Báo cáo khoa học: Identification of the N-terminal region of TjZNT2, a Zrt⁄Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity ppt

Identification of the N-terminal region of TjZNT2,
a Zrt
⁄
Irt-like protein family metal transporter, as a novel
functional region involved in metal ion selectivity
Sho Nishida
1
, Yasuhiro Morinaga
2
, Hitoshi Obata
1
and Takafumi Mizuno
1
1 Graduate School of Bioresources, Mie University, Japan
2 Faculty of Bioresources, Mie University, Japan
Introduction
Heavy metal elements, such as iron, zinc, manganese,
copper, molybdenum, and nickel, are essential in
plants as catalytic cofactors or as structural elements
of numerous proteins. They are transported into cells
across biomembranes by transporter proteins.
Recently, several heavy metal transporter families (e.g.
COPT, NRAMP, P-type ATPase, CDF, and MOT)
have been identified in plants [1–3]. The Zrt ⁄ Irt-like
proteins (ZIPs) constitute one of the most important
metal transporter families. ZIP transporters are pre-
dicted to have eight transmembrane (TM) domains,
with the N-terminal and C-terminal regions exposed to
the extracellular surface, and have been demonstrated
to transport divalent heavy metal ions into the cyto-
plasm [4,5]. To date, many ZIP genes have been iso-

lated and characterized in plants [1,2,6]. Most ZIP
transporters display selectivity for Zn
2+
and ⁄ or Fe
2+
,
but several members are also selective for Cu
2+
or
Co
2+
[7–9]. ZIP transporters play critical roles in the
uptake of these essential metal elements and mainte-
nance of their appropriate distribution in plants.
Most ZIP transporters also transport toxic metal
ions, e.g. Cd
2+
or Hg
2+
, and therefore mediate the
accumulation of these elements in plants [10–13]. In
Keywords
ion selectivity; manganese; metal
transporter; zinc; ZIP family
Correspondence
T. Mizuno, Mie University, Graduate School
of Bioresources, Kurimamachiya-cho 1577,
Tsu, Mie 514-8507, Japan
Fax: +81 59 231 9684
Tel: +81 59 231 9607

E-mail:
(Received 20 April 2010, revised 11
December 2010, accepted 24 December
2010)
doi:10.1111/j.1742-4658.2011.08003.x
The Zrt ⁄ Irt-like protein (ZIP) family of transporter proteins is involved in
the uptake of essential metal elements in plants. Two homologous ZIP
genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that
share high amino acid sequence similarity except at the N-terminus and the
cytoplasmic loop between transmembrane domains III and IV, and that
have been shown to be Zn
2+
and Mn
2+
transporters, respectively. To iden-
tify the region that determines the ion selectivity of these transporters, we
constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed
for the Zn
2+
uptake of yeast cells expressing them. As a result, the extra-
cellular N-terminal ends were identified as regions involved in Zn
2+
selec-
tivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its
N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking
the N-terminal extension was shown to possess Zn
2+
uptake activity. This
suggests that the extended N-terminal region inhibits Zn
2+

transport by
TjZNT2. Further studies showed that it is the first 25 amino acid region of
the N-terminus that is important for the inhibition of Zn
2+
transport. Fur-
thermore, the N-terminal truncated TjZNT2 lacked Mn
2+
uptake activity.
These findings suggest that the N-terminal region is a novel substrate
selector in the ZIP family of transporters.
Abbreviations
HA, hemagglutinin; HRD, histidine-rich domain; LZM, low-zinc medium; sGFP, synthesized green fluorescent protein; TM, transmembrane;
YNB, yeast nitrogen base; ZIP, Zrt ⁄ Irt-like protein.
FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 851
recent years, the accumulation of toxic metals in crops
has become a major issue for food safety and human
health [14]. To develop new technologies for reducing
toxic metal levels in crops, it is essential to understand
the molecular mechanisms of metal selectivity in ZIP
transporters. As a representative study in this area,
Rogers et al. reported that the extracellular loop
between TM II and TM III was involved in the ion
selectivity of the ZIP transporter AtIRT1, a high-affin-
ity Arabidopsis thaliana iron transporter [15].
Previously, we cloned two ZIP genes, TjZNT1 and
TjZNT2, from a nickel hyperaccumulator, Thlaspi
japonicum. TjZNT1 and TjZNT2 have been identified
as excess nickel resistance genes [16,17]. TjZNT1 and
TjZNT2 share high sequence similarity (78% iden-
tity), with the exception of the N-terminus and the

