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GmZIP1 encodes a symbiosis specific zinc transporter in soybean
Sophie Moreau1||, Rowena M. Thomson2||, Brent N. Kaiser3, Ben Trevaskis5, Mary Lou
Guerinot4, Michael K. Udvardi5, Alain Puppo1, David A. Day2*
1Laboratoire de Biologie Végétale et Microbiologie, CNRS FRE 2294, Université de Nice-
Sophia Antipolis, Parc Valrose, 06108 Nice cédex 2, France
2Biochemistry Department, University of Western Australia, Crawley, WA 6009, Australia
3Environmental Biology, RSBS, Australian National University, GPO Box 475, Canberra, ACT
2601, Australia
4Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
5Max Planck Institute of Molecular Plant Physiology, am Mühlenberg 1, 14476 Golm, Germany
|| These authors made equal contributions to the work
*Corresponding Author: Professor David Day
Phone: 61-8-93803325
Fax: 61-8-93801148
Email: [email protected]
Running title: GmZIP1 is a zinc transporter in soybean nodules
GenBank Accession Number for GmZIP1: AY029321
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SUMMARY
The importance of zinc in organisms is clearly established and mechanisms involved in zinc
acquisition by plants have recently received increased interest. In this report, the identification,
characterization and location of GmZIP1, the first soybean member of the ZIP family of metal
transporters, are described. GmZIP1 was found to possess 8 putative transmembrane domains
together with a histidine-rich extra-membrane loop. By functional complementation of zrt1zrt2
yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with
an estimated Km value of 13.8 µM. Cadmium was the only other metal tested able to inhibit zinc
uptake in yeast. An antibody raised against GmZIP1 specifically localized the protein to the
peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction
of the plant with its microsymbiont. The specific expression of GmZIP1 in nodules was
confirmed by Northern blot, with no expression in roots, stems or leaves of nodulated soybean
plants. Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least
some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1. The
orientation of the protein in the membrane, and its possible role in the symbiosis are discussed.
INTRODUCTION
Zinc is an essential micronutrient for all organisms, including plants. More than 3% of the
proteins of S. cereviseae and C. elegans are predicted to contain sequence motifs characteristic of
zinc binding structural domains (1). Zinc deficiency is a widespread micronutrient deficiency
limiting crop production (2). In recent years, genes encoding zinc transporters have been
identified in various organisms (3-11). These studies have shed some light on zinc uptake and
regulation, particularly at the plasma membrane level. However, with the exception of the
recently identified Zrt3p transporter on the vacuole membrane in yeast (9), little is known about
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intracellular zinc transport systems, nor about the mechanisms of the transporters identified. Here
we investigate zinc transport at the symbiotic interface between legumes and rhizobia, which
presents an additional level of complexity.
Many legumes form a symbiosis with nitrogen-fixing soil bacteria (rhizobia) which enables the
plants to utilise atmospheric N2 for growth. Infection of the legume root by rhizobia results in the
formation of specialised organs called nodules which provide the microaerobic conditions
required for operation of the nitrogenase enzyme. Within the infected cells of nodules, the N2-
fixing bacteroids are enclosed in a plant membrane to form an organelle-like structure termed the
symbiosome (12). The envelope of the symbiosome is called the peribacteroid membrane (PBM)
and effectively controls the exchange of metabolites between the symbiotic partners. The PBM,
although originating from the plasma membrane of root cells, evolves over the course of nodule
organogenesis to become a new and specialised membrane containing symbiosis-specific proteins
(see Ref. 13 for a review).
The principle metabolic exchange that occurs between plant and bacteroid is reduced carbon
(usually malate) from the plant for fixed N from the bacteroid, and specific transport mechanisms
have been identified for this exchange (14). However, the bacteroids are dependent on the plant
for all micronutrients and transporters for these must also exist on the PBM. Included in these
micronutrients is the essential metal zinc. Amongst the various transporters identified in other
systems, the ZIP family of zinc transporters was first identified in Arabidopsis and members have
also been identified in other plants (see Ref. 8 for a recent review). In general, the activities of
these transporters have been studied by expressing the proteins in yeast and their activity in the
parent plants has not been ascertained. Here we report the isolation of the first member of the ZIP
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family from soybean and localise it to the peribacteroid membrane of N2-fixing root nodules. The
ability to isolate intact symbiosomes from soybean nodules has allowed us to compare the
activity of GmZIP1 in both its native membrane and in yeast.
EXPERIMENTAL PROCEDURES
Materials
The Saccharomyces cerevisiae strains used were DY1455 (MATα ade2-1oc can1-100 oc his3
leu2 trp1 ura3 gal), DEY1453 (MATα ade2 can1 his3 leu2 trp1 ura3 fet3::HIS3 fet4::LEU2) and
ZHY3 (MATα ade6 can1 his3 leu2 trp1 ura3 zrt1::LEU2 zrt2::HIS3). AtIRT1 cDNA in yeast
expression vector pFL61 (15) is referred to as pAtIRT1.
Yeast, growth and transformation
Cells were grown in yeast extract/peptone/glucose (YPD) or synthetic defined (SD) media (2%
glucose, 0.67% Bacto-yeast nitrogen base; Difco) supplemented with necessary auxotrophic
requirements. Yeast transformations were performed using a lithium acetate based method (16)
and SD media was used to select transformants. Low Zinc Medium without EDTA (LZM-EDTA)
was used for yeast zinc uptake and was prepared as previously described (17).
PCR cloning of GmZIP1
GmZIP1 was cloned using PCR based on observed sequence similarity between AtIRT1
(U27590), AtIRT2 (TO4324), the pea Rit1 (AF065444) and a rice EST (D49213). Near complete
conservation of the amino acid sequence occurs in several short regions such as CFHQMFEGM
(residues 241-249 in Rit1) and MSLMAKWA (residues 341-348 in Rit1, underlined residues not
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conserved). The first set of primers corresponded to these regions, but to avoid the use of
degenerate primers the codons of Rit1 from Pea (the closest relative to soybean) were used.
Using these primers a partial cDNA was amplified from a soybean root nodule cDNA library
(Marathon cDNA Amplification Kit from Clontech). Based on this sequence, gene-specific
primers were designed (5’-RACE primer: 5’-TCT GCG CAA GAA GAT CTA CAA GTG C-3’
and 3’-RACE primer: 5’-CAA TAA GAC CAC TGA TGG CTG CA-3’). Next, 5’- and 3’-RACE
reactions were performed using a soybean root nodule cDNA library. Finally, primers designed
to clone the open reading frame (5’-TTG CCT CTT TCA CTG ATC ACA TG-3’ and 5’-CTC
TCA TTC TAT CTT AAG CCC AT-3’) were used to amplify a full GmZIP1 open reading frame
(based on sequence alignment to other ZIP genes) of 1062 bp. GmZIP1 was cloned into pFL61
yeast expression vector to give pGmZIP1.
