
SpPCS, phytochelatin synthase enzyme; ScYCF1, S. cerevisiae yeast cadmium factor 1; AtHMA4, A. thaliana heavy-metal ATPase 4; HsMRP1, Homo
sapiens multidrug resistance-associated protein 1; PC, phytochelatin; PCS,
phytochelatin synthase enzyme; C. elegans PCS1; BSO, buthionine sulfoximine; ICP, inductively coupled plasma atomic emission spectrometry;
EMM, Edinburgh’s minimal medium; GFP, green fluorescent protein; EGFP,
enhanced GFP; MES, 4-morpholineethanesulfonic acid.
VOLUME 284 • NUMBER 8 • FEBRUARY 20, 2009
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Sandra Pre´ve´ral‡1, Landry Gayet‡1,2, Cristina Moldes‡1, Jonathan Hoffmann‡, Sandra Mounicou§, Antoine Gruet‡,
Florie Reynaud‡, Ryszard Lobinski§, Jean-Marc Verbavatz¶, Alain Vavasseur‡, and Cyrille Forestier‡3
From the ‡Commissariat a` l’Energie Atomique (CEA) Cadarache, Direction des Sciences du Vivant (DSV), Institut de Biologie
Environnementale et Biotechnologie (IBEB), Service de Biologie Ve´ge´tale et de Microbiologie Environnementales, Laboratoire des
Echanges Membranaires et Signalisation, the CNRS, UMR Biologie Ve´ge´tale et de Microbiologie Environnementales, and the
Aix-Marseille Universite´, Saint-Paul-lez-Durance, F-13108, the §Equipe de Chimie Analytique Bio-Inorganique, CNRS UMR 5034,
He´lioparc, 2 av. Pr. Angot, 64053 Pau Cedex 9, and the ¶CEA Saclay, DSV, Institut de biologie et de technologie de Saclay, SB2SM,
F-91191 Gif-sur-Yvette, France
SpHMT1 Requires Glutathione but Not Phytochelatins
FEBRUARY 20, 2009 • VOLUME 284 • NUMBER 8
EXPERIMENTAL PROCEDURES
Yeast Strains, Bacterial Strains, and Growth Conditions—
The S. cerevisiae strains were provided from the EUROSCARF collection. Y0000 (BY4741; MATa; his3D1;
met15D0; ura3D0) and the Scycf1 mutant Y04069 (BY4741;
YDR135c::kanMX4) were grown at 30 °C in complete medium
(yeast extract peptone dextrose; YPD) or in synthetic medium
with dextrose (all amino acids). The transformed yeast were
grown in selective medium (synthetic medium with dextrose
without uracil) containing 2% of dextrose, raffinose, or
galactose.
The S. pombe strains were the previously described the
SpHMT1-deficient ⌬hmt1 strain LK100 and the corresponding
S. pombe wild-type strain Sp223 (h⫺ade6-216, leu1-32, ura4294) (13) as well as the phytochelatin synthase-deficient ⌬pcs
strain Sp27 and the corresponding S. pombe wild-type strain
FY254 (h⫺ ade6-M210 leu1-32 ura4-⌬18 can1-1) (21). Transformation of S. pombe was performed as described previously
(22). Cells were routinely grown at 30 °C in complete (YPD
medium or Edinburgh’s minimal medium (EMM) supplemented appropriately (i.e. without Leu (EMM⫺Leu) as
described previously (23). Medium containing different concentrations of CdCl2 was prepared immediately prior to the
growth experiment. Plates used for the cadmium spot test were
prepared by adding the indicated concentration of CdCl2 to the
minimal plate medium.
Plasmid constructions and gene expression were performed
in the E. coli strains Top10 (Invitrogen). Bacteria cells were
grown at 37 °C in liquid Luria-Bertani (LB) medium supplemented with appropriate antibiotics.