TM III–IV loop. To examine the functions of
TjZNT1 and TjZNT2, these transporters were tagged
with hemagglutinin (HA) at their N-termini to verify
their expression and subcellular localization by immu-
noassay, and were expressed in Saccharomyces cerevi-
siae. The TjZNT1 and TjZNT2 expressed were clearly
different with regard to ion selectivity: TjZNT1 pos-
sesses selectivity for both Zn
2+
and Mn
2+
, and
TjZNT2 possesses selectivity for Mn
2+
[16,17]. This
finding indicates that there are structural differences
involved in the differences in ion specificity between
TjZNT1 and TjZNT2. The TM III–IV loop is one of
the least similar regions between the two proteins,
and has histidine-rich domains (HRDs) that render it
a potential metal-binding site of the ZIP transporter
[18]. Indeed, our recent work showed that the HRDs
of TjZNT1 may be involved in ion selectivity [19],
and the sequences of the HRDs are apparently differ-
ent between TjZNT1 and TjZNT2. Therefore, we
speculated that the difference in ion specificity
between TjZNT1 and TjZNT2 derived from the
TM III–IV loop. We show here, however, that untag-
ged TjZNT1 shows selectivity exclusively for Zn
2+

,
whereas untagged TjZNT2 remains an Mn
2+
trans-
porter, indicating that mutation of the N-terminus
affects the ion selectivity of TjZNT1. This finding
raises the possibility that differences in N-terminal
structure are involved in the differential ion selectivity
between TjZNT1 and TjZNT2.
This study aimed to identify the regions involved in
TjZNT1 and TjZNT2 ion selectivity. We mapped the
regions of interest by use of a series of TjZNT1 and
TjZNT2 chimeric proteins, and found that differences
in Zn
2+
selectivity between the two proteins were asso-
ciated with differences in the extracellular N-terminal
structure. Furthermore, an N-terminal-truncated
TjZNT2 was shown to have Zn
2+
selectivity. Our
findings suggest that the N-terminal region is a novel
ion selection site in the ZIP family of proteins.
Results
Subcellular localization of TjZNT1 and TjZNT2 in
yeast
Initially, differences in subcellular localization between
TjZNT1 and TjZNT2 in yeast cells were studied. ZIP
proteins are generally targeted to the plasma mem-
brane [20,21], with a few exceptions [22,23]. TjZNT1

and TjZNT2 were shown to complement the functions
of plasma membrane transporters of S. cerevisiae,
ZRT1 and SMF1, respectively, indicating that TjZNT1
and TjZNT2 are localized in the plasma membrane.
Protein subcellular localization prediction software,
wolf psort [24], also indicated that TjZNT1 and
TjZNT2 are likely to be targeted to the plasma mem-
brane (data not shown). Previously, we have shown
that TjZNT1 with synthesized green fluorescent protein
(sGFP) fused to the C-terminus (TjZNT1::sGFP) local-
izes not only at the plasma membrane but also at the
endomembrane, owing to heterologous expression, in
yeast cells [19]. To visually confirm the subcellular
localization of TjZNT2 in yeast cells, the sGFP gene
was fused to the 3¢-end of TjZNT2 and driven by the
MET25 promoter in S. cerevisiae. Confocal laser scan-
ning microscopy revealed that TjZNT2::sGFP was
localized to the plasma membrane and endomembrane
(Fig. 1), and showed similar fluorescence patterns to
those of TjZNT1::sGFP. These results indicate that
Fluorescence DIC Overlap
TjZNT2
TjZNT1
sGFP
Fig. 1. Subcellular localization of TjZNT1 and TjZNT2 in yeast cells.
TjZNT1::sGFP, TjZNT2::sGFP and sGFP alone were expressed in
BJ1824 cells, under control of the MET25 promoter. sGFP fluores-
cence images (left), differential interference contrast (DIC) images
(center) and overlapped images (right) are shown. Images were
acquired with a laser scanning confocal microscope. Bar: 5 lm.

N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al.
852 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS
TjZNT1 and TjZNT2 are both targeted to the plasma
membrane in yeast cells, and the different ion specifici-
ties do not therefore derive from different membrane
localizations.
Mapping of the regions responsible for
differences in Zn
2+
selectivity between TjZNT1
and TjZNT2
To determine the regions responsible for Zn
2+
selectiv-
ity, TjZNT1 and TjZNT2 were divided into five parts
on the basis of their amino acid sequence alignment
(Fig. 2), and a series of chimeric constructs composed
of the two genes were made in a systematic manner
with a PCR strategy. In this study, the constructs were
not tagged, because the functions of TjZNT1 and
TjZNT2 may be affected by terminal tagging. As a
rapid approach, the Zn
2+
selectivity of these alleles
was determined by their ability to rescue the yeast zrt1
mutant that lacks the Zn
2+
uptake of ZRT1 and can-
not survive in low-zinc medium (LZM), where Zn
2+

