From the United States Department of
Agriculture/Agricultural Research Service Children's Nutrition
Research Center, Baylor College of Medicine, Houston, Texas 77030, the
¶ Department of Human and Molecular Genetics, Baylor College
of Medicine, Houston, Texas 77030, and
Vegetable and Fruit
Improvement Center, Texas A&M University, College Station, Texas
77845
Received for publication, September 27, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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In plants and fungi, vacuolar transporters help
remove potentially toxic cations from the cytosol.
Metal/H+ antiporters are involved in metal
sequestration into the vacuole. However, the specific transport
properties and the ability to manipulate these transporters to alter
substrate specificity are poorly understood. The Arabidopsis
thaliana cation exchangers, CAX1 and
CAX2, can both transport Ca2+ into the vacuole. There are
11 CAX-like transporters in Arabidopsis; however, CAX2 was
the only characterized CAX transporter capable of vacuolar
Mn2+ transport when expressed in yeast. To determine the
domains within CAX2 that mediate Mn2+ specificity, six CAX2
mutants were constructed that contained different regions of the CAX1
transporter. One class displayed no alterations in Mn2+ or
Ca2+ transport, the second class showed a reduction in
Ca2+ transport and no measurable Mn2+
transport, and the third mutant, which contained a 10-amino acid domain
from CAX1 (CAX2-C), showed no reduction in Ca2+ transport
and a complete loss of Mn2+ transport. The subdomain
analysis of CAX2-C identified a 3-amino acid region that is responsible
for Mn2+ specificity of CAX2. This study provides
evidence for the feasibility of altering substrate specificity in a
metal/H+ antiporter, an important family of transporters
found in a variety of organisms.
The differential partitioning of cations is crucial for life
processes, and transporters play a critical role in maintaining the
proper concentrations of these ions in various cellular compartments (1). The inability of plants to actively avoid toxic concentrations of
particular cations in the environment places a particular importance on
cation transporters. One mechanism employed by plants and fungi to
avoid cation toxicity is the sequestration of cations into large
vacuoles (2). A fundamental question that arises is whether there are
separate vacuolar transporters for each cation, and if not, what is the
metal specificity of a given transporter?
Several types of active transport mechanisms exist in plants to drive
cations out of the cytosol against a steep concentration gradient (3,
4). One important class of transporters is the H+-coupled
cation antiporters, which have been identified at the vacuolar
(tonoplast) membrane and are driven by a proton electrochemical gradient (5-8). These transporters have numerous functions including resetting cytosolic levels of Ca2+ post signaling, vacuolar
sequestration of potentially toxic concentrations of Cd2+,
Zn2+, Mn2+ and other metals, and vacuolar
storage of essential micronutrients such as Zn2+,
Mg2+, and Mn2+. Despite numerous descriptive
reports in whole plants (6, 9, 10) and the recent cloning of several of
these transporters (11-13), there is a dearth of information available
regarding H+-coupled ion selectivity, and much less for the
residues that define specific cation transport (14).
Two Arabidopsis cation exchanger
(CAX)1 genes,
CAX1 and CAX2, were identified that could
suppress mutants of Saccharomyces cerevisiae
defective in vacuolar Ca2+ transport (11). Experiments
using vacuolar membranes from yeast cells expressing CAX1 (11)
demonstrate that this protein has biochemical properties similar to
those of native plant vacuolar H+/Ca2+
exchangers (5, 15). In similar experiments in yeast, CAX2 appears to
have a higher Km for Ca2+ than CAX1 and
a lower capacity for Ca2+ transport (11). CAX2 localizes to
the plant vacuole, and when expressed at high levels in transgenic
plants, increases vacuolar metal transport and causes the plants to
accumulate more Ca2+, Mn2+ and Cd2+
(16). Furthermore these transgenic plants were more tolerant to
Mn2+ in the growth media. These transport properties of
CAX2 suggest the potential for broad substrate specificity among the 11 CAX-like transporters found in the Arabidopsis genome
(17).
Two domains have been described that modulate CAX1 activity (14, 18,
19). The first domain has been termed the Ca2+ domain
(CaD), located between amino acids 87 and 95 in CAX1 (14). This domain
appears to be necessary for Ca2+ transport by CAX1.
Exchanging this 9-amino acid region of CAX1 into CAX2 (giving the
construct CAX2-9) greatly increases its Ca2+ transport
activity but does not appear to alter transport of other metals (14).
The second domain that regulates CAX function has been termed the
regulatory or autoinhibitory domain (18, 19). Sequence analysis
suggests that an N-terminal regulatory domain may be present in all
plant CAX-like transporters (20). The CAX1 and CAX2 open reading frames
contain additional amino acids at the N terminus that were not found in
the original "shorter" N-terminally truncated CAX (sCAX) clones
(sCAX1 and sCAX2) identified by suppression of yeast vacuolar
Ca2+ transport mutants (20, 21). These findings suggest
structural features involved in regulation and Ca2+
transport but do not identify domains that may confer metal specificity among these CAX transporters. The manipulation of CAX transporters, through alteration in expression and substrate specificity, is an
essential component in developing plants with increased tolerance to
metals or removing toxic levels of metals from soil (phytoremediation) (22). For a successful phytoremediation strategy, it is important to
understand what determines the specificity of broad range metal transporters such as CAX2.
In this report, we have characterized the transport properties of the
CAX2 transporter. We identify specific domains within CAX2 that mediate
Mn2+ substrate specificity and alter these domains to
abolish the Mn2+ transport capabilities of CAX2, thus
increasing its metal specificity. These findings serve as a framework
for engineering metal specificity among the various
H+/cation exchangers found in bacteria, fungi, and plants.