Symbiosome isolation and membrane purification
Soybean (Glycine max. cv. Stevens) seeds were inoculated with Bradyrhizobium japonicum
strain USDA 110. Plants were grown in pots as previously described (18). Symbiosomes were
isolated from soybean nodules harvested from 4-5 week old plants and purified in mannitol
medium through a Percoll density gradient, as described before (19). Isolated symbiosomes were
vortexed vigorously to disrupt the PBM and centrifuged at 12 000 x g for 10 min to pellet the
bacteroids. The supernatant was centrifuged at 200 000 x g for an hour. Peribacteroid membranes
were collected from the pellet of this second centrifugation while the supernatant represented the
peribacteroid space fraction (20). Proteins were phenol extracted by the method of Hurkman and
Tanaka (21), concentrated by ammonium acetate / methanol precipitation at -20°C and
resuspended into [65 mM Tris pH 6.8, 2 % SDS, 10 % Glycerol, 50 mM DTT, 10 % β-
Mercaptoethanol, 0.002 % Bromophenol Blue] before SDS-PAGE. Microsomal fractions were
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obtained from nodules ground in [25 mM MES-KOH pH 7.0, 350 mM Mannitol, 3 mM MgSO4,
1 mM PMSF, 1 mM pAB, 10 µM E64, 1 mM DTT], filtered and centrifuged at 20 000 x g for 20
min, in order to pellet the symbiosomes. Supernatant was again centrifuged at 125 000 x g for an
hour and the pellet from this centrifugation contained the soybean nodules microsomes. Proteins
were extracted and concentrated as described above. The same protocol was used to purify
proteins from frozen root after homogenization in [0.7 M Sucrose, 0.5 M Tris, 30 mM HCl, 0.1
M KCl, 1% β-Mercaptoethanol]. Protein concentrations were estimated according to the
Coomassie Plus protein assay reagent kit (Pierce).
Northern and Southern analysis
Poly-A+ RNA was purified from the leaves, stems and roots as well as the nodules of various
aged plants using Dynabeads oligo-dT25 (Dynal). The Northern gel was loaded with 1 µg poly-A+
RNA samples and then run, blotted onto nylon membrane (Hybond N, Amersham), and the
membrane baked according to standard procedures (22). GmZIP1 DNA was DIG-labelled using
the PCR DIG Labelling Mix (Roche). Hybridisation overnight at 55°C was followed by washes
(2 x 15 min at room temperature in 2 x SSC, 1 % SDS and then 2 x 30 min at 68°C in 0.1 x SSC,
1 % SDS). Immunological detection of the probe was accomplished using anti-DIG antibody
conjugated to alkaline phosphatase and the CDP-Star chemiluminescent substrate (Roche).
Total RNAs were isolated from nodules of soybean plants using RNeasy Plant Minikit (Qiagen).
Samples containing 20 µg RNA were denatured, separated onto a 1.2 % agarose 7.4 %
formaldehyde gel, transferred to nylon membrane and baked for 2 hours at 80°C. Equal loading
of RNA in each lane was confirmed by vizualization of ribosomal RNA bands after staining of
the gel with ethidium bromide. [32P-CTP]-labeled riboprobe was synthesized using an in vitro
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transcription system kit (Promega) and ApaI-linearized pGmZIP1 as template. After 12 h
hybridization at 55°C in [50 % formamide, 5 x SSPE, 0.5 % SDS, 0.25 % powdered milk, 10 %
dextran sulfate], the membrane was washed twice for 20 min at 55°C in 2 x SSC 0.1 % SDS and
was then exposed to film (Biomax, Kodak).
Genomic DNA was isolated from the leaves of 28 day old plants using Wizard Genomic DNA
Purification Kit (Promega). Southern blot analysis was performed with 5 µg of genomic DNA
digested with restriction enzymes. DNA was separated on a 0.8 % agarose gel, blotted onto nylon
membrane and the membrane baked for 2 hours at 80°C. GmZIP1 DNA was DIG-labelled as
above. Hybridisation was performed overnight at 42°C, followed by washes (2 x 15 min at 24°C
in 2 x SSC, 0.1 % SDS and then 2 x 30 min at 42°C in 0.5 x SSC, 0.1 % SDS). Detection of
signal was as above using CDP-Star.
Zinc uptake
a) Yeast zinc uptake. ZHY3 yeast strains carrying plasmid pFL61 or pGmZIP1 were grown to
mid-log phase in LZM-EDTA. Cells were harvested, washed once and resuspended in a minimal
volume of LZM-EDTA. Cells were equilibrated at 30°C for 20 min before being mixed with
twice their volume of a radiolabeled 65Zn2+ solution. Uptake solution contained LZM-EDTA pH
4.2, 20 µM ZnCl2 and 200 nCi 65ZnCl2 (New England Biolab). Cells were incubated in a 30°C
water bath for stated amounts of time. Aliquots were collected on glass microfiber filters (GF/F
Whatman) and washed five times with 1 ml of ice-cold SSW pH 4.2 (1 mM EDTA, 20 mM
trisodium citrate, 1 mM KH2PO4, 1 mM CaCl2, 5 mM MgSO4, 1 mM NaCl ; Ref. 23). 65Zn
content of the cells was determined by liquid scintillation counting of the filters. Competition for
Zn2+ uptake by metal ions was measured by adding a 10-fold molar excess of iron, copper, nickel,
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manganese, cobalt, cadmium or molybdenum to 20 µM 65Zn-labeled solution. All metals were
used as their chloride salts and were of analytical reagent grade or equivalent. To study the
competition for zinc uptake by ferrous iron, 10 mM ascorbic acid was also added to the mix and
the sulfate salt of iron was used in this case. When needed, the uptake solution was buffered with
Tris-HCl for pH ranges of 7 to 9 or with citric acid-NaOH for pH3 to pH6. A stock solution of
250 mM ZnCl2 was prepared in 0.02 N HCl. Cell number was determined by measuring the
absorbance of liquid cultures at 600 nm and comparing with a standard curve.
b) Symbiosome zinc uptake. Isolated symbiosomes were diluted to a protein concentration of
1mg/ml and pre-equilibrated at 30°C for 15 min. Symbiosome aliquots were added to a double
volume of assay buffer giving a final concentration of 0.3 mg/ml. Uptake experiments were
conducted as described above for yeast, except that mannitol medium was used instead of LZM
or SSW. When used as the uptake wash buffer, 10 mM nitrilotriacetic acid was added to the
mannitol medium, to chelate loosely bound metals. In all experiments, controls for background
adherence of Zn were performed by measuring uptake at 0°C ; these values were subtracted from
all of the data shown.
Preparation of antiserum to GmZIP1 protein
Two peptides were selected from immunogenic regions of the GmZIP1 protein sequence,
corresponding to the first 10 N-terminal amino acid residues (MKRFHSDSK) and to amino acid
residues 182-195 (HGHVPTDDDQSSELL) present in the loop between transmembrane domains
III and IV. These two peptides are unique when searched against Genbank. Peptides were
synthesized and coupled to KLH (Keyhole Limpet Hemocyanin) as a carrier protein. Rabbits
were primed and boosted three times with the mix of the two coupled peptides, following the
Eurogentec Double X immunization program over a time of 3 months. Preimmune serum and
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antiserum obtained in the final bleed were purified through a HiTrap Protein A column
(Amersham) and used at a 1:1,000 dilution unless otherwise stated.
Western blot analysis
Proteins were separated on 12 % polyacrylamide gels under denaturing conditions (24) and
electrophoretically transferred to Hybond-C nitrocellulose membrane (Amersham). Membranes
were blocked with 1 % Blocking solution (Boehringer) and incubated for 1 hour with a 1:1,000
dilution of primary antibody. Antisera used were anti-GmZIP1 antiserum (described above) or
anti-AtIRT1 antiserum (M.L. Guerinot, unpublished data). After washing off the unbound
antibodies several times with 1 x TBS-Tween 20, the membranes were incubated for 1 hour with
a 1:20,000 dilution of sheep anti-rabbit horseradish peroxidase-conjugated IgG (Boehringer) and
washed several times. Immunodetection was performed with a chemiluminescence Western
blotting kit according to the supplier (Boehringer).
RESULTS
GmZIP1 is a member of a zinc transporter family
Sequence analysis of the soybean cDNA showed that it encodes a protein of 354 amino acid
residues (Fig. 1). A BLAST search on the translated protein sequence indicated strong homology
with several members of the ZIP family as well as with other zinc and iron transporters.
