1

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: dday@cyllene.uwa.edu.au

Running title: GmZIP1 is a zinc transporter in soybean nodules

GenBank Accession Number for GmZIP1: AY029321

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

7

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,

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

10

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11

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.

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

12

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

13

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

14

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

15

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).

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

16

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

17

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

18

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

19

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)

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

20

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.

REFERENCES

1. Clarke, N. D., and Berg, J. M. (1998) Science 282, 2018-2022

2. Ruel, M. T., and Bouis, H. E. (1998) Am. J. Clin. Nutr. 68, 488S-494S

3. Eide, D. J. (1998) Annu. Rev. Nutr. 18, 441-469

4. Fox, T. C., and Guerinot, M. L. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 669-696

5. McMahon, R. J., and Cousins, R. J. (1998) Am. Soc. Nutr. Sci. 3166, 667-670

6. Nelson, N. (1999) EMBO J. 18, 4361-4371

7. Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564

8. Guerinot, M. L. (2000) Biochim. Biophys. Acta 1465, 190-198

9. MacDiarmid, C. W., Gaither, L. A., and Eide, D. (2000) EMBO J. 19, 2845-2855

10. Pence, N. S., Larsen, P. B., Ebbs, S. D., Letham, D. L., Lasat, M. M., Garvin, D. F., Eide, D.,

and Kochian, L. V. (2000) Proc Natl Acad Sci U S A. 97, 4956-4960

11. Assunçao, A. G. L., da Costa Martins, P., de Folter, S., Vooijs, R., Schat, H., and Aarts, M.

G. M. (2001) Plant Cell Environ. 24, 217-226

12. Roth, L. E., Jeon, K., and Stacey, K. (1988) Molecular genetics of plant-microbe interactions.

Eds Palacios R. and Verma D.P.S., APS Press, St Paul, pp. 220-225

13. Whitehead, L.F., and Day, D.A. (1997) Physiol. Plant. 100, 30-44

14. Day, D.A., Kaiser, B.N., Thomson, R., Udvardi, M.K., Moreau, S., and Puppo, A. (2001)

Aust. J. Plant Physiol., 28, 667-674

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

21

15. Minet, M., Dufour, M. E., and Lacroute, F. (1992) Plant J. 2, 417-422

16. Gietz, R. D., and Schiestl, R. H. (1995) Methods Mol. Cell. Biol. 5, 255-269

17. Zhao, H., and Eide, D. (1996) Proc. Natl. Acad. Sci. USA 93, 2454-2458

18. Puppo, A., Dimitrijevic, L., and Rigaud, J. (1982) Planta 156, 374-379

19. Day, D. A., Price, G. D., and Udvardi, M. K. (1989) Aust. J. Plant Physiol. 16, 69-84

20. Herrada, G., Puppo, A., and Rigaud, J. (1992) Biochem. Biophys. Res. Commun. 184, 1324-

1330

21. Hurkman, W. L., and Tanaka, C. K. (1986) Plant Physiol. 81, 802-806

22. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and

Struhl, K. (2000) Current Protocols in Molecular Biology. Wiley, New York

23. Eide, D., and Guarente, L. (1992) J. Gen. Microbiol. 138, 347-354

24. Laemmli, U. K. (1970) Nature 227, 680-685

25. Grotz, N., Fox, T., Connolly, E., Park, W., Guerinot, M. L., and Eide, D. (1998) Proc. Natl.

Acad. Sci. USA 95, 7220-7224

26. Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) Proc. Natl. Acad. Sci. USA 95,

5857-5864

27. Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P., and Bork, P. (2000) Nucleic Acids Res.