Yeast Strains, Vectors, and DNA Manipulations--
K667
(cnb1::LEU2 pmc1::TRP1
vcx1 Site-directed Mutagenesis--
Site-directed mutagenesis was
performed by a Class IIS restriction enzyme-mediated method (27), using
sCAX2 in pBluescript as the template DNA. BsmBI
was the Class IIS restriction enzyme used throughout this study.
CAX2-9 was constructed previously (14). The primers used
were as follows: for CAX2-A, 5'-GAA TTC CGT CTC CTA ATA ATC TCC
AAG AAG TCA TTC TCG GGA CAA AAC TCA ATC TAC TAC TAC CTT TC-3'
(forward), 5'-GAA TTC CGT CTC CAT TAA GTA CGC TGT TCT TTG G-3'
(reverse); for CAX2-B, 5'-GAA TTC CGT CTC CTT TGA CCA ATA ACA AAG
TGG CAG TGG TCA AAT ATT CGT TAC TAG GCT CAA TTC TGT C-3'
(forward), 5'-GAA TTC CGT CTC CCA AAG CGA AAA TTG ATA TGA TC-3'
(reverse); for CAX2-C, 5'-GAA TTC CGT CTC CTG GCA CTT CAC TCT TCT
GTG GAG GAA TCG CGA ATT ACC AAA AAG ACC AAG TCT TTG-3' (forward),
5'-GAA TTC CGT CTC CGC CAA GTA CAA GTA ACA TGT TAG AC-3' (reverse); for
CAX2-D, 5'-GAA TTC CGT CTC CCT ACT TGA AAA ACG GAG AGG CTT CGG
CTG CTG TTT TGT CCG ACA TGC AAC TAG CCC TGT CAA GGT TCA G-3'
(forward), 5'-GAA TTC CGT CTC CGT AGT GAA GAA CAG CCG GGA AGA G-3'
(reverse); for CAX2-E, 5'-GAA TTC CGT CTC CGT TAT GGA CTC ACC GTC
AAT TGT TCG ATG CAC TCG ATG AGG AAT CAA ATC AGA AC-3' (forward), 5'-GAA TTC CGT CTC CTA ACT GGA AGA AGA GGT AAG-3' (reverse); for CAX2-C1, 5'-GAA TTC CGT CTC CTG GCA CTT CAC TCT TTT GTG GTG
GAC TAG TCT TTT ACC-3' (forward; the reverse primer was the same as
CAX2-C reverse); for CAX2-C2, 5'-GAA TTC CGT CTC CTG GCA TCG CGA
ATT ACC AAA AAG ACC AAG TCT TTG-3' (forward), 5'-GAA TTC CGT CTC
CGC CAC CAC AAA AGA AGG CGC AG-3' (reverse). The bold letters in the
primer sequences indicate introduced mutations. All constructs were
sequenced completely and then subcloned into the yeast expression
vector piHGpd.
Construction of C-terminal C-Myc-tagged Fusions--
CAX1-c-Myc
was constructed in a previous study (20). Five tandem copies of the
c-Myc epitope (EQKLISEEDL) were used to produce in-frame fusions of
c-Myc to the C-terminal end of CAX2 constructs. The c-myc
sequence was amplified by PCR from plasmid pT7-5Xmyc using the primers
5'-GAA TTC GGA TCC GGT CGA CGG TAT CG-3' (forward) and
5'-GAA TTC GAG CTC TTA TCC ACC AAC CCG GGG TAC CGA ATT C-3' (reverse).
A BamHI site (underlined) was included in the forward primer
to ligate the tag to the engineered BamHI site at the 3'-end of the CAX2 constructs.
Protein Isolation and Expression Analysis of C-Myc-tagged
Constructs--
Total protein was isolated from yeast expressing
c-Myc-tagged constructs using the glass bead method (26). Protein
concentration was determined by using a protein assay kit
(Bio-Rad). The protein was separated by SDS-PAGE and detected by
anti-c-Myc monoclonal antibody (Berkeley Antibody Co., Richmond, CA) as
described (20).
Assay for Yeast Suppression--
The assay for Ca2+
tolerance on solid agar media was performed as previously
described (11). K667 cultures expressing various constructs were grown
in a selective liquid medium for 16 h, and 4 µl were spotted
onto yeast extract-peptone-dextrose (YPD) agar media containing
appropriate concentrations of CaCl2 or MnCl2. The cultures were then allowed to grow for 48 h at 30 °C.
Preparation of Membrane Vesicles for Ca2+ Transport
Assays--
Yeast membrane microsomes were prepared as described
previously (28).
Measurement of 45Ca2+ and
54Mn2+ Uptake--
For the measurement of
Ca2+ and Mn2+ uptake, membrane vesicles (30-40
µg/ml) were incubated in buffer containing 0.3 M
sorbitol, 5 mM Tris-MES (pH 7.6), 25 mM KCl,
0.1 mM sodium azide, and 0.2 mM sodium
orthovanadate. Vacuolar H+-translocating ATPase-catalyzed
H+ translocation was initiated by the addition of 1 mM MgSO4 and 1 mM ATP. The vesicles
were allowed to reach steady state with respect to pH gradient for 5 min at 25 °C before the addition of 45Ca2+
(6 mCi/ml; American Radiolabeled Chemicals, St. Louis, MO) or 54Mn2+ (6.5 mCi/ml; PerkinElmer Life Sciences).