Consequently, we have named this cDNA GmZIP1. A phylogenetic tree obtained after compiling
GmZIP1 with the sequences of 18 other known plant ZIP members (Fig. 2), revealed that
GmZIP1 is most closely related to AtZIP1, AtZIP3 and AtZIP5, three recently identified zinc
transporters of Arabidopsis (25, 8). According to the SMART (26, 27) and TMMTOP (28)
predictions, GmZIP1 contains eight transmembrane spanning regions, a very short C-terminal tail
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and a predicted extracellular location of both the N- and C-terminal ends. The 20 first amino acid
residues were also predicted to be part of a signal peptide. The extra-membrane loop located
between putative helices III and IV is rich in histidine residues. This feature is one of the
characteristics of the ZIP proteins, together with completely conserved histidine and glycine
residues in helix IV, which are also present in GmZIP1 at positions 212 and 217, respectively.
Moreover, amino acids 207-221 of GmZIP1 give a perfect match with the bona fide signature
sequence of the ZIP family (29). These results, together with the presence of several putative
metal ion binding sequence motifs between helices III and IV, suggest that GmZIP1 can be
considered a new member of the ZIP zinc transporter family and the first one identified in
soybean.
GmZIP1 encodes a zinc transporter
To further characterise this protein and to establish whether it has any metal transporting
capacity, the GmZIP1 cDNA was expressed in S. cerevisiae mutant strains which are unable to
grow on iron- or zinc-limited media, and their growth was monitored on plates (Fig. 3). Although
the DY1455 wild type strain can grow under zinc-deficient conditions, ZHY3 cells are very
sensitive to zinc deprivation because both their high (ZRT1) and low (ZRT2) affinity zinc uptake
systems have been mutated (30). However, GmZIP1-expressing ZHY3 cells were able to grow
on restrictive medium, indicating that GmZIP1 could encode a putative zinc transporter. A
similar experiment was performed using DEY1453 cells which lack both high (FET3) and low
(FET4) affinity iron transporters and cannot grow on iron-limited media. Under the conditions
tested, transformation with GmZIP1 did not restore the growth of the fet3fet4 mutant, suggesting
that GmZIP1 cannot use iron as a substrate. In both sets of experiments, the mutant strains were
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also transformed with pAtIRT1, which has previously been shown to complement both fet3fet4
and zrt1zrt2 cells (31), as a positive control.
In order to quantify the transport of zinc by GmZIP1, uptake assays with 65Zn were performed in
ZHY3 mutant cells. At 30°C, zinc accumulation in GmZIP1-expressing cells was linear for at
least 60 min (Fig. 4A). Cells transformed with the pFL61 empty vector, on the other hand,
showed a very low level of zinc accumulation (data not shown) which presumably represented
residual zinc uptake through other yeast metal ion transporters. Values obtained in these control
experiments were subtracted from all the data presented. No zinc uptake could be detected when
assays were conducted on ice suggesting that the zinc accumulation observed was due to an
internalisation of the metal rather than a non-specific adsorption of zinc to the cell surface. An
absence of uptake was also observed when cells were starved of glucose for an hour prior to
starting the uptake assays (Fig. 4A). However, no change in uptake level was noticed when pH
was varied from 3 to 7 (data not shown), indicating that GmZIP1 activity is not pH-dependent.
pH values higher than 7 are known to lead to the formation of monovalent Zn(OH)+, neutral
Zn(OH)2 and insoluble complexes (32) and, therefore, were not tested. We also investigated the
affinity of the uptake system over a range of zinc concentrations. Uptake was followed over a 20
min period and was found to be concentration-dependent and saturable (Fig. 4B). The transport
kinetic parameters, Km and Vmax, were determined from Lineweaver and Burk data
transformations (Fig. 4B, inset) and were estimated at 13.8 µM and 12.5 fmol per min per 106
cells, respectively.
The specificity of GmZIP1 for zinc or other metals was assessed in competition experiments
performed in the presence of a 10-fold molar excess of other, non-labelled divalent cations.
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Among the metals tested, cadmium alone had a significant inhibitory effect on zinc uptake (Table
1). It is interesting to note that neither Fe(III) nor Fe(II) could compete with zinc, in agreement
with the inability of GmZIP1 to complement the fet3fet4 mutant on iron-limited media.
Tissue-specific expression and localisation of GmZIP in nodules
The results presented above clearly show that the soybean GmZIP1 behaved like a zinc
transporter, when expressed in a heterologous system. We subsequently investigated the role of
GmZIP1 in planta. Poly-A+ RNA was isolated from leaves, stems, roots, and nodules and
analysed on a Northern blot (Fig. 5). Under the conditions used, the probe detected GmZIP
mRNA only in the nodules. The GmZIP transcript signal only appeared in nodules of plants 18
days and older and the abundance of transcripts did not change between nodules of 23 and 42
day-old plants (Fig. 5). No signal was observed in roots, stems, or leaves. This tissue-specific
expression suggests that GmZIP1 is a symbiotic protein that is active in mature, nitrogen-fixing
nodules. Since in Arabidopsis ZIP1 and ZIP3 transcripts are only observed in roots when plants
are starved of zinc (25), and since the plants used in the present study were grown in the presence
of ample zinc, the results suggest either that the nodule infected cell cytosol is depleted of zinc
(perhaps by the bacteroids themselves) or that expression of the symbiotic gene is regulated by
other factors. The answer to this question awaits further experimentation.
In order to analyse the presence of GmZIP1 at the protein level and to localise it within nodules,
we used both an antiserum raised against AtIRT1 and a GmZIP1 specific antibody. The AtIRT1
antiserum reacted with several proteins in a microsomal membrane preparation from nodules (see
experimental procedures) but only a single protein of 34 kDa on the purified PBM (Fig.6A)
which is the predicted size of GmZIP1. While we cannot eliminate the possibility that there is an
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IRT homologue on the PBM, which reacts with AtIRT1 antiserum, this is unlikely since the
primers we used to amplify GmZIP1 from the nodule cDNA library should have amplified IRT
clones also. A stronger reaction against the 34 kDa PBM protein was observed with the GmZIP1
antibody which did not react with protein samples isolated from root microsomes or nodule
microsomes isolated after removing the symbiosomes (Fig. 6B). PBM proteins isolated from
plants of 4, 6 and 7 week-old plants reacted equally with the GmZIP1 antibody (Fig. 6C). The
results shown in Fig. 6 suggest that GmZIP1 is a symbiotic isoform of a larger GmZIP family.
This idea is supported by the results of the Southern blot of soybean genomic DNA (Fig. 7). The
hybridisation pattern seen with the GmZIP1 probe is consistent with the presence of a multigene
family. No cross-reaction of the GmZIP1 antibodies with symbiosome space or bacteroid
proteins was observed, nor between the PBM and the rabbit pre-immune serum (data not shown).
The results of Fig. 6 also show that the PBM preparation was not contaminated significantly by
other membranes from the nodule. In this context it should be noted that we prepare PBM from
purified symbiosomes which are routinely checked by microscopy and marker enzyme assays for
contamination by other plant organelles and membranes (59). This contamination is negligible,
largely because of the rate zonal method of purification of symbiosomes on dense Percoll
gradients (19).