is
limited by EDTA [15]. We confirmed that the wild-
type strain (BY4741) could grow in LZM plates sup-
plemented with 600 lm ZnCl
2
but the zrt1 mutant
could not, and that both strains grew in LZM supple-
mented with 1000 lm ZnCl
2
(data not shown).
TjZNT1 and TjZNT2 also have Cd
2+
uptake activity
(Fig. S1), and the expression of these transporters
increases the Cd
2+
sensitivity of yeast cells. All strains
transformed with the chimeric transporter genes
showed increased sensitivity to Cd
2+
, as did the strains
expressing TjZNT1 or TjZNT2, confirming that these
chimeric transporters were functionally expressed as
metal transporters in yeast (Fig. 3).
Complementation assays showed that a dramatic
change in phenotype occurred only when the N-termi-
nal regions of the two proteins were exchanged. An in-
frame exchange between the extracellular N-termini
(Na region) conferred the ability to complement the
zrt1 mutant on TjZNT2 (Fig. 3), and TjZNT1 with an

in-frame fusion of the TjZNT2 N-terminus maintained
its ability to complement the mutant. Exchange of the
TM III–IV loop (L region), one of the least similar
regions between the two proteins, did not affect
the Zn
2+
selectivity of TjZNT1 or TjZNT2. In-frame
exchange of high-similarity regions (Nb,Ca or Cb -
region) also yielded chimeras that showed no change
in their Zn
2+
selectivity.
Inhibition of TjZNT2 Zn
2+
transport by its
N-terminal region
As described above, differences in ion selectivity
between TjZNT1 and TjZNT2 were speculated to
be caused by differences in the N-terminal regions of
these proteins. TjZNT2 possesses a 36 amino acid
hydrophilic extension at the N-terminus (Fig. 2), and
has a second methionine at position 37 that is in a
similar position to the TjZNT1 start codon. To clarify
the effect of the N-terminal length on Zn
2+
selectivity,
we constructed the N-terminally truncated TjZNT2
mutant and lacked the first 36 amino acids (DN36),
and investigated its Zn
2+

uptake activity. It was found
that DN36 complemented the zrt1 mutant (Fig. 4A),
T
jZNT1 1: MASSPTKILCDAGESDLCRDDAAAFLLKFVAIASIL
T
jZNT2 1:MFFIDVLWKLFPLYLFGSERDYLSETESILKIVPETMAAASSLSILCDAGEPDLCRDDSAAFLLKLVAIASIF
T
jZNT1 37:LAGVAGVAIPLIGKNRRFLQTEGNLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPDYPWSKFPFPGFFA
T
jZNT2 74:LAGAAGVAIPLIGRNRRFLQTDGSLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPEFPWKKFPFPGFFA
T
jZNT1 110:MVAALITLIVDFMGTQYYESKQQRNEVAGGGEAADVVEPGREETS-SVVPVVVERGNDDSKVFGEEDGGGMHI
T
jZNT2 147:MVAALITLLVDFMGTQYYEKKQEREATTHSGEQP SSGPEQSLGIVVPVAGEEGNDE-KVFGEEDSGGIHI
T
jZNT1 182:VGIRAHAAHHRHSHSNGHGTCDGH AHGQSHGHVHVHGSHDVENGARHVVVSQILELGIVSHSIIIGLS
T
jZNT2 216:VGIHAHAAHHTHNHTQGQSSCDGHSKIDIGHAHGHGHGHSHGGLELGNGARHVVVSQVLELGIVSHSIIIGIS
T
jZNT1 250:LGVSQSPCTIRPLIAALSFHQFFEGFALGGCISQAQFKNKSAIIMACFFALTTPIGIGIGTAVASSFNSHSPG
T
jZNT2 289:LGVSQSPCTIRPLIAALSFHQFFEGFALGGCISQAQFKNKPATIMACFFALTTPISIGIGTAVASSFNAHSVG
T
jZNT1 323:ALVTEGILDSLSAGILVYMALVDLIAADFLSKRMSCNLRLQVVSYVMLFLGAGLMSALAIWA
T
jZNT2 362:ALVTEGILDSLSAGILVYMALVDLIAADFLSKMMSCNFRLQIVSYLLLFLGSGLMSSLAIWT
L
Nα Nβ
Cα
Cβ

Fig. 2. Sequence alignment between TjZNT1 and TjZNT2. The TM regions predicted by TOPPRED 2 ( />are underlined. TjZNT1 and TjZNT2 were divided into five parts: Na1 ⁄ Na2 (1–7 amino acids ⁄ 1–44 amino acids), Nb1 ⁄ Nb2 (8–128 amino
acids ⁄ 45–165 amino acids), L1 ⁄ L2 (129–223 amino acids ⁄ 166–266 amino acids), Ca1 ⁄ Ca2 (224–321 amino acids ⁄ 267–362 amino acids), and
Cb1 ⁄ Cb2 (322–384 amino acids ⁄ 363–423 amino acids). These regions are shown as gray arrows above the sequences.
S. Nishida et al. N-terminus of TjZNT2 is involved in ion selectivity
FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 853
and measurement of the
65
Zn uptake of cells showed
that the
65
Zn accumulation of cells expressing DN36
was 10-fold greater than that of control cells (Fig. 4B).
These results indicate that truncated TjZNT2 possesses
the ability to transport Zn
2+
, and that the extended
N-terminal region inhibits that ability.
To determine more precisely the region involved in
Zn
2+
transport inhibition, three versions of the N-ter-
minally truncated TjZNT2 mutants (DN10, DN15, and
DN25) were constructed. DN10 was capable of comple-
menting Zn
2+
uptake of the zrt1 mutant strain,
although the cell growth observed under zinc-deficient
conditions was considerably lower than that observed
with cells expressing DN36 (Fig. 4A). The DN10 Zn
2+