The final concentration of Ca2+ and Mn2+ in the
reaction mixture was 10 µM and 1 mM,
respectively. At the indicated times, aliquots (70 µl) of the
reaction mix were removed and filtered through premoistened 0.45-µm
pore size cellulose acetate GS-type filters (Millipore, Bedford, MA).
After a 1-ml wash with ice-cold wash buffer (0.3 M
sorbitol, 5 mM Tris-MES, pH 7.6, 25 mM KCl, and
0.1 mM CaCl2 or MnCl2 as
appropriate), the filters were air-dried, and radioactivity was
determined by liquid scintillation counting. For metal
competition experiments, CAX2, but not CAX1, CAX3, or CAX4, Suppresses the Mn2+
Sensitivity of a Yeast Mutant--
Yeast strains lacking functional
calcineurin (for example cnb strains) display increased
Mn2+ sensitivity due, in part, to decreased activity of the
Golgi Ca2+/Mn2+-ATPase PMR1 (23, 29, 30).
Furthermore, the additional lack of the vacuolar
Ca2+/H+ antiporter VCX1 (cnb vcx1
strains) slightly exacerbates the Mn2+ sensitivity of
calcineurin mutants (30), indicating that VCX1 may transport
Mn2+ in addition to Ca2+. Yeast mutants deleted
in VCX1 and the vacuolar Ca2+-ATPase PMC1 are sensitive to
high concentrations of Ca2+ in the media (23). Expression
of VCX1 and N-terminally truncated CAX2 can suppress both the
Mn2+ and Ca2+ sensitivity of a yeast mutant
(K667, pmc1 vcx1 cnb1) defective in calcineurin and the
vacuolar Ca2+ transporters (16). As shown previously,
N-terminal truncations of CAX1 can strongly suppress the
Ca2+ sensitivity of these strains, whereas N-terminal
truncations of CAX4 only weakly suppress the Ca2+
phenotype, and N-terminal truncations of CAX3 are unable to suppress the Ca2+ phenotype (11, 31, 32). We were interested in
testing whether these characterized CAX transporters could suppress the
Mn2+ sensitivity of this yeast strain. As shown in Fig.
1, these CAX transporters cannot suppress
the Mn2+ sensitivity phenotype of the yeast mutant.
Additionally, the mung bean Ca2+/H+ antiporter,
VCAX1 (28), could not suppress Mn2+ sensitivity (data not
shown). Only N-terminally truncated CAX2-expressing yeast mutants were
capable of growing on plates containing 10 mM
MnCl2 (Fig. 1).
Construction of CAX1/CAX2 Chimeras--
Utilizing
chimeric CAX constructs in which parts of the CAX1 and CAX3
transporters were exchanged, a 9-amino acid region between putative
transmembrane domains (TMD) 1 and 2 of CAX1 has been identified as
important in mediating Ca2+/H+ transport (14).
We have termed this domain the CaD. Swapping of the equivalent 9 amino
acids of CAX3 into CAX1 abolished CAX1-mediated Ca2+/H+ transport. Alternatively, swapping of
the CAX1 CaD into CAX3 allowed this chimeric CAX transporter to
transport Ca2+ (14). As CAX2 is the only plant CAX
transporter characterized to date with the ability to suppress the
Mn2+ sensitivity of calcineurin-deficient yeast mutants
(Fig. 1), a similar series of chimeric CAX constructs utilizing CAX2
should delineate the Mn2+ specificity domain(s) of this
transporter. We chose to make chimeric constructs between CAX1 and CAX2
because each construct should maintain the ability to transport
Ca2+. Thus, the fidelity of each construct could be rapidly
assessed through suppression of Ca2+ sensitivity, and we
could then test for alterations in Mn2+ suppression.
Initially we planned a systematic approach to identify CAX2
Mn2+ specificity domains, similar to that used to identify
the CaD of CAX1 (14). We chose to divide CAX1 and CAX2 into four
segments of approximately equal size and exchange each segment to
create eight different chimeric constructs (33). Although the
construction of the chimeric clones was successful, Ca2+
antiport activity was abolished for some of the constructs, indicating that protein stability was affected (data not shown). Therefore, an
alternative approach was used.
At the amino acid level, CAX2 is 43% identical (56% similar) to the
CAX1 open reading frame. A sequence comparison among CAX1, CAX2, CAX3,
and CAX4 identified five short domains, each consisting of 9-15
amino acids, which had very low sequence similarity between CAX1 and
CAX2 (Fig. 2). We have designated these
domains A to E. The A domain (amino acids 65-73 of CAX1) is present at
the start of TMD 1, the B domain (amino acids 150-160 of CAX1) is present between TMD 3 and 4, the C domain (amino acids 175-184 of
CAX1) is present in TMD 4, the D domain (amino acids 219-233 of CAX1)
is present between TMD 5 and 6, and the E domain (amino acids 257-265
of CAX1) is present between TMD 6 and the acidic motif. There is very
little variation among CAX1, CAX3, CAX4, and the mung bean VCAX1 in
domains A, B, C, and E, whereas domain D and the region corresponding
to the CAX1 9-amino acid CaD is very divergent among all CAX sequences
(Fig. 2). We created five mutants in CAX2 that contain the
corresponding CAX1 A, B, C, D, or E domain. These mutants were
designated CAX2-A, CAX2-B, CAX2-C, CAX2-D, and CAX2-E, respectively.