The presence of GmZIP1 on the PBM having been established, we further investigated the
capacity of symbiosomes to take up zinc. As shown in Fig.8A, purified symbiosomes were able
to accumulate zinc, supplied as a 20 µM radiolabelled zinc chloride solution, and zinc uptake was
linear for up to two min. Zinc uptake by isolated symbiosomes responded to the concentration
added and showed saturation kinetics (Fig. 8B). The apparent Km was 91 µM, somewhat higher
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than that observed with GmZIP1-expressing yeast cells. Over the concentration range tested,
there was no indication of more than one transport activity. Since other, as yet unidentified, metal
ion transporters could contribute to zinc uptake across the PBM, the GmZIP1 antiserum was
employed to confirm the involvement of GmZIP1. Isolated symbiosomes were pre-incubated
with GmZIP1 antibody 30 min prior to mixing with the radiolabelled solution. The interaction of
the antibody with GmZIP1 resulted in a 35 % (SE ± 11, n=15) inhibition of zinc uptake, using a
pre-incubation of the symbiosomes with the pre-immune fraction of the serum as a control (Fig.
9). This result indicates that a significant proportion of the zinc uptake observed in symbiosomes
is due to GmZIP1.
The effect of divalent cations on zinc uptake by symbiosomes was analysed by incubating the
organelles with a 10-fold excess of competitor metal together with 20 µM 65Zn. The results were
very similar to those obtained with yeast expressing GmZIP1; Cd2+ was able to severely inhibit
the accumulation of zinc in symbiosomes leading to a 70 % decrease in 5 min (Table 1). Cu2+
was also found to compete with zinc and caused a 30 % decrease in Zn uptake. Nonetheless, the
lack of complete inhibition by the GmZIP antibody, and the fact that Cu inhibited Zn uptake
partially into symbiosomes but not into yeast, may reflect the presence of multiple transport
systems for Zn on the PBM.
DISCUSSION
The PCR approach employed allowed the identification of GmZIP1, the first soybean member of
the zinc- and iron-transporter family. Southern blotting indicated the presence of other members
of a soybean ZIP family, but GmZIP1 was immunolocalised specifically to the PBM. The
inhibition of zinc uptake into symbiosomes by the antibody demonstrates that the activity
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measured in symbiosomes was at least partially due to the protein encoded by the cDNA.
GmZIP1 possesses putative sites of glycosylation, identified using the PROSITE program, and
proteins of the PBM are known to be highly glycosylated on their peribacteroid space side. An
interaction between these sugar groups facing the PBS and the bacteroid membrane has been
suggested and could help to synchronise the development of both the PBM and the bacteroid
membrane (33). GmZIP1 mRNA appears to be expressed in nodules after the onset of nitrogen
fixation, suggesting that the transporter could play a housekeeping role in zinc metabolism of
soybean nodules.
GmZIP1 shares both structural and functional homology with the zinc transporters of
Arabidopsis. Based on phylogenetic analysis, GmZIP1 is most closely related to AtZIP1, AtZIP3
and AtZIP5. The energy-dependent and pH-independent nature of GmZIP1-mediated uptake is
also reminiscent of zinc uptake mediated by AtZIP1 and AtZIP3. The Km value of 13 µM
calculated for GmZIP1 expressed in yeast is very close to the values found for AtZIP1 (13 µM)
and AtZIP3 (14 µM; Ref. 25) and is consistent with the level of zinc in soil. It has been estimated
that plants growing in a non-polluted soil and under neutral pH could be exposed to a zinc
concentration of about 50 µM (34).
GmZIP1 is predicted to have eight transmembrane segments, typical of the yeast ZRT and the
plant ZIP proteins. These are distinct from another family of zinc transporters whose members
only possess six transmembrane domains. This group includes, among others, the Zn/Cd and
cobalt yeast transporters and ZAT, another Arabidopsis zinc transporter. The latter group are
thought to be involved either in zinc efflux from cells or in zinc sequestration by vacuoles (35).
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Nramp proteins have also been identified in plants and these can transport zinc as well as other
divalent cations (36-39).
It is interesting to note the presence of an ATP synthase signature sequence on GmZIP1,
between amino acid residues 329 and 338 (identified by a PROSITE search, with a probability of
finding the exact motif of 0.8247E-06). This signature has been selected from the consensus
pattern identified in the CF(o) subunit of several ATP synthases and this subunit is known to be a
key component of the proton channel (40). According to this analysis, Arg333 in GmZIP1 would
correspond to the arginine residue important for the proton translocating activity of ATP
synthases (41). It is possible that H+ movement is also catalysed by GmZIP1 and could be linked
electrochemically to zinc movement across the PBM. No such motif was found on AtZIP1 and 3,
perhaps indicating a different transport function of GmZIP1.
The PBM is energised by a H+-pumping ATPase which generates a membrane potential positive
on the inside of the symbiosome (and an acidic interior if permeant anions are present; Ref. 42).
In this respect, the symbiosome resembles a vacuole. Studies with tonoplast vesicles have
suggested that zinc (and other divalent metal ions) enter the vacuole in exchange for H+ via an
antiport mechanism (34), perhaps catalysed by a homologue of the ZAT1 protein identified in
Arabidopsis (43). It is possible that Zn/H+ antiport also occurs across the PBM. Indeed, in
addition to the identified ZIP, a member of the ZAT family may also be present on the PBM
since there are many similarities between the symbiosome and the vacuole of plants. However,
when we tested the effect of ATP with or without permeant anions on Zn uptake by isolated
symbiosomes the results were variable with, on average, a small inhibition seen in the presence
of ATP. Dissipation of the membrane potential by permeant anions had no significant effect on
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Zn uptake (results not shown). Similar results were observed with ferric citrate uptake into
isolated symbiosomes (44). In this context, it is interesting to note that a human member of the
ZIP family, hZIP2, has been shown to be energy independent with a proposed Zn/HCO3 symport
mechanism (7).
The proposed mechanism of zinc transport via GmZIP1 raises an interesting problem with
respect to the orientation of the protein when expressed on the plasma membrane in yeast.
Clearly, in this situation, GmZIP1 catalyses uptake of zinc into the cell. However, uptake into
isolated symbiosomes is equivalent to export from the plant cell. If GmZIP1 has the same
physical orientation in the two membranes, which is likely considering that the secretory pathway
is thought to mediate protein insertion into the PBM, then GmZIP1 must be able to catalyse bi-
directional transport of zinc. This is not unusual for a carrier, but if Zn uptake into yeast is linked
directly to the proton gradient then GmZIP must be able to catalyse Zn/H+ symport as well as
antiport. Alternatively, zinc uptake could be linked to the membrane potential or pH gradient via
other ion movements. Further experiments on the two systems may provide new insights into the
mechanism of zinc transport in plants.
The difference in apparent Km of zinc uptake by the two systems (13 and 91 µM for the yeast and
symbiosome, respectively), may reflect different binding affinities on the two sides of the
transporter. Although, both values still fall within the scale of a low affinity plant system (45,
46) it is also possible that the higher Km reflects the participation of other transporters in Zn
movement across the PBM. That is, the true affinity of GmZIP1 may be higher than that
measured with isolated symbiosomes. The Zn concentration in the cytosol of nodule infected
cells is unknown but the calculated Km of GmZIP1 is similar to that calculated for Zn uptake into
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oat root tonoplast vesicles (34) . Nonetheless, it should be considered that GmZIP1 may also
function to export Zn from the symbiosome in vivo.