Plasmid Constructs—Plasmid pART-SpHMT1 contains the
complete hmt1 open reading frame (13). Plasmids pARTSpHMT1 and pEGFP-N2 (BD Biosciences) were used to generate a SpHMT1-EGFP-N2 fusion by the “splicing by overlap
extension” technique as already described (7). To generate a
plasmid expressing SpHMT1-GFP in E. coli, the pUCSpHMT1-GFP was constructed in two steps. First, two primers
(SpHMT1–7, 5⬘-CTCTAGAGAAGGAGATATACATATGGTTCTACGTTACAACAGCC-3⬘; SpHMT1– 8, 5⬘-GGTAGGCCTTTAGTATGGAATC-3⬘) were designed to amplify by
PCR (annealing temperature of 50 °C, elongation time of 1 min)
a 400-bp fragment containing the first 379 bp of hmt1, the
appropriate 5⬘-ribosomal-binding site (RBS) sequence
upstream of hmt1 open reading frame start codon, and an XbaI
restriction site in 5⬘ position. PCR product was sequenced,
digested by XbaI, and cloned into the XbaI-SmaI digested plasmid pUC19 (New England Biolabs) to obtain the pUC-PCRRBS-SpHMT1. Finally, a 2.8-kb StuI-SacI fragment from
pART-SpHMT1GFP was subcloned into the same sites of pUCPCR-RBS-SpHMT1 to obtain pUC-SpHMT1-GFP. All plasmids were confirmed by sequencing.
Plasmids pYES2 expressing EGFP-N2, YCF1, or YCF1EGFP-N2 were previously described (7). The plasmid pYES
HMT1-EGFP-N2 was generated by HindIII-SacI digestions
of pUC-HMT1-GFP and pYES2. The plasmid pUCSpHMT1K623M-GFP was the result of a directed mutagenesis
using the Stratagene QuikChange II XL site-directed mutagenJOURNAL OF BIOLOGICAL CHEMISTRY
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involved in arsenic, antimony, mercury, and lead detoxification
(references cited within Ref. 7).
A second strategy of cadmium detoxification is found in
plants (8), with the exception of mosses, and in the fission
yeast Schizosaccharomyces pombe (9). In that case, cytoplasmic cadmium is chelated by enzymatically synthesized thiol
peptides, phytochelatins of the structure (␥-Glu-Cys)n-Gly,
where n equals 2–11 (10). These peptides are produced upon
cadmium exposure by the constitutively expressed phytochelatin synthase enzyme (11), the structure of which has been
recently resolved (12). The complex phytochelatin-cadmium
(PC-Cd) is then transferred to the vacuole by undetermined
transporter(s) in plants and by the ABC half-transporter,
SpHMT1, in S. pombe (13, 14). It is interesting to note that in
S. pombe, a mutation of the phytochelatin synthase enzyme,
SpPCS, or of the SpHMT1 transporter led to similar
cadmium-hypersensitive phenotypes (15). This indicates
that the transfer of PC-Cd complexes from the cytoplasm
to the vacuole is essential in cadmium resistance in this
organism.
In mammals, besides GSH, cadmium can be detoxified after
association with metallothioneins, a superfamily of ubiquitous
cysteine-rich low molecular weight proteins. These biomolecules, discovered as cadmium-containing proteins in horse
kidney, have extremely high metal and sulfur contents (up to
10% w/w) (16).
More recently, it appears that cadmium can be taken up, as a
free metallic cation, by energized transporters from the P1bATPases family. For instance, CadA and AtHMA4 are efficient
cadmium transporters, respectively, in Listeria monocytogenes
and in Arabidopsis thaliana (17, 18). However, if the detoxification activity of ScYCF1 (or HsMRP1 in humans) is GSH-dependent, cadmium transporters from the P1b-ATPases family
are functional in the absence of GSH.
This scheme, discriminating different strategies engaged
by plants and animals in cadmium detoxification, has
recently been ruled out by the discovery of a functional phytochelatin synthase enzyme in the worm Caenorhabditis
elegans that is able to complement the cadmium sensitivity
of an S. pombe PCS knock-out strain (19). CePCS1-deficient
worms were found markedly more sensitive to cadmium
intoxication, leading to the first demonstration of the PCmediated detoxification of cadmium in an animal (19). In
fact, potential phytochelatin synthase orthologs have been
found in a large list of eukaryotes outside the plant kingdom
(20). The identification of an ortholog of the ABC transporter SpHMT1 in C. elegans, CeHMT-1, has completed the
homology between the S. pombe and C. elegans pathways for
cadmium detoxification (15, 19). Strikingly, CeHMT-1-deficient worms were found strongly more sensitive to cadmium
than CePCS1-deficient worms (19), suggesting that the role
of CeHMT-1 was not limited to the transport of PC-Cd in
cadmium tolerance.
In the present study, we show that SpHMT1 overexpression
is able to confer cadmium tolerance in organisms devoid of
phytochelatins, such as S. cerevisiae and Escherichia coli. This
function requires the presence of glutathione in the cell and a
functional ATP-binding domain in the protein.
SpHMT1 Requires Glutathione but Not Phytochelatins
4938 JOURNAL OF BIOLOGICAL CHEMISTRY
band-pass filter at 510 –570 nm (EGFP-N2 excitation peak at
488 nm, emission peak at 507 nm). Images were captured with
a Zeiss AxioCam camera and its dedicated software.