uptake activity was 40% or less than that of DN36
(Fig. 4B). The strain expressing DN15 also showed sig-
nificantly lower cell growth, and the Zn
2+
uptake
activity was about 80% when compared with DN36.
There was no significant difference in Zn
2+
uptake
activity between DN25 and DN36. These results
suggest that the first 25 amino acids are involved in
inhibiting Zn
2+
transport.
Involvement of the TjZNT2 N-terminus in Mn
2+
selectivity
We examined the influence of N-terminal truncation of
TjZNT2 on Mn
2+
selectivity. TjZNT2 complemented
Mn
2+
uptake in the smf1 mutant, which lacks the
Mn
2+
uptake of SMF1 and cannot survive in
low-manganese medium [17], but DN36 did not show
complementary Mn
2+

uptake (Fig. 5A). Manganese
accumulation of an smf1 strain expressing TjZNT2
was significantly higher than that of the vector control
cells, whereas a strain expressing DN36 showed no
increase in manganese accumulation (Fig. 5B). The
strain expressing DN36 had an increased zinc content
pKT10
TjZNT1
TjZNT2
TjZNT1–L2
TjZNT2–L1
ZnCl
2
CdCl
2
1000 µM 600 µM 50 µM
D
600
=10
–1
10
–2
10
–1
10
–2
10
–1
10
–2

TjZNT1–N 2
TjZNT2–N 1
TjZNT1–N 2
TjZNT2–N 1
TjZNT2–C 1
TjZNT1–C 2
TjZNT1–C 2
TjZNT2–C 1
Fig. 3. Mapping of the regions responsible for differences in Zn
2+
selectivity between TjZNT1 and TjZNT2. Chimera cDNAs were pro-
duced with the overlap-PCR technique. The chimeric constructs are
depicted as topology models; black and gray models represent
TjZNT1 and TjZNT2, respectively. The pKT10 vector alone, TjZNT1,
TjZNT2 or chimeric constructs were expressed in the zrt1 strain.
Yeast cells were grown on LZM supplemented with 600 ⁄ 1000 l
M
ZnCl
2
or YNB medium supplemented with 50 lM CdCl
2
. Plates
were incubated at 30 °C for 5 days.
N36
N10
N15
N25
pKT10
ZnCl
2

CdCl
2
1000 µM 600 µM 50 µM
D
600
=
10
–1
10
–2
10
–1
10
–2
10
–1
10
–2
TjZNT2
TjZNT1
N36 N25TjZNT2
65
Zn uptake (pmol 10
–6
cells·min
–1
)
N10 N15pKT10
a
a

b
b
d
c
1.0
0.8
0.6
0.4
0.2
0
1.2
1.4
1.6
TjZNT1
e
AB
Fig. 4. The effect of the TjZNT2 N-terminal truncation on Zn
2+
uptake activity. (A) TjZNT1, TjZNT2 or truncations (DN10, DN15, DN25, or
DN36) were expressed in the zrt1 strain. Cells were grown on LZM supplemented with 600 ⁄ 1000 l
M ZnCl
2
or YNB medium supplemented
with 50 l
M CdCl
2
. Plates were incubated at 30 °C for 5 days. (B) Yeast cell Zn
2+
uptake was measured with
65

Zn. Cells were incubated in
LZM-EDTA containing 10 l
M ZnCl
2
at 30 °C for 10 min. Data are means ± standard deviations of four independent experiments. Different
letters indicate statistically significant differences (P < 0.01) between the strains, based on ANOVA (Tukey’s HSD).
N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al.
854 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS
as compared with the vector control strain, whereas
the iron content was not significantly different among
the strains (Fig. 5C,D). These data suggest that the
Mn
2+
selectivity of TjZNT2 is lost by N-terminal
truncation.
Discussion
The focus of the present study was to identify a novel
region involved in ion selectivity in the ZIP family of
transporters by structurally comparing TjZNT1 and
TjZNT2, which display differential ion selectivities. To
determine the region responsible for differences in ion
selectivity, TjZNT1 and TjZNT2 chimeric genes were
generated. We confirmed that these chimeric transport-
ers were functionally expressed in yeast cells through
assay of their Cd
2+
uptake, although this result does
not guarantee that the chimeras are properly folded and
fully active. Thus, it should be noted that the results
from these chimeras could reflect artefacts. However,