28, 231-234

28. Tusnády, G.E., and Simon, I. (1998) J. Mol. Biol. 283, 489-506

29. Eng, B. H., Guerinot, M. L., Eide, D., and Saier, M. H. Jr (1998) J. Membrane Biol. 166, 1-7

30. Zhao, H., and Eide, D. (1996) J. Biol. Chem. 271, 23203-23210

31. Korshunova, Y. O., Eide, D., Clark, W. G., Guerinot, M. L., and Pakrasi, H. B. (1999) Plant

Mol. Biol. 40, 37-44

32. Reid, R. J., Brookes, J. D., Tester, M. A., and Smith, F. A. (1996) Planta 198, 39-45

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

22

33. Robertson, J. G., and Lyttleton, P. (1984) J. Cell Sci. 69, 147-157

34. Gonzalez, A., Koren'kov, V., and Wagner, G. J. (1999) Physiol. Plant. 106, 203-209

35. Guerinot, M. L., and Eide, D. (1999) Curr. Opin. Plant Biol. 2, 244-249

36. Belouchi, A., Kwan, T., and Gros, P. (1997) Plant Mol. Biol. 33, 1085-1092

37. Alonso, J.M., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J.R. (1999) Science 284,

2148-2152

38. Curie, C., Alonso, J.M., Le Jean, M., Ecker, J.R., and Briat, J.F. (2000) Biochem. J. 347, 749-

755

39. Thomine, S., Wang, R., Ward, J.M., Crawford, N.M., and Schroeder, J.I. (2000) Proc.Natl.

Acad. Sci. USA 97, 4991-4996

40. Lewis, M. L., Chang, J. A., and Simoni, R. D. (1990) J. Biol. Chem. 265, 10541-10550

41. Cain, B. D., and Simoni, R. D. (1989) J. Biol. Chem. 264, 3292-3300

42. Udvardi, M. K., and Day, D. A. (1997) Annu. Rev. Plant Physiol. Plant Molec. Biol. 48, 493-

523

43. van der Zaal, B. J., Neuteboom, L. W., Pinas, J. E., Chardonnens, A. N., Schat, H., Verkleij,

J. A. C., and Hooykaas, P. J. J. (1999) Plant Physiol. 119, 1047-1055

44. Le Vier, K. D., Day, D. A., and Guerinot, M. L. (1996) Plant Physiol. 111, 893-900

45. Hart, J. J., Norvell, W. A., Welch, R. M., Sullivan, L. A., and Kochian, L. V. (1998) Plant

Physiol. 118, 219-226

46. Hacisalihoglu, G., Hart, J. J., and Kochian, L. V. (2001) Plant Physiol. 125, 456-463

47. Pence, N. S., Larsen, P. B., Ebbs, S. D., Letham, D. L. D., Lasat, M. M., Garvin, D. F., Eide,

D., and Kochian, L. V. (2000) Proc. Natl. Acad. Sci. USA 97, 4956-4960

48. Eckhardt, U., Marques, A.M., and Buckhout, T.J. (2001) Plant Mol. Biol. 45, 437-448

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

23

49. Capela, D., Barloy-Hubler, F., Gouzy, J., Bothe, G., Ampe, F., Batut, J., Boistard, P., Becker,

A., Boutry, M., Cadieu, E., Dréano, S., Gloux, S., Godrie, T., Goffeau, A., Kahn, D., Kiss, E.,

Lelaure, V., Masuy, D., Pohl, T., Portetelle, D., Pühler, A., Purnelle, B., Ramsperger, U., Renard,

C., Thébault, P., Vandenbol, M., Weidner, S., and Galibert, F. (2001) Proc. Natl. Acad. Sci. USA

98, 9877-9882

50. Santos, R., Herouart, D., Puppo, A., and Touati, D. (2000) Mol. Microbiol. 38, 750-759

51. Rogers, E. E., Eide, D. J., and Guerinot, M. L. (2000) Proc. Natl. Acad. Sci. USA 97, 12356-

12360

52. Huang, C., Barker, S. J., Langridge, P., Smith, F. W., and Graham, R. D. (2000) Plant

Physiol. 124, 415-422

53. Cakmak, I., and Marschner, H. (1986) Physiol. Plant. 68, 483-490

54. Webb, M. J., and Loneragan, J. F. (1988) Soil Sci. Soc. Am. J. 52, 1676-1680

55. Leidi, E. O., and Rodriguez-Navarro, D. N. (2000) New Phytol. 147, 337-346

56. Summers, M. L., Botero, L. M., Busse, S. C., and McDermott, T. R. (2000) J. Bacteriol. 182,

2551-2558

57. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680

58. Kumar, S., Tamura, K., Jacobsen, I., and Nei, M. (2000) Pennsylvania State University,

University Park, PA and Arizona State University, Tempe, AZ

59. OuYang, L-J, Udvardi, M.K. and Day, D.A. (1990) Planta 182, 437-444.

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

24

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

25

(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.

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

26

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

27

(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.

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

28

% 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 %.

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

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----

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

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

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

����������������� �

������������������

������������������

�� �������������� �

����������������� �

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

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:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from