Of a spectrum of different metals tried, GmZIP1-dependent zinc uptake in yeast was inhibited
only by cadmium. This was also observed with purified symbiosomes. This inhibitory effect of
cadmium on zinc uptake is not restricted to GmZIP1. Other transporters are known to present this
dual Zn/Cd uptake capacity (47) and this can probably be accounted for by the very high
electronic homology between zinc and cadmium. In the symbiosome, Cu was also able to
compete with Zn transport to some extent (Table 1) and it is possible that in vivo GmZIP1 can
transport both ions. In fact, Eckhardt et al. (48) have shown that LeIRT1 and LeIRT2 can
complement a Cu mutant of yeast. In this context, it is interesting to note that a putative Cu/Zn
superoxide dismutase, SMc02597, has been identified recently on the chromosome of
Sinorhizobium meliloti (49) and SOD enzymes are thought to play key roles in bacteroid-plant
interactions (50). Nodules contain very high concentrations of iron and the inability of iron to
inhibit zinc uptake may, therefore, also be an important feature of GmZIP1.
Stabilisation of the metal via an electronic interaction with amino acid residues of GmZIP1 could
play an important role in the specificity of the transporter. Rogers et al (51) recently showed that
replacement of key aspartate (D100 and D136) residues of AtIRT1 with alanine, converted the
transporter into a form only able to take up zinc, while the wildtype enzyme also catalysed Fe and
Mn uptake. Interestingly, unlike strains carrying the D100A allele, the strain carrying the D136A
allele was no longer sensitive to 0.2 µM cadmium, indicating that the strain carrying this allele
transports less cadmium than strains carrying either the wild type IRT1 or the D100A allele.
GmZIP1 has both of these conserved aspartate residues and nonetheless is unable to transport Fe
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but is sensitive to cadmium. While the results of Rogers et al (51) indicate that the transport of
different metals are physically separable, it is also clear that substrate selectivity involves more
than just a few key amino acids. Each member of this transporter family must be analysed
separately to achieve precise engineering of activities. It would certainly be interesting to perform
a similar mutagenesis study on GmZIP1 and to analyse the effects of a modified specificity of the
metal transporter upon the symbiosis. For this purpose, residues Asp-104 or Asp-140 of GmZIP1
could be good candidates, as they are the soybean equivalents of Asp-100 or Asp-136 in AtIRT1.
Besides being a vital micronutrient for all organisms because of its cofactor role in many
enzymes, zinc is thought to play a role in signal-transduction and in gene regulation. In plants,
zinc has been shown to have a major role in the regulation of genes encoding high affinity
phosphate transporters in roots (52). This role is specific to zinc, as it cannot be replaced by Mn,
for example, and seems very important, as this tight control of P uptake is lost under Zn
deficiency (52-54). In this context, it has been established that nodulation and N2 fixation have a
high P requirement. At low nitrate concentration, increasing amounts of P promoted both nodule
formation and N2 fixation (55). At the microsymbiont level, the high P concentration present in
nodules (20-100 mM) switches exopolysaccharide production of Sinorhizobium meliloti from
galactoglucan (EPS II) to succinoglycan (EPS I). The lon mutant of S. meliloti, shown to
constitutively express EPS II, only forms pseudo-nodules, delayed in appearance and unable to
fix N2 (56). By controlling the P status in nodules, zinc could play a critical role in nodulation
and symbiosis.
Acknowledgments – We thank Dr. David Eide (University of Missouri-Columbia) for providing
the DEY1453 and ZHY3 yeast mutants, Dr. Emmanuel Lesuisse (Institut Jacques Monod – Paris)
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for the DY1455 yeast strain, and Joanne Castelli for expert technical assistance. This research
was supported by the Australian Research Council (DAD), the CNRS PICS program #637 (SM,
AP) and the Department of Energy, grant #DE-FG07-97ER20292 (MLG). RT was supported by
an Australian Postgraduate Research Award.
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FIGURES
Fig. 1 Predicted amino acid sequence of GmZIP1 and alignment with selected members of
the ZIP gene family. The multiple alignment was performed with ClustalW (57). Fully
conserved residues are boxed in black while semi-conservative substitutions are boxed in gray.
Putative transmembrane domains for GmZIP1, as defined by TMMTOP (28), are indicated with
bars and Roman numerals. Arabidopsis MIPS identification numbers are as follows: IRT1
(At4g19690), IRT2 (At4g19680), IRT3 (At1g60960), ZIP1 (At3g12750), ZIP2 (At5g59520),
ZIP3 (At2g32270), ZIP4 (At1g10970), ZIP5 (At1g05300), ZIP6 (At2g30080), ZIP7 (Genbank
AAD32923.1; MIPs number not yet assigned), ZIP8 (Genbank AB019224; gene is incorrectly
listed as a pseudogene), ZIP9 (At4g33020), ZIP10 (At1g31260), ZIP11 (At1g55910),
ZIP12(At5g62160). Other Genbank accession numbers are: Lycopersicon esculentum LeIRT1
(AF136579), LeIRT2 (AF136579) and Pisum sativum RIT1 (AF065444).
Fig. 2 Phylogenetic tree of selected ZIP transporters. Alignment of full length sequences and
sequence identifiers are as described in the legend for Fig.1. The tree and bootstrap values were
calculated using the neighbour joining algorithm implemented in MEGA version 2.0 (58). Values
indicate the number of times (in percent) that each branch topology was found in 500 replicates
of a bootstrap analysis, assuming a gamma distribution for amino acid substitutions.
Fig. 3 Complementation of yeast uptake-deficient strains on selective media. Yeast cells
were transformed with the empty vector pFL61 or with a vector encoding AtIRT1 or GmZIP1.
Yeast cultures were adjusted to the indicated OD600 and 2 µl were spotted on SD medium
supplemented with the indicated concentration of iron (panel A, iron-deficient mutant) or zinc
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(panel B, zinc-deficient mutant). Pictures were taken after incubating the plates at 30°C for 3
days. wildtype = DY1455, fet3fet4 mutant = DEY1453, zrt1zrt2 mutant = ZHY3.
Complementation is indicated by (+), inability to complement is indicated by (-).
Fig. 4 Zinc accumulation in GmZIP1-expressing ZHY3 cells. A. Temperature and energy-
dependency of zinc uptake. Zinc uptake experiments were performed in LZM-EDTA medium
containing 20 µM 65ZnCl2. Cell suspensions and reaction mixes were pre-equilibrated at 30°C
(■ ) or on ice (�), at least 20 min before starting the experiment. For glucose starvation
experiments (�), yeast cells were pre-incubated 1 hour at 30°C, in LZM-EDTA without glucose.
Zinc uptake was then performed at 30°C in the same medium. Assays were done in triplicate and
the uptake was repeated twice. Some error bars do not extend outside the data point symbol. B.
Concentration curve. 20-min zinc uptake was measured in the presence of increasing amounts
of ZnCl2 in the reaction medium (LZM-EDTA). Inset shows Lineweaver-Burk plot of the uptake
data with calculated Km = 13.8 µM and Vmax = 12.5 fmol/min/106 cells. Assays were done as in
A. and experiments were repeated 4 times. A representative experiment is shown.
Fig. 5 Tissue-specific expression of GmZIP and expression during nodule development. Left
and right panels: Northern blot analysis was performed using 1 µg poly A+ RNA isolated from
soybean nodules of 11-, 14-, 18- and 23-day old soybean plants as well as 23 day old soybean
roots, stems and leaves. Hybridisation was with DIG-labelled GmZIP1 DNA.
Central panel: Northern blot was performed with 20 µg of total RNA isolated from nodules of
23-, 28-, 35-, 42-day old soybean plants. Hybridisation was with a 32P-labelled GmZIP1
riboprobe.