the approach lead to the identification of the extracellu-
lar N-terminus as a substrate selector in TjZNT2. On
alignment of the amino acid sequences of reported ZIP
transporters, the length and sequence of the N-terminal
region varied between members (data not shown),
suggesting a relationship between N-terminal structure
and ion specificity in other ZIP transporters. To our
knowledge, this is the first report showing involve-
ment of the N-terminus in ion specificity in ZIP trans-
porters.
TjZNT2 possesses a 36 amino acid hydrophilic exten-
sion at its extracellular N-terminus that is not present
in the sequence of TjZNT1. The present work provides
several pieces of evidence for the involvement of this
N-terminal region in the ion selectivity of TjZNT2 and
related proteins. First, truncation of the extended
N-terminus confers the ability to transport Zn
2+
on
TjZNT2, which is not a property of the native protein.
The TjZNT2 N-terminus therefore behaves as an ion
transport autoinhibitory domain. Further studies
showed that it is the first 25 amino acids that are
important for this inhibition. Second, native TjZNT2
can transport Mn
2+
and Cd
2+
, whereas the truncated
TjZNT2 lacks Mn

2+
transport activity. This indicates
that the N-terminal region does not inactivate TjZNT2,
but affects the ion selectivity of the protein. Finally,
tagging with HA at the N-terminus of TjZNT1 was
found to alter the ion selectivity of the protein, suggest-
ing that modification of the N-terminal sequence
directly affects the conformation of the ion-selectivity
domain in ZIP transporters clustered with TjZNT2.
As the N-terminal region contains no known metal-
binding motifs, we speculate that it either interacts with
other ion-selectivity regions in the protein, altering their
AB
pKT10
pKT10
TjZNT1
TjZNT2
N36
Wild-type
smf1
MnCl
2
0 µM 50 µM
D
600
=
10
–1
10
–3

10
–2
10
–1
10
–3
10
–2
pKT10 TjZNT2 N36
Zn accumulation (ng· g
–1
· DW)
Mn accumulation (ng· g
–1
· DW)
Fe accumulation (ng· g
–1
· DW)
350
300
200
100
0
a a
b
50
150
250
CD
100

80
60
40
20
0
aaa
120
pKT10 TjZNT2
N36
pKT10 TjZNT2 N36
12
10
8
6
4
2
0
a
b
c
Fig. 5. The effect of the TjZNT2 N-terminal
truncation on Mn
2+
uptake activity. (A) Wild-
type (BY4741) and smf1 strains were grown
on YNB medium supplemented with 2%
galactose, 20 m
M EGTA, 0 ⁄ 50 lM MnCl
2
and 50 mM Mes at pH 6.0. Plates were

incubated at 30 °C for 5 days. (B–D) Cells
were cultured at 30 °C to exponential phase
in YNB medium supplemented with 50 l
M
MnCl
2
and 50 mM Mes at pH 6.0. Data are
means ± standard deviations of four inde-
pendent experiments. Different letters
indicate statistically significant differences
(P < 0.01) between the strains, based on
ANOVA (Tukey’s HSD). DW, dry weight.
S. Nishida et al. N-terminus of TjZNT2 is involved in ion selectivity
FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 855
conformation, or it constitutes a part of the ion-selec-
tivity domain with other neighboring regions. The lat-
ter proposal is supported by the presence of several
charged residues that could directly interact with the
substrate in the 25 amino acid N-terminus. Fusion
of the TjZNT2 N-terminal region with the N-terminus
of TjZNT1 did not inhibit the latter’s ability to trans-
port Zn
2+
(Fig. 3). This suggests that the N-terminus
of TjZNT2 interacts specifically with other regions in
the protein that are not present in TjZNT1. In addi-
tion, the exchange of the Nb,L,Ca and Cb regions
between TjZNT1 and TjZNT2 did not cancel the auto-
inhibition of Zn
2+

transport of TjZNT2 suggesting
that the N-terminus may interact with multiple regions.
To obtain further evidence, we are presently assessing
the alteration of ion selectivity by use of a synthetic
peptide to mimic the extracellular N-terminus of
TjZNT2 as a biological approach. Further studies are
in progress to reveal the ion selection mechanism for
the N-terminus in greater detail.
Two further regions were considered as candidates
for mediating the difference in ion selectivity between
TjZNT1 and TjZNT2: the TM III–IV loop, one of the
least similar regions; and the TM II–III loop, identified
as an ion-selective region in AtIRT1. However,
exchanging these regions between TjZNT1 and
TjZNT2 did not affect their Zn
2+
selectivity. As
described above, the difference in ion selectivity
between TjZNT1 and TjZNT2 primarily derives from
the N-terminus. Indeed, there are no differences in ion
specificity between TjZNT1 and truncated TjZNT2,
and we conclude that these loops do not play a major
role in ion selectivity in the case of TjZNT1 and
TjZNT2, although they may play secondary roles in
combination with the N-terminal region.
Here, we report that the ion selectivity of a ZIP
transporter can be controlled by modification of the
N-terminus. Our findings may have relevance in the
development of techniques for the reduction of toxic
metal levels in crops, or the generation of plants that