Electron Microscopy—Bacteria transformed with pUC19 or
pUC-SpHMT1-GFP were fixed overnight in 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline and
extensively washed in phosphate-buffered saline. Cell pellets
were infiltrated in a 2% agar gel and embedded in Unicryl.
Eighty nm-tick sections were cut, collected on EM grids, preincubated in 20 mM Tris buffer (pH 7.5) containing 0.1% bovine
serum albumin, 0.1% fish gelatin, 0.05% Tween 20 (buffer-T),
and followed by a 90-min incubation in the same buffer-T containing a 1:90 dilution of anti-GFP polyclonal antibodies
(AbCam antibody ab290). Sections were washed three times in
buffer-T and then incubated in a 1:25 dilution of 10-nm goldconjugated secondary antibodies for 45 min (Amersham Biosciences). After washing in Tris, sections were stained with uranyl acetate and lead citrate and photographed on a FEI CM12
microscope (FEI, Eindhoven, The Netherlands). For statistical
analysis, pictures were randomly taken of each of the two samples and of the gold particles located along the plasma membrane, and cytoplasmic background labeling was counted in
⬃80 different cells from each sample.
Membrane Extraction and Western Blot Analysis—Transformed bacteria were grown in 5 ml of LB ampicillin and subcultured in 1 liter of LB for 4 h at 37 °C. The culture was stopped
when the A600 reached 1.5. Bacteria were collected by low speed
centrifugation (4000 ⫻ g, 15 min at 4 °C). All following steps
were carried at 4 °C. Pellets were resuspended in the buffer 50
mM Tris-HCl, 5 mM MgCl2, 1 mM dithiothreitol, 5 ␮M leupeptin, 5 ␮M pepstatin A, and 1 mM phenylmethylsulfonyl fluoride.
Bacteria were lysed by two successive passages through a
French press (18,000 p.s.i.). EDTA was then added at 10 mM.
Unbroken bacteria and membrane residues were removed by a
30-min centrifugation at 15,000 ⫻ g. Membranes were collected by centrifugation at 100,000 ⫻ g and washed in 15 ml of
the buffer 50 mM Tris-HCl, pH 8, 5 ␮M leupeptin, 5 ␮M pepstatin A, and 1 mM phenylmethylsulfonyl fluoride and centrifuged
again at 100,000 ⫻ g. The pellet was finally suspended in 50 mM
Tris-MES, pH 8, 300 mM sucrose. Membrane extracts were
denaturated at room temperature and subjected to SDS-PAGE
electrophoresis. After transfer onto polyvinylidene difluoride
membrane, proteins were detected with an anti-GFP antibody
(monoclonal antibody JL-8, BD Biosciences; dilution 1:5000) as
already described (7).
Periplasmic Extraction—The periplasm extraction was done
as already described (24). Briefly, bacteria transformed with
pUC19 or pUC-SpHMT1-GFP grown to saturation in LB
medium supplemented with 100 ␮M ampicillin were subcultured at a starting A600 of 0.1 into 50 ml of LB with 50 ␮M
cadmium and grown overnight. The culture was stopped when
the A600 reached 2.6. Bacteria were collected by low speed centrifugation (4000 ⫻ g, 15 min at 4 °C). Pellets were resuspended
in 1.5 ml of Tris-HCl, pH 8, by briefly vortexing, and 600 ␮l of
CHCl3 was added. After brief vortexing, the tubes were maintained at room temperature for 15 min, and then 5 ml of 10 mM
Tris-HCl, pH 8, was added. Intact cells were separated by centrifugation (6000 ⫻ g, 20 min). The supernatant containing the
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esis kit with two primers: SpHMT1-F-K623M, 5⬘-TCAGGTGGTGGGATGTCTACAATTATG-3⬘, and SpHMT1-RK623M, 5⬘-CATAATTGTAGACATCCCACCACCTGA-3⬘.
This plasmid was used to create pART SpHMT1K623M-GFP by
BglII-ScaI digestion in the pART SpHMT1-GFP and pYES2
HMT1K623M-EGFP-N2 after ligation of HindIII-SacI fragment
with the pYES2 digested by the same enzymes. Plasmid pYES2
HMA4-EGFP-N2 was previously described (18).
Growth Inhibition by Cadmium and Determination of Metal
Content—To examine growth in liquid medium, yeasts grown
to saturation in EMM⫺Leu medium were subcultured at a
starting A600 of 0.1 into the same medium containing different
concentrations of CdCl2, and the extent of growth after 48 h
was determined by measuring the A600. The growth rate of each
culture was also determined by taking A600 readings at multiple
intervals throughout the entire 48-h period. To examine
growth on plates, cells were grown overnight to saturation in
EMM⫺Leu or S-URA 2% raffinose medium. This overnight
culture was diluted in EMM⫺Leu or S-URA 2% galactose
medium to an A600 of 0.4, which in turn was diluted in 10-fold
increments. Two microliters of each 10-fold dilution was spotted onto EMM⫺Leu or S-URA 2% galactose plates containing
different concentrations of CdCl2 and incubated for 7 or 3 days
at 30 °C. In the case of E. coli, single colonies of bacteria transformed with pUC19 or pUC-SpHMT1-GFP were grown to saturation in LB medium (37 °C, 180 rpm) and were subcultured at
a starting A600 of 0.05 into the same medium containing different concentrations of CdCl2. The growth rate of each culture
was determined by taking A600 readings at multiple intervals
throughout the entire 6-h period. Determination of cadmium
content was realized by induced coupled plasma experiments.
After centrifugation, pellets were washed three times with 10
mM EDTA, dried for 48 h at 50 °C, and mineralized. The metal
content was determined using ICP (ICP-OES Vista-MPX, Varian). Metal resistance assays in the presence of BSO were carried out as described previously (7).
Localization of SpHMT1—The localization of the SpHMT1EGFP-N2 fusion protein was examined in LK100 S. pombe and
in the Y04069 S. cerevisiae transformed strains. After overnight
culture, yeast cells (0.8 OD) were resuspended in 1 ml of selective medium with galactose containing 8 ␮M FM4-64 (Red Synaptracer 3-2 Interchim FP-41109A) After 15 min at 30 °C, cells
were centrifuged and washed for 2 h at 30 °C under agitation
with 1 ml of selective medium containing galactose. Yeast cells
were washed two times in phosphate-buffered saline and resuspended in 1 ml of water before observation on a glass slide. The
observations were done with a confocal laser scanning microscope (Leica TCS SP2 AOBS) fitted with a krypton/argon laser
at ⫻100 magnification. Excitations were performed at 488 or
568 nm for EGFP-N2 (excitation peak at 488 nm and emission
peak at 507 nm) and FM4-64 (excitation peak at 515 nm, emission peak at 640 nm), respectively. The fluorescence was collected through 510 –570 and 660 – 800 nm for EGFP and FM464, respectively. The localization of SpHMT1 in bacteria was
conducted with log-phase cells expressing GFP-tagged
SpHMT1. Cells were examined at 100-fold magnification on a
poly-lysine-coated slide by using a Nikon Optiphot-2 fluorescence microscope. The fluorescence was collected through a
SpHMT1 Requires Glutathione but Not Phytochelatins
periplasm was collected, and 1 ml was used to determine the
cadmium periplasm content by ICP.
RESULTS AND DISCUSSION
Overexpression of SpHMT1 in S. pombe Rescues Cadmium
Tolerance in a Sphmt1-deleted Strain—As already reported by
Ortiz et al. (13, 14), an S. pombe mutant having a defect in the
Sphmt1 gene and unable to accumulate PC-Cd complexes in
the vacuole was found to be cadmium-sensitive. This defect was
rescued by expression of the wild-type SpHMT1 protein in the
mutant (Fig. 1A). Three independent clones of S. pombe strains
expressing a fusion of SpHMT1 with the green fluorescent protein also exhibited an enhanced cadmium tolerance when compared with the Sphmt1 deletion strain (Fig. 1A). Thus, the
C-terminal fusion of GFP did not impair the protein function as
already observed for other ABC transporters such as HsMRP1
or ScYCF1 (7, 25). The fusion protein co-localized with the
vacuolar marker FM4-64 (Fig. 1B), in accordance with the location of the transporter at the vacuolar membrane previously
deduced from membrane fractionation (13). A similar vacuolar
location has been observed for CeHMT-1 in S. pombe using a
similar GFP fusion strategy (19). The protein was detected in
Western blots using anti-GFP antibodies at the 112 kDa
expected molecular mass (Fig. 1C). We recently reported that
ScYCF1 confers a tolerance to other metal(loid)s besides cadmium (7). Surprisingly, among the metal(loid)s tested,
FEBRUARY 20, 2009 • VOLUME 284 • NUMBER 8
SpHMT1 was able to confer a tolerance to cadmium but not to
Sb(III), AgNO3, As(III), As(V), CuSO4, or HgCl2 (supplemental
Fig. 1).
Overexpression of SpHMT1 Complements Cadmium Tolerance of an S. pombe Strain Devoid of Phytochelatins—To determine whether SpHMT1 is an exclusive PC-Cd transporter, we
used the Sp254 S. pombe strain deleted for the PCS gene, Sp27.
This strain is unable to synthesize phytochelatins from glutathione and is highly sensitive to cadmium (21). In this genetic
background, overexpression of SpHMT1 was able to completely restore cell tolerance to cadmium in the drop test as well
as in liquid media experiments (Fig. 1, D and E). The cadmium
resistance was not restored when the ⌬pcs strain was transformed with the empty vector pART (Fig. 1E). These results
indicate that the lack of phytochelatins can be compensated by
SpHMT1 overexpression and strongly suggest that SpHMT1
can contribute to cadmium tolerance in S. pombe independently of any phytochelatin synthesis. To confirm this hypothesis, expression of SpHMT1 was investigated in organisms naturally devoid of PC.
SpHMT1 Rescues Cadmium Tolerance in the Hypersensitive
⌬ycf1 Strain of S. cerevisiae—In S. cerevisiae, the cadmium
detoxification strategy differs from the one reported in
S. pombe (5, 13, 14). There is no PCS gene ortholog in the
genome of S. cerevisiae, and cadmium detoxification occurs
JOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 1. SpHMT1 overexpression confers cadmium tolerance to ⌬pcs or ⌬SpHMT1 fission yeast cells. A, overexpression of SpHMT1 or SpHMT1-GFP
allowed to recover cadmium tolerance in the wild-type (WT) yeast (Sp223) or in the mutant strain LK100 (⌬hmt1). B, in confocal microscopy, SpHMT1-GFP
fluorescence co-localized with the vacuolar marker FM4-64. The panels are as follows: wild-type cells overexpressing HMT1-GFP observed in bright light (␣),
HMT1-GFP revealed by the GFP green labeling (␤), vacuolar membranes revealed by the FM4-64 red labeling (␹), and an overlay showing the yellow colocalization of HMT1-GFP to the vacuolar membrane (␦). C, SDS-PAGE electrophoresis followed by a Western blot analysis using anti-GFP antibodies on cell
lysate from a wild-type strain (Sp223) or a ⌬hmt1 strain (LK100), overexpressing (⫹) or not (⫺) SpHMT1-GFP. D, overexpression of SpHMT1-GFP allowed
recovery of a wild-type cadmium tolerance in the ⌬pcs context (Sp27). E, dose-dependent growth inhibition induced by cadmium for the different yeast strains.
Wild-type and ⌬pcs strains were transformed either by pART (open circle and open square for wild-type and ⌬pcs strains, respectively) or by pART-SpHMT1-GFP
(closed circle and closed square for wild-type and ⌬pcs strains, respectively). Experiments were done at least in triplicate, and a typical result is shown.
SpHMT1 Requires Glutathione but Not Phytochelatins
mainly by the formation of bis(glutathionato)-cadmium complexes in the cytoplasm followed by their transfer into the vacuole by the full-sized ABC transporter ScYCF1 (5). In accordance, overexpression of ScYCF1 in the S. cerevisiae wild-type
context allowed an increase in cadmium tolerance (Fig. 2A).
Remarkably, overexpression of SpHMT1 was even more efficient, allowing a yeast cadmium tolerance up to 200 ␮M. The
ability of SpHMT1 to confer cadmium tolerance was even more
pronounced in the cadmium-hypersensitive Scycf1 mutant
context (Fig. 2A). In this strain, overexpression of SpHMT1 was
able to induce a cadmium resistance greater than the one
observed after overexpression of the homologous transporter,
ScYCF1. A Western blot analysis revealed that the fusion protein SpHMT1-GFP was reproducibly and largely more detected
than ScYCF1-GFP based on a similar quantity of proteins
loaded on gel (Fig. 2B). This result might argue with the great
efficiency of SpHMT1 in cadmium detoxification. In contrast
with its location at the vacuolar membrane in S. pombe, in
S. cerevisiae, SpHMT1-GFP was located in vesicles surrounding the main vacuole (Fig. 2C). Experiments conducted here,
with the two major yeast models that diverged ⬃400 million
years ago (26), S. cerevisiae using GSH to complex cadmium
and S. pombe using PC, confirm that SpHMT1 activity in cadmium tolerance is independent of PC. Altogether these results
demonstrate that SpHMT1 can use another substrate than PC
for the cadmium resistance activity. To investigate further possible substrates coming from the thiol pathway, a S. cerevisiae
yeast strain depleted of GSH was used.
4940 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 3. A K623M mutation in SpHMT1 results in a dominant negative
form of the transporter. A, upper panel, overexpression of the K623M
mutated form of SpHMT1 conferred cadmium sensitivity to a wild-type
S. pombe strain (Sp223). Middle and lower panel, overexpression of the K623M
mutated form of SpHMT1 suppressed the tolerance given by overexpression of SpHMT1 in the ⌬hmt1 background (LK100) or in the S. cerevisiae
⌬ycf1 background (Y04). Experiments were done at least in triplicate, and
a typical result is shown. B, SDS-PAGE electrophoresis followed by a Western blot analysis using anti-GFP antibodies on cell lysate from a wild-type
strain (Sp223, left panel) or a ⌬hmt1 strain (LK100, right panel), overexpressing (HG) or not (⫺) SpHMT1-GFP and overexpressing three independent clones of SpHMT1K623M-GFP (lanes 1–3).
Cadmium Tolerance Conferred by SpHMT1 Depends on Glutathione Synthesis—The comparison of the action mechanism
of ScYCF1, the activity of which is strictly coupled to the presence of GSH and of SpHMT1, depending on the presence of PC,
lead us to evaluate the hypothesis that GSH might be a candidate as a co-substrate substitute for PC in cadmium transport
by SpHMT1. This hypothesis was tested by a direct application
in the culture medium of DL-BSO, an inhibitor of ␥-glutamyl
cysteine synthetase (27). In S. cerevisiae, after application of 5
mM BSO, the GSH pool was reduced to 32% of its initial content
after 6 h and to 50% after 24 h (7). In the absence of metal,
application of 5 mM BSO had no effect on growth of the different yeast strains studied (Fig. 2D). In the presence of 5 mM BSO
combined with 50 or 100 ␮M cadmium, the growth of wild-type
strain was diminished by about 30 or 50%, respectively, as
already reported (7). Similarly, in the presence of BSO, the cadmium tolerance of Scycf1 strains expressing ScYCF1-GFP or
SpHMT1-GFP was diminished. In contrast, cadmium tolerance resulting from an overexpression of the P1B-ATPase
AtHMA4, (the plasma membrane Arabidopsis Cd-ATPase)
was unaffected in the presence of BSO (Fig. 2D) (18). Thus, it is
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FIGURE 2. SpHMT1-GFP confers cadmium (Cd) tolerance to the S. cerevisiae ⌬ycf1 yeast strain. A, heterologous overexpression of SpHMT1-GFP
conferred a higher cadmium tolerance to a wild-type S. cerevisiae yeast strain
(Y00) than overexpression of the endogenous transporter ScYCF1. GFP was
expressed as a control. Similar results were obtained in the ⌬ycf1 context (Y04
strain) B, SDS-PAGE electrophoresis followed by a Western blot analysis using
anti-GFP antibodies on cell lysate of S. cerevisiae yeast strains overexpressing
SpHMT1-GFP or ScYCF1-GFP. The expression level of SpHMT1-GFP was found
to be strongly higher than that of ScYCF1-GFP. C, in confocal microscopy,
SpHMT1-GFP fluorescence in S. cerevisiae cells localized in vesicles surrounding the vacuoles. D, sensitivity to the glutathione inhibitor DL-BSO of the different yeast strains overexpressing either the GFP as a control or ScYCF1-GFP,
SpHMT1-GFP, or AtHMA4-GFP. Experiments were done at least in triplicate,
and a typical result is shown.
SpHMT1 Requires Glutathione but Not Phytochelatins
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expressed in a prokaryote, E. coli
DH10B, fully devoid of PC. Under
fluorescence microscopy, only cells
transformed with pUC-SpHMT1GFP were exhibiting a green fluorescent signal (Fig. 4A). Proteins
from the soluble and microsomal
fractions were extracted, separated
by SDS-PAGE, blotted, and immunodetected with an anti-GFP antibody (Fig. 4B). A 112-kDa apparent
polypeptide was detected in the
SpHMT1-GFP microsomal fraction
that is consistent with the predicted
molecular mass of SpHMT1-GFP. A
putative dimeric complex was
detected at 250 kDa as well as bands
at around 65 kDa that could be proFIGURE 4. SpHMT1-GFP is fully translated in E. coli cells and confers cadmium tolerance. A, the green teolytic fragments from SpHMT1,
fluorescence of GFP was observed in bacteria overexpressing SpHMT1-GFP. Bacteria overexpressing pUC19 or as already described in the natural
pUC-SpHMT1-GFP were observed in bright light (panels a and c, respectively) or under fluorescence micros- host (13). Finally, taking advantage
copy (panels b and d, respectively). B, immunodetection by Western blot analysis of SpHMT1-GFP in the membrane extract of E. coli cells transformed with pUC19 (empty vector) or pUC-SpHMT1-GFP. C, electron micro- of the C-terminal EGFP tag and
graph of E. coli cells overexpressing pUC19 (left panel) or pUC-SpHMT1-GFP (right panel). The preferential 10-nm gold-conjugated secondary
localization of SpHMT1-GFP at the periplasmic membrane is revealed after labeling with anti-GFP polyclonal
immunocytolocalizaantibodies and the use of 10-nm gold-conjugated secondary antibodies. D, growth kinetics of E. coli expressing antibodies,
either pUC19 (left panel) or pUC-SpHMT1-GFP (right panel) in the presence of increasing cadmium (Cd) concen- tion of the transporter was investitrations. E, dose-response curve to cadmium in E. coli cells expressing either pUC19 (open squares) or pUC- gated by electronic microscopy in
SpHMT1-GFP (closed squares). Experiments were done at least in triplicate, and a typical result is shown. F, measurement of cadmium accumulation in E. coli periplasm by ICP. The cadmium quantity is 4-fold higher in the E. coli (Fig. 4C). Although a mean of
presence of HMT1-GFP than in empty vector.
1.6 ⫾ 0.4 plasma membrane gold
particles per ␮m2 was observed in
likely that SpHMT1, similar to ScYCF1, is able to use glutathi- 86 independent cells expressing the empty vector, this number
was significantly increased to 3.84 ⫾ 0.4 (p ⫽ 2.4 10⫺4) in 78
one as a co-substrate in cadmium transport.
SpHMT1 Detoxification Activity Depends on ATP Hydrolysis bacteria expressing pUC-HMT1-GFP. These data demonstrate
and Multimerization—The transport activity of ABC trans- that in a heterologous system such as E. coli, without any
porters is linked to the binding of ATP and its hydrolysis at the eukaryotic signal peptide, HMT1-GFP can be expressed, preflevel of nucleotide-binding domains(28). To ensure that cad- erentially in the periplasmic membrane.
mium tolerance conferred by SpHMT1 overexpression was due
SpHMT1 Enhances E. coli Cadmium Tolerance—In DH10B
to a transport activity, a SpHMT1K623M variant of the protein, E. coli cells transformed by pUC19, the growth was drastically
unable to bind ATP, was produced. Overexpression of this var- affected when cadmium concentration was over 25 ␮M (Fig.
iant did not confer cadmium tolerance to Sphmt1 or Scycf1 4D). Conversely, overexpression of SpHMT1-GFP induced cell
deletion strains (Fig. 3A), demonstrating that the binding of tolerance up to 100 ␮M cadmium (Fig. 4, D and E). Similar
ATP by the protein is crucial in the cadmium resistance proc- results were obtained using other E. coli strains, DH5 and
ess. Using Western blot, we confirmed that the SpHMT1K623M JC7623 (data not shown). Because E. coli does not naturally
variant was properly expressed at a level comparable with the produce PC, these results confirm that SpHMT1 can contribute
wild-type version of SpHMT1 (Fig. 3B). Moreover, it is inter- to cadmium tolerance by another way than PC-Cd transport. In
esting to note that SpHMT1K623M had a dominant negative yeast, cadmium tolerance conferred by SpHMT1 results from
effect when overexpressed in the wild-type S. pombe strain (Fig. cadmium sequestration into the vacuole. In E. coli, the cad3A). ABC transporters are generally multimeric proteins, and mium content in the periplasmic space of SpHMT1-GFP-overthe simplest explanation of this dominant negative effect is that expressing bacteria was found 3-fold the one in control bacteria
this non-functional polypeptide invalidates the transport activ- (Fig. 4F), suggesting that in E. coli, SpHMT1 conferred cadity of the multimeric protein. The fact that both S. cerevisiae mium tolerance through cadmium sequestration into the
and S. pombe yeast cells transformed by the SpHMT1K623M periplasmic space. Analysis by exclusion chromatography of
variant plasmid were sensitive to cadmium intoxication cadmium complexes in periplasmic spaces of pUC SpHMT1strongly suggests that the resistance given by the wild-type GFP-transformed bacteria and pUC19-transformed bacteria
SpHMT1 is not due to a chelation process but rather due to an revealed a similar profile, the only difference being a higher
level of the different complexes in SpHMT1-GFP-expressing
active transport mechanism.
SpHMT1 Is Expressed at the Periplasm of E. coli Cells—Be- bacteria. GSH-Cd complexes were resolved but did not form
cause a recent study has shown that a few amounts of PC were the major cadmium ligand. In the two genetic backgrounds, the
synthesized in S. cerevisiae (29), SpHMT1-GFP was also majority of cadmium was associated to a large peak revealing a
SpHMT1 Requires Glutathione but Not Phytochelatins
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idation of cadmium sequestration in
the vacuole. Thus, invalidation of
this transporter results in critical
levels of cadmium in the cytoplasm,
leading to a strong toxicity.
Altogether these results demonstrate that SpHMT1 is a polyvalent
transporter that can take in charge
different forms of cadmium complexes, as illustrated on Fig. 5. It is
the first study showing that an ABC
transporter (HMT1) can accommodate glutathione, as well as phytochelatin-cadmium complexes. This
information would help identify the
transporter in charge of cadmium
detoxification in plants, a protein
that has long been sought after by
different groups for many years
based on its ability to use phytochelatin-cadmium complexes. Our
FIGURE 5. Scheme of the known mechanisms of cadmium detoxification by ABC transporters in various
organisms. Depending on the organism, cadmium is detoxified either by excretion or by sequestration. The results challenge the dogma of the
cationic metal can be directly excreted by the ATPase AtHMA4 in the plant A. thaliana (18). Most of the time, two separate cadmium detoxificacadmium is associated to glutathione (GS2) or PC. In the first case, Cd-GS2 complexes might be excreted by the
human HsMRP1 (6) or sequestrated by the yeast ScYCF1 (5). In the latter case, Cd-PC complexes are probably tion pathways and demonstrate that
sequestrated in C. elegans by C. elegans heavy metal tolerance factor 1 (CeHMT1) (19) and in S. pombe by a common highly conserved cadSpHMT1 (13). Here, we demonstrate that SpHMT1 does not require PC but needs glutathione. The biosynthesis mium detoxification mechanism
of glutathione and phytochelatins was blocked by the external application of BSO.
has been selected during the evolution from bacteria, including plants
high molecular mass complex (7 kDa), the nature of which was and yeast, to humans. Moreover, besides the fact that cadmium
is implicated in cancer in humans (in liver and kidney), the
not resolved.
Conclusion—We have shown that SpHMT1 is able to rescue nature of the human transporter responsible for cadmium
the cadmium-sensitive phenotype of an S. pombe mutant defi- detoxification remains an open question. Our results would
cient for the PCS gene, suggesting that PC-Cd is not the only lead to building new models of heavy metal detoxification to
substrate of SpHMT1. When heterogously overexpressed in prevent/cure diseases linked to the exposition of humans to this
the Scycf1 deletion mutant of S. cerevisiae, which is cadmium- toxin.
hypersensitive and devoid of PCS, SpHMT1 also allowed recovery of a wild-type phenotype. The complementation of the Acknowledgments—We thank D. Ortiz and S. Clemens for providing,
Scycf1 strain was found BSO-sensitive, suggesting that cad- respectively, the LK100 and Sp27 S. pombe strains as well as the cormium conjugated to glutathione is a substrate of SpHMT1. responding wild-type strains. Dr. P. Richaud and P. Auroy (CEA
Overexpression of a SpHMT1K623M variant exhibiting a point Cadarache, France) are acknowledged for help in ICP analysis.
mutation in the nucleotide-binding domain led to a suppression of the cadmium tolerance, disclosing a possible cadmium
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FEBRUARY 20, 2009 • VOLUME 284 • NUMBER 8
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Sandra Prévéral, Landry Gayet, Cristina
Moldes, Jonathan Hoffmann, Sandra
Mounicou, Antoine Gruet, Florie Reynaud,
Ryszard Lobinski, Jean-Marc Verbavatz,
Alain Vavasseur and Cyrille Forestier
J. Biol. Chem. 2009, 284:4936-4943.
doi: 10.1074/jbc.M808130200 originally published online December 2, 2008
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Membrane Transport, Structure, Function,
and Biogenesis:
A Common Highly Conserved Cadmium
Detoxification Mechanism from Bacteria to
Humans: HEAVY METAL TOLERANCE
CONFERRED BY THE ATP-BINDING
CASSETTE (ABC) TRANSPORTER
SpHMT1 REQUIRES GLUTATHIONE
BUT NOT METAL-CHELATING
PHYTOCHELATIN PEPTIDES