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Fig. 6 Immunolocalization of ZIP proteins to soybean membrane fractions. A. AtIRT1
antibody detects several soybean ZIP proteins. AtIRT1 antibody bound to several soybean ZIP
nodule microsome proteins (Lane 1; 2-min exposure) and less strongly to a protein on the
peribacteroid membrane (Lane 2; 10-min exposure). B. Immunolocalization of GmZIP1
protein is specific to the peribacteroid membrane of soybean nodules. The polyclonal
antibody raised in rabbit against GmZIP1 (see Experimental Procedures) was used to detect
immuno-reactive proteins isolated from peribacteroid membranes (Lane 1), nodule (Lane 2) or
root microsomal fractions (Lane 3). Cross-reactions were revealed by chemiluminescence and 2-
min exposure on a luminometer. C. GmZIP1 protein is found in the peribacteroid membrane
of plants from 4-7 weeks of age. Anti-GmZIP1 antibody was used to detect GmZIP1 protein
from peribacteroid membrane isolated from plants aged 4 weeks (Lane 1), 6 weeks (Lane2) and 7
weeks (Lane 3). Luminometer exposure was for 5-min. Sizes of molecular weight markers are
indicated on the right.
Fig. 7 Indication of a ZIP multigene family in soybean. Southern blot analysis was performed
with 5 µg of genomic DNA from soybean leaves. Genomic DNA was digested with BglII (lane
1), EcoRI (lane 2), Hind III (lane 3) or NotI (lane 4), separated on a 0.8 % agarose gel, blotted
onto nylon membrane and hybridised with DIG-labelled GmZIP1 DNA. The series of molecular
weight markers are shown in kilobase pairs.
Fig. 8 Zinc accumulation in isolated symbiosomes. A. Time-course accumulation of zinc in
symbiosomes. Percoll gradient-isolated symbiosomes were incubated at 30°C in mannitol buffer
supplemented with 20 µM 65ZnCl2. Experiments were performed on 2 independent preparations,
with triplicates for each data point. Average values are shown with error bars representing SE
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(n=6). 8B. Concentration-dependent Zn2+ uptake by symbiosomes. Five-minute uptake
experiments were performed at 30°C using substrate concentrations from 5 to 300 µM total Zn.
Values from control experiments conducted on ice were subtracted. The inset graph is a
Lineweaver-Burk plot of the data giving a calculated Km = 91 µM. The data are from two
independent experiments done in triplicate; the values and error bars are the mean and SE (n = 5
or 6), respectively.
Fig. 9. Effect of GmZIP1 antiserum on zinc uptake into isolated symbiosomes. Five-minute
uptake experiments were performed using symbiosomes isolated from 4-5 week-old plants.
Symbiosomes were preincubated with either preimmune serum or GmZIP1 antiserum before
measuring the rate of uptake of 65Zn. The data represent averages +/- SE (n=15) and results from
Student’s t-test give p=0.035.
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% inhibition of zinc uptake
Metal tested Yeast Symbiosomes
Zn
Cd
Cu
Fe
Mn
Ni
Co
Ca
66.6
51.8
8.4
7.5
<1
<1
<1
n/a
68.2
69.5
32.3
2.4
19.8
15.5
6.3
22.9
Table 1. Effect of divalent metal competition on yeast and symbiosomes zinc uptake. A 10-
fold excess of non-labelled metal was added to the incubation mix containing 20 µM 65ZnCl2.
Experiments were performed in triplicate and repeated twice. (n/a : not available) Standard errors
were always <15 %.
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I ____________ GmZIP1 1 ----------MKRFHSDSKFFTFSILIFLVVLPTLVVA------ECTCDREDE----ERDKSKALRYKIAALVSILVAGA IRT3 1 MFFVDVLWKLVPLYLFGSETKSLSATESILQIVPEAMAA--TSSNVLCNASE-S-DLCRDDSAAFLLKFVAIASILLAGA ZIP4 1 MIFVDVLWKLFPLYSFGSGRD--SLSESILQIIPETMAS--STTKILCDAGE-S-DLCRDDSAAFLLKFVAIASILLAGA ZIP9 1 MSLLQDFWQFLRPLSSGLTAK------LTQFIFACNKT---ESFTTSCDSGD-S-DPCRDDAAALTLKFAAMASILISGA IRT1 1 ------------------MKTIFLVLIFVSFAISPATS----TAPEECGSES-A-NPCVNKAKALPLKVIAIFVILIASM ZIP8 1 -----------MATTTQHMNQIFLVLLLISFAISPAIS----TVPKECETDS-T-DSCIDKTKALPLKIVAIVAILVTSM IRT2 1 ---------------MATTKLVYILLILFTFTVSPAIS----TAPEHCDSGF-D-NPCINKAKALPLKIVAIVAILTTSL LeIRT2 1 --------------MSDYNFKHIAIIFILISIFIPRVL----SVVEDCGAQE-D-NSCVNKSKALPLKIIAIVSILITSM LeIRT1 1 --------------MANYNFKYIAIFLLLISILAPRVL----SVVEDCGAEE-D-NSCVNKSKALPLKIIAIVSILITSM RIT1 1 ---------------MANPVTKQKLISIVFILITLFTS----QALADCETES-T-NSCNNKEKALSLKIIAIFSILVTSM ZIP10 1 ----------MTKSHVIFSASIALFLLLSISHFPGALS----QSNKDCQSKS-N-YSCIDKNKALDLKLLSIFSILITSL ZIP7 1 -------MAYSKACYKLTTITILLLSFTLPSLAGNAEN----ADVSECKAESGD-LSCHNNKEAQKLKIIAIPSILVASM ZIP1 1 ----------MSECGCFSATTMLRICVVLIICLHMCCA------SSDCTSHDDP-VSQDEAEKATKLKLGSIALLLVAGG