For these studies we have also used the previously constructed CAX2-9
construct, which contains the CAX1 9-amino acid CaD (14). All of the
mutants were N-terminally truncated, i.e. constructed
without the first 42 amino acids of CAX2. Because we had previously
found that some CAX1/CAX2 chimeric clones had altered protein
stability, it was very important to verify that each construct was
expressed at approximately equal levels in yeast, and so we tagged each
chimeric construct with a C-terminal c-Myc epitope and analyzed protein
expression (Fig. 3). Each chimera was
expressed similarly to CAX1 and CAX2.
Altered Ca2+- and Mn2+-sensitive Yeast
Growth by CAX2 Mutants--
As we anticipated, each of these
constructs could suppress the Ca2+ sensitivity of the yeast
mutants relatively equally when grown on 200 mM
CaCl2 (Fig. 4). However, when
the yeast strains expressing these constructs were grown on higher
levels of Ca2+ (250 mM CaCl2), we
observed growth differences. Although the CAX2-C-, CAX2-D-, CAX2-E-,
and CAX2-9-expressing strains grew in a manner similar to CAX2
strains, the CAX2-A- and CAX2-B-expressing strains were significantly
reduced in their growth under these media conditions (Fig. 4). In media
containing 10 mM MnCl2, yeast strains harboring
the CAX2, CAX2-9, CAX2-D, and CAX2-E constructs all grew (Fig.
5). Like strains expressing CAX1, those
expressing CAX2-A, CAX2-B, and CAX2-C completely failed to suppress
Mn2+ sensitivity. Even at lower MnCl2
concentrations (5 mM), the CAX2-A, CAX2-B, and CAX2-C
strains were unable to grow (Fig. 5).
Ca2+ and Mn2+ Transport--
To examine
the Ca2+ transport properties of these three
Mn2+-negative mutants directly, Analysis of the CAX2 C-Domain--
The CAX2-C-expressing strains
have a specific defect in Mn2+ tolerance, as the growth of
these strains is largely indistinguishable from CAX2- on
Ca2+-containing media (Fig. 4) despite the lack of growth
on Mn2+-containing media (Fig. 5). The inability of the
CAX2-A- and CAX2-B-expressing strains to suppress Mn2+
sensitivity also implicates these domains as being involved in Mn2+ transport (Fig. 5). However, CAX2-A- and
CAX2-B-expressing strains also exhibited diminished growth on
Ca2+-containing media and decreased
Ca2+/H+ antiport activity (Figs. 4 and
6A), suggesting a nonspecific reduction in transport
capabilities. To examine which amino acids were involved in determining
the Mn2+ specificity of CAX2-C, we divided the CAX2 C
domain into two regions, each containing 3 different amino acids than
those present in the CAX1 C domain. The CAX2-C1 chimera contains the 3 amino acids TSL from CAX1, replacing amino acids CAF of CAX2, and the CAX2-C2 construct contains the 3 amino acids IAN from CAX1, replacing the amino acids LVF of CAX2 (Fig. 2). Yeast strains expressing the
CAX2-C2 construct were indistinguishable from CAX2, as these strains
could suppress both the Ca2+ and Mn2+
sensitivity of K667 yeast (Fig.
7A). Yeast strains expressing CAX2-C1 were able to strongly suppress the Ca2+ sensitivity
phenotype, but there was no growth of these strains on
Mn2+-containing media. When the transport properties of
CAX2-C1 were determined by direct Cation Selectivity Comparisons of CAX2 Mutants--
To further
analyze the altered transport properties of the CAX2 mutants,
competition experiments were performed. This approach allowed us to
determine the effect of the domain swapping between CAX1 and
CAX2 on cation selectivity. Previously, CAX2 has been shown to suppress yeast mutants
sensitive to high Mn2+concentrations, localize to the plant
vacuolar membrane, and increase Ca2+, Cd2+, and
Mn2+ accumulation and vacuolar transport when expressed
ectopically in tobacco (16). In this study we confirm by direct
transport measurements that CAX2 is a Mn2+/H+
antiporter. Although we have not determined the Km
for Mn2+ of CAX2, experimental observations indicate that
it has a low affinity for Mn2+. CAX2 Ca2+
transport activity could be measured using 10 µM
45CaCl2. However, to measure Mn2+
transport activity by CAX2, 1 mM
54MnCl2 had to be used (Fig. 6), as activity
was very weak when measured using 100 µM
54MnCl2 and could not be detected using 10 µM 54MnCl2. Furthermore, a 10×
excess of nonradioactive CaCl2 was able to inhibit 50% of
10 µM 45Ca2+ transport activity
mediated by CAX2, whereas only a 100× excess of nonradioactive
MnCl2 was able to inhibit 50% of activity (Fig. 8).
Similarly, measurements of vacuolar cation/H+ antiport
activity from oat root indicate that
H+-dependent transport of Mn2+ is
less efficient than that of Ca2+, Cd2+, or
Zn2+ (9). However, CAX2-expressing plants were slightly
more tolerant to Mn2+ in the growth media (16),
demonstrating that despite being a low affinity antiporter, CAX2 can be
an important component in providing Mn2+ tolerance. Here we
demonstrate that CAX1, CAX3, and CAX4 cannot suppress the
Mn2+ sensitivity of a yeast mutant (K667; pmc1 vcx1
cnb1), which suggests that ectopic expression of these
transporters in plants will cause altered accumulation, transport, and
possibly tolerance to a different spectrum of cations than CAX2. These
findings also suggest that unique amino acids in the CAX2 transporter
confer Mn2+ specificity and that the CAX2 transporter is a
valuable biological tool for studying the metal specificity of vacuolar
metal/H+ transporters.
Other antiporters are used by a variety of organisms to remove metals
from the cytoplasm. The CDF family of transporters is found in bacteria
and eukaryotes (17). They transport heavy metals including
Ni2+, Co2+, Cd2+, and
Zn2+ and exhibit an unusual degree of sequence divergence.
The mechanisms of energy coupling are not well understood, but these
proteins probably function by H+ antiport for metal efflux.
For example, Zn2+ transport into vacuolar membrane vesicles
by the S. cerevisiae CDF transporter ZRC1 is
ATP-dependent, requiring a H+ gradient
generated by the V-ATPase (35). Cd2+, Zn2+, and
Co2+ efflux in many bacteria is mediated by
cation/H+ antiport. For example, in Ralstonia
sp., metal/H+ antiport is catalyzed by CzcD and the
CzcCBA protein complex (36, 37). However, the overall sequence
and topology of CDF and CzcCBA proteins do not resemble the CAX transporters.
Little is known regarding the substrate specificity of divalent
cation/H+ transporters from any organism, much less the
domains that confer this specificity. For example, a spontaneous mutant
yeast strain, due to a point mutation in the yeast
Ca2+/H+ exchanger VCX1, has increased
Mn2+ resistance compared with wild type yeast (38).
However, no ion competition studies or direct Mn2+
transport measurements have been done on the native or mutated forms of
the transporter. Mutation analysis of a bacterial cation/H+
antiporter, CzcA, identified two conserved Asp residues and a Glu
residue within a transmembrane span that are essential for metal
resistance and antiport activity, but no alteration in metal specificity was observed (39). Recently it has been described that the C-terminal tail of a cyanobacterial
Na+/H+ antiporter has a role in determining ion
specificity (40). Exchange of the C-terminal tail between similar
Na+/H+ antiporters from Aphanothece
halophytica and Synechocystis sp. PCC 6803 greatly
affected ion specificity, particularly with respect to
Li+/H+ antiport activity (40). However, this
may have little significance to CAX2, as these antiporters have very
little sequence similarity with the CAX-like antiporters and are
predominantly alkali metal transporters rather than divalent heavy
metal transporters.
A 9-amino acid domain located between putative membrane spanning
domains 1 and 2 was identified recently as an important domain in
mediating CAX1 specificity toward Ca2+. Inserting this CaD
of CAX1 into CAX2 (CAX2-9) increases Ca2+ transport of
this chimeric construct but does not appear to alter substrate
specificity globally (14). As we demonstrate here, K667 yeast strains
expressing CAX2-9 maintain CAX2-like suppression of the
Mn2+ sensitivity phenotype, further implicating another
domain in the mediation of CAX2 Mn2+ specificity.
Utilizing CAX2 chimeric constructs in a robust yeast assay for
Mn2+ and Ca2+ tolerance, we identified a domain
that appears to be involved specifically in Mn2+ transport.
We have termed this domain, located in TMD 4 at amino acids 177-186 of
CAX2, the C-domain (Fig. 2). When the CAX1 C-domain was inserted into
CAX2 (CAX2-C) and expressed in yeast, the strains strongly suppressed
the yeast Ca2+ sensitivity in a manner similar to CAX2
(Fig. 4); however, these strains failed to suppress the
Mn2+ sensitivity (CAX1-like phenotype). All of the yeast
growth differences on both Ca2+ and Mn2+ media
were confirmed in direct measurements of 45Ca2+
and 54Mn2+ transport and ion competition
studies (Figs. 6, 7B, and 8). CAX2 transported
54Mn2+, whereas CAX2-C and CAX1 did not.
Furthermore, excess nonradioactive Mn2+ inhibited
CAX2-mediated 45Ca2+ transport but did not
inhibit CAX1 or CAX2-C 45Ca2+ transport (Fig.
8). Strains expressing the CAX2-C1 mutant, which contains only the CAF
to TSL change, had the same phenotype as the CAX2-C mutant. Moreover,
direct 54Mn2+ transport measurements confirmed
that the lack of growth on Mn2+-containing media of yeast
expressing CAX2-C1 was due to the loss of
Mn2+/H+ antiport activity by CAX2, resulting
from the CAF to TSL change. Of all the domains studied in these
experiments (domains A to E and the 9-amino acid domain), only
the C domain is located within a TMD. Because of this transmembrane
localization, we tentatively suggest that these CAF residues within the
CAX2 TMD4 may be part of a pore that confers Mn2+ specificity.
It is interesting to note that the CAX2 CAF residues are also found in
the deduced amino acid sequences of the putative Arabidopsis CAX5 (Arabidopsis genome initiative number At1g55730) and
CAX6 (At1g55720) transporters, as well as ZCAX2
(GenBankTM accession number AB044567) from Zea
mays, suggesting that these CAX transporters may also transport
Mn2+. Additionally, the yeast
Ca2+/H+ antiporter VCX1 contains LCF residues
at this domain, hinting that this region is more similar to CAX2 than
to CAX1.
This putative Mn2+ domain may also be involved in
Zn2+ transport. In competition studies,
45Ca2+ transport mediated by CAX2, CAX2-A, -B,
-D, -E, and CAX2-9 was inhibited by 100× excess Zn2+,
whereas CAX1- and CAX2-C-mediated 45Ca2+
transport was not (Fig. 8). The inability of CAX2 to suppress the
Zn2+ sensitivity of the zrc1 cot1 S. cerevisiae
mutant (data not shown) may infer that CAX2 does not transport
Zn2+. However, another putative vacuolar Zn2+
transporter, ZAT1 of Arabidopsis, failed to suppress the
Zn2+ sensitivity of this strain but was able to suppress a
Schizosaccharomyces pombe Zn2+-sensitive mutant
(41). Future direct measurements of Zn2+ transport in yeast
strains expressing CAX2, CAX2-C, CAX2-C1, and CAX2-C2 will need to be
performed to confirm whether this observed inhibition of
Ca2+ transport by Zn2+ was in fact due to
Zn2+/H+ antiport by CAX2, and if so, to
distinguish the Mn2+ and Zn2+ domains of CAX2.
The competition experiment also gave an indication that CAX1 but not
CAX2 may transport Ni2+. Furthermore, it appears that none
of the domains analyzed are involved in Ni2+ transport.
Given that both CAX1 and CAX2 can strongly suppress the K667 yeast
Ca2+ sensitive phenotype, we were surprised that the CAX2-A
and CAX2-B constructs demonstrated weaker Ca2+ suppression
(Fig. 4) and reduced Ca2+/H+ antiport activity
(Fig. 6) as well as the loss of Mn2+/H+
antiport, as inferred from the lack of Mn2+ suppression
(Fig. 5). Both proteins appeared to be expressed at high levels in
yeast (Fig. 3), but perturbations in protein folding or membrane
topology may not allow these proteins to be fully functional. These
alterations in transport have also abolished the Mn2+
competition of CAX2-A- and CAX2-B-mediated Ca2+ transport,
and therefore we cannot completely rule out the possibility that
these domains play some role in Mn2+ transport. Thus, using
this chimeric gene approach, we cannot precisely assess the roles of
these domains in metal specificity. However, the difference between
these constructs and CAX2-C is noteworthy.
Many transition metal transporters, such as those of the Nramp and ZIP
transporter families, appear to have broad substrate ranges (1, 42).
However, there has been little research into the understanding of the
molecular mechanisms determining substrate specificity and how it can
be altered. Other techniques have been used to understand and alter
transport specificity in different types of metal transporters. The
Arabidopsis plasma membrane transporter, IRT1, transports
Cd2+, Fe2+, Mn2+, and
Zn2+ (42). Replacing Asp residues at either position 100 or
136 with an Ala eliminates transport of both Fe2+ and
Mn2+ (43). Similarly, replacement of a Glu residue with Ala
at position 103 eliminates the ability of Zn2+ transport by
IRT1 but retains the transport of other metals. A number of other
residues in or nearby transmembrane domains appear to be
essential for IRT1 function. Mutagenesis of a charged residue within a
membrane-spanning domain in the yeast Golgi
Ca2+/Mn2+-ATPase PMR1 has normal
Ca2+ transport but a 60-fold reduction in the apparent
affinity for Mn2+ (44). Substitution of Ser with Ala at
position 775 in membrane-spanning region 5 in a
Na+/K+-ATPase causes a 30-fold decrease in
K+ but not Na+ affinity (45, 46). Increased
Na+ affinity has also been reported by a point
mutation in a wheat K+-Na+ transporter (47).
Future studies, focusing on altering other regions in CAX2, may
delineate other domains involved in metal specificity.
In summary, we have shown that it is possible to generate a
metal/H+ antiporter that is more specific to a particular
metal, in this case Ca2+, by mutation of a discrete amino
acid domain. The CAX2 constructs described here demonstrate the
validity of altering the metal transport profile of a CAX family
member. Examples of how such transporters could prove beneficial
include making transgenic crop plants that express high levels of CAX2
variants that no longer transport Mn2+. Unlike CAX1 ectopic
expression, CAX2 expression is not deleterious to plant growth (48).
CAX2 variants could be used to accumulate higher levels of a
Ca2+ but not accumulate unwanted metals, thereby boosting
the bioavailable Ca2+ in foods. In future studies we may be
able to use a similar mutagenic approach to increasing the specificity
toward other, more toxic metals. Alternatively, ectopic expression of
CAX2 variants that have increased metal transport could be used to help
remove toxic metals from soils. The first step toward these
applications is expressing these CAX2 variants in plants and analyzing
the changes in metal accumulation and vacuolar metal transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(23)) was the S. cerevisiae strain used to
express wild type and mutant genes. The wild type or mutant genes were
propagated in pBluescript II SK(+) (Stratagene, La Jolla, CA), and
inserts were transferred to the shuttle vector piHGpd (24) for the
expression in the yeast. The plasmids were introduced into yeast by the
lithium acetate/single-stranded DNA/polyethylene glycol transformation
method (25). Standard techniques were used to manipulate the DNA used
in this study (26). All of the CAX1 and CAX2 clones used in this study
were identical to the N-terminally truncated clones originally
identified (11). Thus the proteins encoded by sCAX1 and
sCAX2 lacked the first 36 and 42 amino acids, respectively.
pH-dependent 10 µM 45Ca2+ uptake was measured
at a 10-min time point in the presence of 100 µM or
1 mM nonradioactive metals.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (57K):
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Fig. 1.
Ca2+ and
Mn2+ tolerance of a yeast mutant by
Arabidopsis cation/H+
antiporters. Suppression of Ca2+ and
Mn2+ sensitivity of the pmc1 vcx1 cnb yeast
mutant (K667) by various CAX transporters. Saturated liquid cultures of
K667 containing the indicated plasmids were diluted to the cell
densities as indicated and then spotted onto medium permissive for
growth of strains harboring the plasmid ( His) and medium
that selects for the presence of plasmid-borne vacuolar
Ca2+ or Mn2+ transport (YPD medium containing
175 mM CaCl2 or 10 mM
MnCl2). The strains grown at 30 °C on
His medium and
YPD with metal medium were photographed after 1 and 2 days,
respectively. A representative experiment is shown.