accumulate specific toxic metals for phytoremediation.
We plan to confirm the generality of N-terminus
involvement in the ion selectivity of ZIP transporters.
The present study has also provided important clues
regarding the function of TjZNT2. TjZNT1 and
TjZNT2 are closely clustered with Thlaspi caerulescens
TcZNT1 and TcZNT2, as well as Arabidopsis halleri
ZIP4 and IRT3, which have all been suggested to play
a critical role in metal accumulation in hyperaccumula-
tor species [25–30]. It has also been suggested that
TjZNT2 is involved in nickel hyperaccumulation in
T. japonicum [17], but the true function of TjZNT2 in
T. japonicum remains unclear. Currently, we are trying
to determine the function of TjZNT2 through the
study of transcript regulation as a molecular biological
approach and the determination of the N-terminal
sequence as a biochemical approach.
Experimental procedures
DNA manipulations
The primers used in this study are summarized in Table S1.
TjZNT1 and TjZNT2 chimeras were produced by a stan-
dard overlap extension technique. PCR products were
cloned into the pTAII vector (Toyobo Co., Ltd, Osaka,
Japan), and subcloned into the pKT10-Gal-HA-BS yeast
expression vector at the EcoRI–SalI sites to eliminate HA
tags. Without a stop codon, TjZNT2 was subcloned into
pSNM4 [19] at the EcoRI–PvuII site to express protein
fused with sGFP at the C-terminus. All PCR-derived DNA
clones were sequenced. Escherichia coli and yeast transfor-
mations were performed with standard methods.

Yeast strains and growth conditions
The strains used in the metal uptake assays were: BY4741
(MATa his3 leu2 met15 ura3), zrt1 (BY4741 zrt1::KanMX4),
smf1 (BY4741 smf1::kanMX4), and the BJ1824 strain
(MATa leu2 ura3 trp1 pep4 cir
+
). The BY4741 series was
obtained from the European S. cerevisiae Archive for Func-
tional Analysis (Frankfurt, Germany). The strains were
grown in yeast nitrogen base (YNB) medium (0.67% yeast
nitrogen base without amino acids) supplemented with
appropriate amino acids.
Complementation tests of metal uptake mutants
and Cd
2+
sensitivity test
For the complementation test for the zrt1 mutant, LZM
[31] supplemented with ZnCl
2
(0 or 600 lm) and adjusted
to pH 6.0 was used. The complementation test for the smf1
mutant was performed following the methods of Thomine
et al. [32], using medium containing 20 lm EGTA, MnCl
2
(0 or 50 lm), and 50 mm Mes (pH 6.0). For the Cd
2+
sen-
sitivity test, YNB medium supplemented with 50 mm Mes
and 50 lm CdCl
2

at pH 6.0 was used. Yeast cultures at the
exponential phase were diluted and then spotted onto each
assay plate. Cells were observed following a 5–7-day incu-
bation at 30 °C. All solid plate media contained 2% agar.
All assay media contained 2% galactose to induce Gal1
promoter expression.
sGFP observation
Strains transformed with TjZNT1-pSNM4 and the pSNM4
empty vector were generated as described previously [19].
N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al.
856 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS
The strains were cultured to an exponential phase in YNB
medium at 30 °C, and imaged with a laser scanning confo-
cal microscope (FV 1000; Olympus, Tokyo, Japan).
Metal accumulation assay
The Cd
2+
accumulation assay was performed as described
in our previous report [19], with the exception that the
Cd
2+
concentration in the cells was not corrected for dry
weight but for cell number. For the Mn
2+
accumulation
assay, yeast cells were cultured to exponential phase
(D
600 nm
of 0.8–0.9) in YNB medium supplemented with
50 lm MnCl