ZIP3 1 ----------MKTKNVKLLFFFFSVSLLLIAVVNAAEGHSHGGPKCECSHEDD----HENKAGARKYKIAAIPTVLIAGI ZIP5 1 ---------MRITQNVKLLLFFFFFISFLFIAVSAGE-----S-KCECSHEDD----EANKAGAKKYKIAAIPSVLAAGV ZIP12 1 ----------MSRFRKTLVSAFVLCLVIFPLLVSAAEE------ENQCGGSK-G-GSAAEKASALKYKIIAFFSILIAGV ZIP2 1 -------MALSSKTLKSTLFFLSIIFLCFSLILAHGGID---DGDEEEETNQPPPATGTTTVVNLRSKSLVLVKIYCIII ZIP11 1 --------------MSRSLVFFFLFLVLVVPCLSHGTGG---DHDDDEASHVKS--------SDLKSKSLISVKIACLVI ZIP6 1 -------------------------MASCVTGTEAAIR------AAACRDGE----------EASHLKIVAVFAIFLTSV II III _________ _____________________ ______________ GmZIP1 61 IGVCIPLLGKVISA-LSPEKDTFFIIKAFAAGVILSTGFIHVLPDAFENLTSPCLKE-HPWG---EFPFTGFVAMCTAMG IRT3 77 AGVTIPLIGRNRRF-LQTDGNLFVTAKAFAAGVILATGFVHMLAGGTEALKNPCLPD-FPWS---KFPFPGFFAMIAALI ZIP4 75 AGVAIPLIGRNRRF-LQTEGNLFVAAKAFAAGVILATGFVHMLAGGTEALSNPCLPD-FPWS---KFPFPGFFAMVAALA ZIP9 70 AGVSIPLVG---TL-LPLNGGLMRGAKAFAAGVILATGFVHMLSGGSKALSDPCLPE-FPWK---MFPFPEFFAMVAALL IRT1 57 IGVGAPLFSRNVSF-LQPDGNIFTIIKCFASGIILGTGFMHVLPDSFEMLSSICLEE-NPWH---KFPFSGFLAMLSGLI ZIP8 64 IGVAAPLFSRYVTF-LHPDGKIFMIIKCFASGIILGTGFMHVLPDSFEMLSSPCLED-NPWH---KFPFTGFVAMLSGLV IRT2 60 IGVTSPLFSRYISF-LRPDGNGFMIVKCFSSGIILGTGFMHVLPDSFEMLSSKCLSD-NPWH---KFPFAGFVAMMSGLV LeIRT2 61 IGVCLPLVTRSIPA-LSPERNLFVIVKAFAAGIILATGFMHVLPDSFDMLSSSCLKE-NPWH---KFPFTGFVAMLSAIV LeIRT1 61 IGVCLPLVTRSIPA-LSPERNLFVIVKAFAAGIILATGFMHVLPDSFDMLSSSCLKE-HPWH---KFPFTGFVAMLSAIV RIT1 60 IGVCLPLVSRSVPA-LSPDGNLFVIVKCFAAGIILGTGFMHVLPDSFDMLWSDCLQE-KPWH---EFPFSGFAAMISAVV ZIP10 65 IGVCLPFFARSIPA-FQPEKSHFLIVKSFASGIILSTGFMHVLPDSFEMLSSPCLND-NPWH---KFPFAGFVAMMSAVF ZIP7 69 IGVSLPLFSRSIPA-LGPDREMSVIVKTLASGVILATGFMHVLPDSFDDLTSKCLPE-DPWQ---KFPFATFITMISALL ZIP1 64 VGVSLPLIGKRIPA-LQPENDIFFMVKAFAAGVILCTGFVHILPDAFERLSSPCLED-TTAG---KFPFAGFVAMLSAMG ZIP3 67 IGVLFPLLGKVFPS-LRPETCFFFVTKAFAAGVILATGFMHVLPEAYEMLNSPCLTS-EAW----EFPFTGFIAMIAAIL ZIP5 62 IGVMFPLLGKFFPS-LKPETTFFFVTKAFAAGVILATGFMHVLPEGYEKLTSPCLKG-EAW----EFPFTGFIAMVAAIL ZIP12 63 FGVCLPIFG------LKTESNFFMYVKAFAAGVILATGFVHILPDATESLTSSCLGE-EPPWG--DFPMTGLVAMAASIL ZIP2 71 LFFSTFLAGVSPYF-YRWNESFLLLGTQFSGGIFLATALIHFLSDANETFRGLKHK---------EYPYAFMLAAAGYCL ZIP11 56 IFVLTFISGVSPYF-LKWSQGFLVLGTQFAGGVFLATALMHFLSDADETFRGLLTAE-GESEPSPAYPFAYMLACAGFML ZIP6 40 FGVWGPVLLAKYFHGKPLYDKAILVIKCFAAGVILSTSLVHVLPEAFESLADCQVSSRHPWK---DFPFAGLVTMIGAIT ____ GmZIP1 136 TLMVDTYATAYFKKHHHSQ---D-EATDVEKE-------------------SGHEGHVHLHTHATHGHAHGHVP-----T IRT3 152 TLFVDFMGTQYYERKQEREASESVEPFGREQSPGIVVPMIGEGTNDGKVFGEEDSGGIHIVGIHAHAAHHRHSHPPGHDS ZIP4 150 TLLVDFMGTQYYERKQERNQAATEAAAGSEE--IAVVPVVGERVTDNKVFGEEDGGGIHIVGIRAHAAHHRHSHSNSHGT ZIP9 142 TLLADFMITGYYERKQEKMMNQSVESLGTQVS---VMSDPGLES--GFLRDQEDGGALHIVGMRAHAEHHRHSLSMGAEG IRT1 132 TLAIDSMATSLYTSKNAVGIMP--------------------------------HGHGHGHGPANDVTLPIKE------- ZIP8 139 TLAIDSIATSLYTKKAVADDSE--------------------------------ERTTPMIIQIDHLPLTTKER------ IRT2 135 TLAIDSITTSLYTGKNSVGPVPDEEYGI-DQE-------------------KAIHMVGHNHSHGHGVVLATKD------- LeIRT2 136 TMAIDSIATSMYSKKHRAGLVNPETGG---AD-------------------QEMGAVNGGHSHHHHGSLSTKDG------ LeIRT1 136 TMAIDSIATSLYSKKHNGGVVNPEG-----DQ-------------------EMAVAGNHVHSHHHHGSLSTKDG------ RIT1 135 TMMVDSLATSYYTQKGKKGVIIPAEG-------------------------EVGDQEMGAVHAGHHHHYQVKTE------ ZIP10 140 TLMVDSITTSVFTKSGRKDLRADVAS----VE-------------------TPDQEIGHVQVHGHVHSHTLPHN------ ZIP7 144 VLMIESFAMCAYARRTSKREGEVVPLENGSNS-------------------VDTQNDIQTLENGSSYVEKQEKV------ ZIP1 139 TLMIDTFATGYYKRQHFSNNHGSKQVNVVVDE-------------------EEHAGHVHIHTHASHGHTHG--------- ZIP3 141 TLSVDTFATSSFYKSHCKAS---KRVSDGETG-------------------ESS-------------------------- ZIP5 136 TLSVDSFATSYFHKAHFKTS---KRIGDGEEQ-------------------DAGGGGGGGDELGLHVHAHGHTHGIVGVE ZIP12 134 TMLIESFASGYLNRSRLAKEGKTLPVSTG-GE-------------------EEHAHTGSAHTHASQGHSHGSLLIP---- ZIP2 141 TMLADVAVAFVAAGSNNNHVGASVGE----SR-------------------EDDDVAVKEEGRREIKSGVDVSQ------ ZIP11 134 TMLADSVIAHIYSKTQN-------------------------------------DLELQGEDKSNQRS---A-------- ZIP6 117 ALLVDLTASEHMGHGGGGGGDGGMEYMPVG----------------------KAVGGLEMKEGKCGADLEIQENSE----
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IV V ____________________ __________ GmZIP1 188 DDD---------------------------QSSELLRHRVISQVLEVGIIVHSIIIGISLGASESPKTIRPLMAALIFHQ IRT3 232 CEGHSKIDIGHAHAHGHGHGHGHGHVHGGLDAVNGARHIVVSQVLELGIVSHSIIIGLSLGVSQSPCTIRPLIAALSFHQ ZIP4 228 CD------G-------HAHGHSHGHMHGNSDVENGARHVVVSQILELGIVSHSIIIGLSLGVSQSPCTIRPLIAALSFHQ ZIP9 217 FEALSKRSG------VSGHGHGHSHGHGDVGLDSGVRHVVVSQILEMGIVSHSIIIGISLGVSHSPCTIRPLLLALSFHQ IRT1 173 DDS---------------------------SNAQLLRYRVIAMVLELGIIVHSVVIGLSLGATSDTCTIKGLIAALCFHQ ZIP8 181 SST---------------------------CSKQLLRYRVIATVLELGIIVHSVVIGLSLGATNDTCTIKGLIAALCFHQ IRT2 188 -------------------------------DGQLLRYQVIAMVLEVGILFHSVVIGLSLGATNDSCTIKGLIIALCFHH LeIRT2 188 VE-----------------------------GTKLLRYRVIAMVLELGIIVHSIVIGISLGASNNTCTIKGLVAALCFHQ LeIRT1 186 LD-----------------------------GKKLLRYRVIAMVLELGIIVHSIVIGLSLGASSNTCTIKGLVAALCFHQ RIT1 184 GE-----------------------------ESQLLRYRVIAMVLELGIVVHSIVIGLAMGSSNNTCSIKGLVAALCFHQ ZIP10 191 LH-----------------------------GENDKELGSYLQVLELGIVVQSIVIGLSVGDTNNTCTIKGLVAALCFHQ ZIP7 199 NED---------------------------KTSELLRNKVIAQILELGIVVHSVVIGLAMGASDNKCTVQSLIAALCFHQ ZIP1 191 -------------------------------STELIRRRIVSQVLEIGIVVHSVIIGISLGASQSIDTIKPLMAALSFHQ ZIP3 173 VDS---------------------------EKVQILRTRVIAQVLELGIIVHSVVIGISLGASQSPDAAKALFIALMFHQ ZIP5 194 SGE---------------------------SQVQLHRTRVVAQVLEVGIIVHSVVIGISLGASQSPDTAKALFAALMFHQ ZIP12 190 -QD---------------------------DDHIDMRKKIVTQILELGIVVHSVIIGISLGASPSVSTIKPLIAAITFHQ ZIP2 192 -------------------------------ALIRTSGFGDTALLIFALCFHSIFEGIAIGLSDTKSDAWRNLWTISLHK ZIP11 166 ---------------------------------TTETSIGDSILLIVALCFHSVFEGIAIGISETKSDAWRALWTITLHK ZIP6 171 ------------------------------EEIVKMKQRLVSQVLEIGIIFHSVIIGVTMGMSQNKCTIRPLIAALSFHQ VI VII __________ __________________ _______________ GmZIP1 241 FFEGMGLGSCITQANFK--KLSITLMGLVFALTTPMGIGIGIGITK--VYDENSPTALIVEGIFNAASAGILIYMALVDL IRT3 312 FFEGFALGGCISQAQFR--NKSATIMACFFALTTPIGIGIGTAVAS--SFNSHSVGALVTEGILDSLSAGILVYMALVDL ZIP4 295 FFEGFALGGCISQAQFR--NKSATIMACFFALTTPLGIGIGTAVAS--SFNSHSPGALVTEGILDSLSAGILVYMALVDL ZIP9 291 FFEGFALGGCVAEARLT--PRGSAMMAFFFAITTPIGVAVGTAIAS--SYNSYSVAALVAEGVLDSLSAGILVYMALVDL IRT1 226 MFEGMGLGGCILQAEYT--NMKKFVMAFFFAVTTPFGIALGIALST--VYQDNSPKALITVGLLNACSAGLLIYMALVDL ZIP8 234 MFEGMGLGGCILQAEYT--NVKKFVMAFFFAVTTPSSIALGIALSS--VYKDNSPTALITVGLLNACSAGLLIYMALVDL IRT2 237 LFEGIGLGGCILQADFT--NVKKFLMAFFFTGTTPCGIFLGIALSS--IYRDNSPTALITIGLLNACSAGMLIYMALVDL LeIRT2 239 MFEGMGLGGCILQAEYK--FLKKTLMAFFFAVTTPFGIALGMALST--TYEETSPRALITVGLLNASSAGLLIYMALVDL LeIRT1 237 MFEGMGLGGCILQAEYK--FMKKAIMAFFFAVTTPFGIALGIALST--TYEENSPRALITVGLLNASSAGLLIYMALVDL RIT1 235 MFEGMGLGGCILQAEYK--FVKKAIMVFFFSITTPLGIAIGIAMSS--NYKENSPKALITVGLLNGSSAGLLIYMALVDL ZIP10 242 MFEGMGLGGCILQAEYG--WVKKAVMAFFFAVTTPFGVVLGMALSK--TYKENSPESLITVGLLNASSAGLLIYMALVDL ZIP7 252 LFEGMGLGGSILQAQFK--SKTNWTMVFFFSVTTPFGIVLGMAIQK--IYDETSPTALIVVGVLNACSAGLLIYMALVNL ZIP1 240 FFEGLGLGGCISLADMK--SKSTVLMATFFSVTAPLGIGIGLGMSSGLGYRKESKEAIMVEGMLNAASAGILIYMSLVDL ZIP3 226 CFEGLGLGGCIAQGKFK--CLSVTIMSTFFAITTPIGIVVGMGIAN--SYDESSPTALIVQGVLNAASAGILIYMSLVDL ZIP5 247 CFEGLGLGGCIAQGNFN--CMSITIMSIFFSVTTPVGIAVGMAISS--SYDDSSPTALIVQGVLNAASAGILIYMSLVDF ZIP12 242 LFEGFGLGGCISEAKFR--VKKIWVMLMFFALTAPIGIGIGIGVAE--IYNENSPMALKVSGFLNATASGILIYMALVDL ZIP2 241 VFAAVAMGIALLKLIPKRPFFLTVVYSFAFGISSPIGVGIGIGINA----TSQGAGGDWTYAISMGLACGVFVYVAVNHL ZIP11 213 IFAAIAMGIALLRMIPDRPLFSSITYSFAFAISSPIGVAIGIVIDA----TTQGSIADWIFALSMSLACGVFVYVSVNHL ZIP6 221 IFEGLGLGGCIAQAGFK--AGTVVYMCLMFAVTTPLGIVLGMVIFAATGYDDQNPNALIMEGLLGSFSSGILIYMALVDL
VIII _____ ____________________ GmZIP1 317 LAADFMN-----PRMQKSGSLRLGANLSLLLGAGCMSLLAKWA- IRT3 388 IAADFLS-----TKMRCNFRLQIVSYVMLFLGAGLMSSLAIWA- ZIP4 371 IAADFLS-----KRMSCNLRLQVVSYVMLFLGAGLMSALAIWA- ZIP9 367 IAADFLS-----KKMSVDFRVQVVSYCFLFLGAGMMSALAIWA- IRT1 302 LAAEFMG-----PKLQGSIKMQFKCLIAALLGCGGMSIIAKWA- ZIP8 310 LAAEFMG-----SMLQRSVKLQLNCFGAALLGCGGMSVLAKWA- IRT2 313 LATEFMG-----SMLQGSIKLQIKCFTAALLGCAVMSVVAVWA- LeIRT2 315 LAADFMG-----DKLQGSVKLQIKSYMAVLLGAGGMSLMAKWA- LeIRT1 313 LAADFMG-----DKLQGSVKLQIKSYMAVLLGAGGMSVMAIWA- RIT1 311 LAADFMS-----RRMQGSIKLQLKSYVAVFLGAGGMSLMAKWA- ZIP10 318 LAADFMG-----QKMQRSIKLQLKSYAAVLLGAGGMSVMAKWA- ZIP7 328 LAHEFFG-----PKIQGNIKLHVLGYVATFTGAAGMSLMAKWA- ZIP1 318 LATDFMN-----PRLQSNLWLHLAAYLSLVLGAGSMSLLAIWA- ZIP3 302 LAADFTH-----PKMQSNTGLQIMAHIALLLGAGLMSLLAKWA- ZIP5 323 LAADFMH-----PKMQSNTRLQIMAHISLLVGAGVMSLLAKWA- ZIP12 318 VAPLFMN-----QKTQSSMKIQVACSVSLVVGAGLMSLLAIWA- ZIP2 317 ISKGYK------PREECYFDKPIYKFIAVFLGVALLSVVMIWD- ZIP11 289 LAKGYR------PNKKVHVDEPRYKFLAVLFGVVVIAIVMIWDT ZIP6 299 IALDFFHNKMLTTCGESGSRLKKLCFVALVLGSASMSLLALWA-
Figure 1
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Michael K. Udvardi, Alain Puppo and David A. DaySophie Moreau, Rowena M. Thomson, Brent N. Kaiser, Ben Trevaskis, Mary Lou Guerinot,
GmZIP1 encodes a symbiosis specific zinc transporter in soybean
published online November 12, 2001J. Biol. Chem.
10.1074/jbc.M106754200Access the most updated version of this article at doi:
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