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Fig. 2.
Alignment of deduced amino acid sequence of
polypeptides encoded by Arabidopsis thaliana CAX1,
CAX2, CAX3, and CAX4 and the
targets of CAX2 mutagenesis. Five clusters of amino acids in CAX2
that are different from those of CAX1 were selected for mutagenesis to
their respective CAX1 sequences (designated A to
E, bold overlines). The 9-amino acid region
reported in a previous study (14) was included in this investigation.
Alignments were performed using a program by Corpet (49).
Consensus amino acid residues are boxed in black
(identical) or gray (similar). Gaps introduced to maximize
the alignments are denoted by hyphens. The 11 putative
transmembrane spans (M1-M11) predicted for CAX1 and the
central hydrophilic motif rich in acidic residues are
overlined. The translation starting sites for the
N-terminally truncated constructs used in this study (sCAX1,
sCAX2, sCAX3, and sCAX4) are indicated by
arrows. CAX4 was truncated by the addition of a Met residue
before Ala39 as described previously (32). The
GenBankTM accession numbers for CAX1 to CAX4 are AF461691,
AF424628, AF256229, and AF409107, respectively.
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Fig. 3.
Expression levels of CAX1, CAX2, and CAX2
chimeric constructs. Western blot showing relative expression
levels of various constructs with C-terminal c-Myc tags used in the
tolerance assay. Equal amounts of total protein isolated from yeast
strains expressing each c-Myc-tagged construct as indicated were
separated by SDS-PAGE, blotted, and then subjected to Western blot
analysis using an anti-c-Myc monoclonal antibody.
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Fig. 4.
The effect of CAX1 domains mobilized into
CAX2 on the suppression of Ca2+ sensitivity in
yeast. Suppression of Ca2+ sensitivity of the
pmc1 vcx1 cnb yeast mutant, K667, by various CAX constructs.
The same assay conditions were used as described in the legend for Fig.
1, except yeast growth is shown on YPD containing 200 mM
and 250 mM CaCl2. A representative experiment
is shown.
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Fig. 5.
Identification of domains in CAX2 that are
necessary for conferring Mn2+ tolerance in
yeast. Suppression of Mn2+ sensitivity of the
pmc1 vcx1 cnb yeast mutant, K667, by various CAX constructs.
The same assay conditions were used as described in the legend for Fig.
1, except yeast growth is shown on YPD containing 5 mM and
10 mM MnCl2. A representative experiment is
shown.
pH-dependent
10 µM 45Ca2+ uptake into yeast
membrane vesicles was measured (Fig.
6A). Ca2+ antiport
activity mediated by CAX2-A and CAX2-B was significantly lower than for
CAX2 (57.4 and 33.4% of CAX2 activity, respectively), whereas
Ca2+ antiport activity mediated by CAX2-C was not
significantly different from CAX2. The ability to suppress the
Mn2+ sensitivity of the yeast mutants infers
Mn2+ transport capability. However, it is important to
demonstrate directly that these CAX2 constructs mediate
Mn2+/H+ antiport activity in yeast. Therefore,
we measured
pH-dependent 1 mM
54MnCl2 uptake into microsomal vesicles
isolated from K667 yeast strains expressing various CAX transporters.
Mn2+/H+ transport activity was observed in
membrane vesicles from CAX2- and CAX2-E-expressing strains (Fig.
6B), but no Mn2+/H+ antiport
activity was detectable in vesicles from CAX1- or CAX2-C-expressing yeast (data not shown). The Mn2+/H+ antiport
activity measured from CAX2-E vesicles was modestly greater than the
activity of CAX2 (Fig. 6B), confirming the slight increase
in yeast growth of CAX2-E strains compared with CAX2 strains on high
Mn2+ media (Fig. 5).
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Fig. 6.
Direct
45Ca2+ and
54Mn2+ transport of CAX2
and CAX2 chimeric constructs. A, a single 10-min time
point measurement of pH-dependent 10 µM
45Ca2+ uptake in microsomes extracted from
yeast expressing wild type CAX2, CAX2-A, CAX2-B, or CAX2-C. Results are
shown following subtraction of the protonophore gramicidin background
values. B, a time course of 1 mM
54Mn2+ transport into microsomes extracted from
yeast strains expressing the wild type CAX2 or CAX2-E. Solid
line, uptake in the absence of 5 µM gramicidin;
broken line, uptake in the presence of 5 µM
gramicidin. The data in A and B represent the
means (±S.E.) of two replications and are representative of at least
three independent experiments.
pH-dependent
45Ca2+ and 54Mn2+
uptake measurements into membrane vesicles prepared from
CAX2-C1-expressing yeast, CAX2-C1 was found to have significant
Ca2+/H+ antiport activity, which was
indistinguishable from that of CAX2, but no
Mn2+/H+ antiport activity (Fig.
7B).
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Fig. 7.
Identification of amino acids in the C domain
of CAX2 that are responsible for the loss of Mn2+
tolerance. A, suppression of Ca2+ and
Mn2+ sensitivity of the pmc1 vcx1 cnb yeast
mutant, K667, by CAX2, CAX2-C1, and CAX2-C2 constructs. The same assay
conditions were used as described in the legend for Fig. 1, except
yeast growth is shown on YPD containing 200 mM
CaCl2 and YPD containing 10 mM
MnCl2. The CAX2 C domain consists of 10 amino acids of
which only the first 3 and the last 3 are heterologous between CAX1 and
CAX2 (see Fig. 2). The first 3 amino acids (C1 domain) were changed to
the equivalent amino acids in CAX1 (to give CAX2-C1), and the last 3 amino acids (C2 domain) were changed to the equivalent amino acids in
CAX1 (to give CAX2-C2). A representative experiment is shown.
B, single time point measurements of
pH-dependent 10 µM
45Ca2+ uptake (left) and
pH-dependent 1 mM
54Mn2+ uptake (right) into
microsomal vesicles extracted from K667 yeast expressing wild type CAX2
or CAX2-C1. Ca2+ uptake was measured at 10 min, and
Mn2+ uptake was measured at 30 min. The results are shown
following subtraction of the protonophore gramicidin background values.
The data represent the means (±S.E.) of three to six replicates.
pH-dependent 10 µM 45Ca2+ uptake was measured at
a single 10-min time point into yeast microsomal vesicles isolated from
strains expressing CAX1, CAX2, and the six CAX2 mutants.
Ca2+ uptake determined in the absence of excess
nonradioactive metal (control) was compared with Ca2+
uptake determined in the presence of two concentrations (10× and
100×) of various nonradioactive metals (Fig.
8). Inhibition of Ca2+ uptake
by nonradioactive Ca2+ was used as an internal control, and
as expected, Ca2+ uptake by each CAX transporter was
strongly inhibited by excess Ca2+. Nonradioactive
Ca2+, particularly the 10× concentrations, did not
completely inhibit Ca2+ uptake, further highlighting the
low Ca2+ affinity of the CAX transporters. We have
previously demonstrated that tobacco plants ectopically expressing CAX2
have significantly increased vacuolar transport of Cd2+
(16). Ca2+ uptake by CAX1 and CAX2 were both strongly
inhibited by Cd2+, and this Cd2+ inhibition was
consistently observed in all of the six CAX2 mutants. However, this
inhibition was only significant for all CAX constructs at the higher
Cd2+ concentration. In agreement with the yeast suppression
and 54Mn2+/H+ antiport data, excess
nonradioactive Mn2+ inhibited Ca2+ transport by
CAX2 but not by CAX1. Of the CAX2 mutants, Mn2+ inhibition
was only observed for CAX2-D, CAX2-E, and CAX2-9, but not for CAX2-A,
CAX2-B and CAX2-C, confirming the yeast suppression data. Two other
divalent cations, Zn2+ and Ni2+, were also
tested. No significant Ca2+ uptake inhibition by
Zn2+ was observed for CAX1, whereas for CAX2, the degree of
Ca2+ uptake inhibition by Zn2+ was similar to
that observed for Mn2+. These results indicate that CAX2
may also be able to transport Zn2+. To test this
theory further, CAX2 was expressed in a highly Zn2+-sensitive yeast double mutant (zrc1 cot1)
lacking the vacuolar cation diffusion facilitator (CDF) transporters
ZRC1 and COT1 (34). The results were inconsistent, and it was concluded
that CAX2 was unable to significantly suppress the Zn2+
sensitivity of the double mutant (data not shown). The highest concentration of excess Zn2+ was able to inhibit
Ca2+ uptake to varying degrees by every CAX2 mutant except
CAX2-C. However, except for CAX2-9, no inhibition of Ca2+
uptake was seen by the lower Zn2+ concentration for any of
the CAX2 mutants. Ni2+ inhibited Ca2+ uptake by
CAX1 only when added at the higher concentration, but no significant
inhibition by Ni2+ was observed for CAX2 or any CAX2
mutant.
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Fig. 8.
Inhibition of Ca2+
uptake by CAX1, CAX2, and CAX2 derivatives into yeast
endomembrane vesicles in the presence of other metals.
Uncoupler-sensitive ( pH-dependent) uptake of 10 µM 45Ca2+, estimated as the
difference between uptake with and without 5 µM
gramicidin, was measured in the absence (control) or presence of 10×
or 100× nonradioactive CaCl2, MnCl2,
CdCl2, ZnCl2, or NiCl2 after 10 min. Ca2+ uptake values are shown following subtraction of
the gramicidin background values and expressed as percentages of the
control in the absence of any excess nonradiolabeled metals.
"C" denotes the control samples. The data represent the
means of two to eight replications from two to four independent
membrane preparations, and the bars indicate S.E. In the
summary table, + and
signs indicate the presence or absence of
competition by each cation, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Ning-Hui Cheng for critical reading of the manuscript and Michael Grusak for radioactive manganese.
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FOOTNOTES |
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* This work was supported by the United States Department of Agriculture/Agricultural Research Service under Cooperative Agreement 58-6250-6001 and by National Institutes of Health Grants CHRC 5 P30 and 1R01 GM57427.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.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030. Tel.: 713-798-7011; Fax: 713-798-7078; E-mail: kendalh@bcm.tmc.edu.
Published, JBC Papers in Press, December 22, 2002, DOI 10.1074/jbc.M209952200
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ABBREVIATIONS |
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The abbreviations used are: CAX, cation exchanger; CaD, Ca2+ domain; CDF, cation diffusion facilitator; MES, 4-morpholineethanesulfonic acid; sCAX, N-terminally truncated CAX; YPD, yeast extract-peptone-dextrose medium; TMD, transmembrane domain.
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