2
and 50 mm Mes at pH 6.0. The Mn
2+
con-
centration was determined as described previously [16].
Measurements of mineral concentrations were made by
inductively coupled plasma atomic emission spectrometry
(ICPS-7500; Shimadzu Corp., Kyoto, Japan).
65
Zn uptake assay
The
65
Zn uptake assay was conducted following the method
of Eide et al. [33], except that
65
ZnCl
2
and LZM-EDTA
(LZM without EDTA) were substituted for
59
FeCl
3
and
low-iron medium–EDTA, respectively. LZM-EDTA was
supplemented with 2% galactose and 10 lm ZnCl
2
, and
adjusted to pH 6.0. Cells in the YNB medium (2% galac-
tose) at the exponential phase (D
600 nm

of 0.8–0.9) were
harvested, washed twice, and resuspended in an ice-cold
assay buffer (LZM-EDTA). Then, the attenuance of the cell
suspensions was measured. Cells were incubated in the
assay buffer containing
65
ZnCl
2
for 10 min at 30 °C and
for 2 min on ice, collected on Ultrafree MC (pore size,
0.5 lm; Millipore, Billerica, MA, USA), and washed with
ice-cold SSW (1 mm EDTA, 20 mm Na
3
-citrate, pH 4.2,
1mm KH
2
PO
4
,1mm CaCl
2
,5mm MgSO
4
, and 1 mm
NaCl).
65
Zn concentration was determined on a c-counter.
Acknowledgements
We are grateful to D. R. Fernando (University of Mel-
bourne) for critical reading of the manuscript. This
work was supported, in part, by a Grant-in-Aid for

JSPS Fellows 09J05716 (S. Nishida) and a Grant-in-
Aid for Young Research (B) 18780045 (T. Mizuno and
S. Nishida) from the Japan Society for the Promotion
of Science.
References
1 Colangelo E & Guerinot M (2006) Put the metal to the
petal: metal uptake and transport throughout plants.
Curr Opin Plant Biol 9, 322–330.
2 Kra
¨
mer U, Talke I & Hanikenne M (2007) Transition
metal transport. FEBS Lett 581, 2263–2272.
3 Tomatsu H, Takano J, Takahashi H, Watanabe-Takah-
ashi A, Shibagaki N & Fujiwara T (2007) An Arabidop-
sis thaliana high-affinity molybdate transporter required
for efficient uptake of molybdate from soil. Proc Natl
Acad Sci USA 104, 18807–18812.
4 Eide D (2006) Zinc transporters and the cellular traf-
ficking of zinc. Biochim Biophys Acta 1763, 711–722.
5 Guerinot M (2000) The ZIP family of metal transport-
ers. Biochim Biophys Acta 1465, 190–198.
6Ma
¨
ser P, Thomine S, Schroeder J, Ward J, Hirschi K,
Sze H, Talke I, Amtmann A, Maathuis F, Sanders D
et al. (2001) Phylogenetic relationships within cation
transporter families of Arabidopsis. Plant Physiol 126,
1646–1667.
7 Eide D, Broderius M, Fett J & Guerinot M (1996)
A novel iron-regulated metal transporter from plants

identified by functional expression in yeast. Proc Natl
Acad Sci USA 93, 5624–5628.
8 Korshunova Y, Eide D, Clark W, Guerinot M &
Pakrasi H (1999) The IRT1 protein from Arabidopsis
thaliana is a metal transporter with a broad substrate
range. Plant Mol Biol 40, 37–44.
9 Wintz H, Fox T, Wu Y, Feng V, Chen W, Chang H,
Zhu T & Vulpe C (2003) Expression profiles of Arabid-
opsis thaliana in mineral deficiencies reveal novel trans-
porters involved in metal homeostasis. J Biol Chem 278,
47644–47653.
10 Liu Z, Li H, Soleimani M, Girijashanker K, Reed JM,
He L, Dalton TP & Nebert DW (2007) Cd
2+
versus
Zn
2+
uptake by the ZIP8 HCO
3
)
-dependent symporter:
kinetics, electrogenicity and trafficking. Biochem Bio-
phys Res Commun 365, 814–820.
11 Connolly E, Fett J & Guerinot M (2002) Expression of
the IRT1 metal transporter is controlled by metals at
the levels of transcript and protein accumulation. Plant
Cell 14, 1347–1357.
12 Nakanishi H, Ogawa I, Ishimaru Y, Mori S & Nishiza-
wa NK (2006) Iron deficiency enhances cadmium
uptake and translocation mediated by the Fe

2+
trans-
porter OsIRT1 and OsIRT2 in rice. Soil Sci Plant Nutr
52, 464–469.
13 Clemens S (2006) Toxic metal accumulation, responses
to exposure and mechanisms of tolerance in plants.
Biochimie 88, 1707–1719.
14 Codex Alimentarius Commission (2006) Joint FAO ⁄
WHO Food Standards Programme, Report of the
Twenty-eighth Session, Geneva.
15 Rogers E, Eide D & Guerinot M (2000) Altered selec-
tivity in an Arabidopsis metal transporter. Proc Natl
Acad Sci USA 97, 12356–12360.
16 Mizuno T, Usui K, Nishida S, Unno T & Obata H
(2007) Investigation of the basis for Ni tolerance con-
ferred by the expression of TjZnt1 and TjZnt2 in yeast
strains. Plant Physiol Biochem 45, 371–378.
S. Nishida et al. N-terminus of TjZNT2 is involved in ion selectivity
FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 857
17 Mizuno T, Usui K, Horie K, Nosaka S, Mizuno N &
Obata H (2005) Cloning of three ZIP ⁄ Nramp trans-
porter genes from a Ni hyperaccumulator plant Thlaspi
japonicum and their Ni
2+
-transport abilities. Plant
Physiol Biochem 43, 793–801.
18 Eng B, Guerinot M, Eide D & Saier MJ (1998)
Sequence analyses and phylogenetic characterization
of the ZIP family of metal ion transport proteins.
J Membr Biol 166, 1–7.

19 Nishida S, Mizuno T & Obata H (2008) Involvement of
histidine-rich domain of ZIP family transporter TjZNT1
in metal ion specificity. Plant Physiol Biochem 46, 601–
606.
20 Ishimaru Y, Suzuki M, Kobayashi T, Takahashi M,
Nakanishi H, Mori S & Nishizawa N (2005) OsZIP4, a
novel zinc-regulated zinc transporter in rice. J Exp
Bot 56, 3207–3214.
21 Vert G, Grotz N, De
´
dalde
´
champ F, Gaymard F, Gueri-
not M, Briat J & Curie C (2002) IRT1, an Arabidopsis
transporter essential for iron uptake from the soil and
for plant growth. Plant Cell 14, 1223–1233.
22 MacDiarmid C, Gaither L & Eide D (2000) Zinc trans-
porters that regulate vacuolar zinc storage in Saccharo-
myces cerevisiae. EMBO J 19, 2845–2855.
23 Kuma
´
novics A, Poruk K, Osborn K, Ward D & Kap-
lan J (2006) YKE4 (YIL023C) encodes a bidirectional
zinc transporter in the endoplasmic reticulum of Sac-
charomyces cerevisiae. J Biol Chem 281, 22566–22574.
24 Horton P, Park K, Obayashi T, Fujita N, Harada H,
Adams-Collier C & Nakai K (2007) WoLF PSORT:
protein localization predictor. Nucleic Acids Res 35,
W585–587.
25 Hammond J, Bowen H, White P, Mills V, Pyke K,

Baker A, Whiting S, May S & Broadley M (2006) A
comparison of the Thlaspi caerulescens and Thlaspi ar-
vense shoot transcriptomes. New Phytol 170, 239–260.
26 Assunc¸ a
˜
o AGL, Da Costa Martins P, De Folter S,
Vooijs R, Schat H & Aarts MGM (2001) Elevated
expression of metal transporter genes in three accessions
of the metal hyperaccumulator Thlaspi caerulescens.
Plant Cell Environ 24, 217–226.
27 Talke I, Hanikenne M & Kra
¨
mer U (2006) Zinc-depen-
dent global transcriptional control, transcriptional
deregulation, and higher gene copy number for genes in
metal homeostasis of the hyperaccumulator Arabidopsis
halleri. Plant Physiol 142, 148–167.
28 van de Mortel J, Almar Villanueva L, Schat H,
Kwekkeboom J, Coughlan S, Moerland P, Ver Loren
van Themaat E, Koornneef M & Aarts M (2006) Large
expression differences in genes for iron and zinc homeo-
stasis, stress response, and lignin biosynthesis distin-
guish roots of Arabidopsis thaliana and the related
metal hyperaccumulator Thlaspi caerulescens. Plant
Physiol 142, 1127–1147.
29 Milner M & Kochian L (2008) Investigating heavy-
metal hyperaccumulation using
Thlaspi caerulescens as a
model system. Ann Bot 102, 3–13.
30 Weber M, Harada E, Vess C, Roepenack-Lahaye E &

Clemens S (2004) Comparative microarray analysis of
Arabidopsis thaliana and Arabidopsis halleri roots identi-
fies nicotianamine synthase, a ZIP transporter and other
genes as potential metal hyperaccumulation factors.
Plant J 37, 269–281.
31 Zhao H & Eide D (1996) The yeast ZRT1 gene encodes
the zinc transporter protein of a high-affinity uptake
system induced by zinc limitation. Proc Natl Acad Sci
USA 93, 2454–2458.
32 Thomine S, Wang R, Ward J, Crawford N &
Schroeder J (2000) Cadmium and iron transport by
members of a plant metal transporter family in
Arabidopsis with homology to Nramp genes. Proc Natl
Acad Sci USA 97, 4991–4996.
33 Eide D, Davis-Kaplan S, Jordan I, Sipe D &
Kaplan J (1992) Regulation of iron uptake in
Saccharomyces cerevisiae. The ferrireductase and Fe(II)
transporter are regulated independently. J Biol Chem
267, 20774–20781.
Supporting information
The following supplementary material is available:
Fig. S1. Cadmium accumulation of yeast strains
expressing TjZNT1 and TjZNT2.
Table S1. Primers used for the PCR amplifications.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al.
858 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS