©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of a Putative Arabidopsis thaliana Copper Transporter and Its Yeast Homologue (*)

(Received for publication, July 18, 1995; and in revised form, August 28, 1995)

Karlheinz Kampfenkel (§) Sergei Kushnir (¶) Elena Babiychuk (**) Dirk Inzé (1) Marc Van Montagu

From the Laboratorium voor Genetica and theLaboratoire Associé de l'Institut National de la Recherche Agronomique (France), Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

At the molecular level, little is known about the transport of copper across plant membranes. We have isolated an Arabidopsis thaliana cDNA by complementation of a mutant (ctr1-3) of Saccharomyces cerevisiae defective in high affinity copper uptake. This cDNA codes for a highly hydrophobic protein (COPT1) of 169 amino acid residues and with three putative transmembrane domains. Most noteworthy, the first 44 residues display significant homology to the methionine- and histidine-rich copper binding domain of three bacterial copper binding proteins, among these a copper transporting ATPase. Mutant yeast cells expressing COPT1 exhibit nearly wild type behavior with regard to growth on a nonfermentable carbon source and resistance to copper and iron starvation. Expression of COPT1 is also associated with an increased sensitivity to copper toxicity. Additionally, COPT1 shows significant homology to an open reading frame of 189 amino acid residues on yeast chromosome VIII. This gene (CTR2) may encode an additional yeast metal transporter able to mediate the uptake of copper. A mutation in CTR2 displays a higher level of resistance to toxic copper concentrations. Overexpression of CTR2 provides increased resistance to copper starvation and is also associated with an increased sensitivity to copper toxicity. The amino acid sequence of CTR2, like Arabidopsis COPT1, contains three potential transmembrane domains. Taken together, the data suggest that a plant metal transporter, which is most likely involved in the transport of copper, has been identified.


INTRODUCTION

Copper is a constituent of a great number of proteins and as such an essential micronutrient for plants. Copper-containing proteins act as terminal oxidase, mono- and dioxygenases, in the elimination of superoxide radicals as well as in electron transfer reactions, most notably in the process of respiration and photosynthesis(1) .

At the molecular level, our knowledge on the copper uptake in roots of higher plants is very rudimentary(2) . For the time being, neither a copper transporter of the cytoplasmic membrane nor of the inner membranes of mitochondria or chloroplasts has been identified. A number of uptake studies is consistent with a Michaelis-Menten saturation kinetic of copper transport across plant membranes(3, 4) . However, as pointed out by Graham(5) , it is not yet clear whether this is a protein-mediated diffusion process or an active transport.

To date, the only biochemically characterized eukaryotic copper tranporter known is the CTR1 protein that is required for high affinity copper uptake in Saccharomyces cerevisiae(6, 7) . The CTR1 gene encodes a plasma membrane protein of 406 amino acid residues and three potential transmembrane helices. The amino terminus of CTR1, which is unusually rich in methionine and serine residues, contains a repetitive motif that is also present in bacterial proteins involved in the handling of copper(6) . The analysis of CTR1 mutants revealed an unexpected relation between the uptake of copper and the ability of cells to accumulate iron. Copper is strictly required for high affinity iron uptake (6, 8) by a specific ferrous iron transporter(9) . As a prerequisite to permit continuous high affinity iron transport, an oxidation step of the ferrous to the ferric iron form is necessary. This oxidation is catalyzed through the transmembrane FET3 protein, a copper-containing oxidase(8, 10) , thus most likely explaining this copper requirement. While CTR1 mutants are impaired in the uptake of both copper and iron, FET3 mutants are defective in iron uptake only. This intimate connection between the uptake of these two metals is further demonstrated by the FRE1 gene(11, 12) . FRE1 encodes a multispanning plasma membrane protein that is required for reduction of ferric iron to ferrous iron, which is the actual transport form into the cell(13) . In fact, not only is the expression of FRE1 negatively regulated by both iron and copper(14) , but the FRE1 protein functions as a copper(II) reductase as well(15) .

Yeast mutants have proven to be a valuable tool in identifying new genes from homologous and heterologous sources via functional complementation. This approach was also effective in the identification and characterization of a number of plant membrane proteins that catalyze the transport of inorganic ions or organic molecules across plant membranes such as potassium channels(16, 17) , potassium transporters(18) , ammonium transporters(19) , sucrose transporters (20) , and amino acid permeases(21, 22) . This let us question whether yeast would be a good model organism to study copper and iron transport across plant membranes at the molecular level as well. In fact, the physiology of iron uptake by yeast is similar to the uptake of iron by roots of dicots and nongraminaceous monocots(23) . These plants, like yeast, reduce ferric iron before transport via a plasma membrane-bound iron(III) reductase and appear to have a ferrous iron transporter as well(24) . This suggests that yeast could be a suitable model organism to study the transport of iron and probably of copper as well, e.g. via the functional complementation of mutants perturbed in copper and iron acquisition across plant membranes.

We have isolated a cDNA from Arabidopsis thaliana that suppresses the mutant phenotype of a high affinity copper transport-deficient strain of S. cerevisiae. Characterization of this cDNA, the derived protein (designated COPT1 for Copper Transporter 1), and a corresponding gene (named CTR2) from yeast are described.


EXPERIMENTAL PROCEDURES

Enzymes and Biochemicals

Restriction endonucleases and DNA-modifying enzymes were purchased from Boehringer Mannheim. [alpha-P]dCTP (3000 Ci/mmol), [alpha-S]dATP (1000 Ci/mmol), and [-P]ATP (3000 Ci/mmol) were obtained from Amersham. Bacteriophage DNA and basal medium Eagle vitamin solution were from Life Technologies, Inc. Amino acids to supplement auxotrophies were from Sigma. Yeast nitrogen base without amino acids was from Difco Laboratories. The iron chelator 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4`,4"-disulfonic acid (ferrozine, monosodium salt) and the copper chelator bathocuproinedisulfonic acid (BCS, (^1)disodium salt) were from Fluka. Copper(II) sulfate-pentahydrate was obtained from Merck.

Strains and Growth Conditions

For functional complementation, S. cerevisiae strain 83 (MATa leu2-3, 112 gcn4-101 his3-609 ura3-52 ctr1-3), which is defective in high affinity copper uptake(6) , was used. As a related wild type strain CM3262 (MATa ino1-13 leu2-3, 112 gcn4-101 his3-609 ura 3-52) (6) was used. Strain KK3 (ctr2::HIS3, this study) is a derivative of strain CM3262. Strain KK4 (ctr1-3 ctr2::HIS3, this study) was derived from strain 83. The construction of the disruption mutants is described below. Escherichia coli strain XL1-Blue (F` (proAB lacI^qZDelta M15, Tn10 (tet^r)) recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac) (Stratagene) was used for cloning, maintenance, and propagation of plasmids. For growth of yeast under nonselective conditions, YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 2% (w/v) glucose) was used. Ura transformants were selected on SDC medium (0.67% (w/v) yeast nitrogen base without amino acids, 0.3% (w/v) casamino acids, 2% (w/v) glucose). The ability to grow with a nonfermentable carbon source was assessed by replacing the glucose in YPD with 3% (v/v) glycerol (YG medium). For growth of yeast under moderate iron starvation, the YNBFC medium (6) was used. This is a modified SD medium made from its components(25) , omitting iron and copper and adding MES buffer (50 mM (pH adjusted to 6.3 with KOH)). Vitamins were supplemented by adding 50 ml of basal medium Eagle vitamin solution per liter(11) . Nutrients required for growth of auxotrophic strains were added at the final concentrations indicated by Sherman et al.(25) . Uracil was omitted when selection for presence of a plasmid was necessary. To achieve more severe iron starvation, the ferrous iron chelator ferrozine was added to YNBFC at a concentration of 1 mM(6) . To obtain more severe copper starvation, the copper chelator BCS was added to YNBFC(7) . Copper-rich medium was obtained by adding CuSO(4) to YNBFC upon autoclaving. Yeast cells were grown at 30 °C either in liquid culture with vigorous shaking or on agar plates (media supplemented with 18 g of agar per liter). E. coli cells were grown in TY medium (8 g of bactotryptone, 5 g of yeast extract, and 5 g of NaCl per liter) with or without ampicillin (50 mg/liter).

Preparation of Library DNA and Yeast Transformation

The cDNA expression library derived from whole Arabidopsis seedlings in the yeast expression vector pFL61 (26) was a gift from Dr. Michèle Minet (CNRS, Gif-sur-Yvette, France). The library was electroporated into E. coli XL1-Blue cells and amplified by growing 3 times 10^6 colonies on TY agar plates in the presence of ampicillin. After overnight growth, cells were scraped off in a minimal volume of TY and harvested by centrifugation. Plasmid DNA was extracted by the alkaline lysis procedure and purified by CsCl centrifugation according to Sambrook et al.(27) . Yeast transformation was performed by the method of Dohmen et al.(28) . Transformants were selected on SDC, washed from the plates with water, and, in case of complementation of strain 83 (ctr1-3), subsequently selected on YG plates. Colonies able to grow were selected in liquid SDC, and plasmid DNA was isolated and transformed into E. coli as described(29) .

Plasmids and DNA Constructions

Genomic DNA from yeast strain CM3262 was isolated as described by Ausubel et al.(29) . The CTR2 gene has been amplified with Taq polymerase via the polymerase chain reaction (PCR) using the oligonucleotides 5`-gaattcaaaaagaattcGCACACGCGTGGC-3` (sequence given in capitals corresponds to the sense strand from position -47 to -35 relative to the start codon, and lower case letters correspond to EcoRI site) and 5`-aagcttaaaaagctTACGAAAAATCCCGG-3` (sequence in capitals is complementary to the sense strand from position 624 to 638, and lower case letters correspond to HindIII site). The CTR2 gene has been originally given the name YHR185c(30) , and the nucleotide sequence is available in the GenBank under the accession number U00030. The PCR product (0.715 kb) was gel purified, cleaved with EcoRI and HindIII, and cloned into the EcoRI- and HindIII-cut pBluescript II KS (Stratagene). The CTR2 nucleotide sequence was determined from the resulting plasmid, pKK35, on both strands using vector-specific primers (Stratagene) and found to be identical with the published sequence(30) . Plasmid pKK35 was cleaved with BglII at codon 120 of CTR2 and ligated with the HIS3 gene, cut out as a BamHI fragment (1.7 kb) from plasmid pFL39(31) . From the resulting plasmid, pKK37, the ctr2::HIS3 construct was cut out with BamHI and ApaI and used to transform strains CM3262 and 83. Homologous recombinants were selected on SD plates (25) omitting histidine. The genomic locus was examined by PCR on isolated genomic DNA to verify the correct integration. To overexpress CTR2 under the control of the phosphoglycerate kinase gene (PGK) promoter, a SmaI-HincII fragment of pKK35 was cloned into pFL61(26) , cleaved with NotI, and blunt ended with the Klenow fragment of deoxyribonuclease I in the presence of deoxynucleotides. The transcription polarity of the insert was examined by restriction analysis. The resulting plasmid has been named pKK52. Plasmid pKK31 is a pFL61 (26) derivative and contains the COPT1 cDNA under the control of the PGK promoter.

DNA Sequence Analysis

Double-stranded DNA was sequenced with the dideoxy chain termination method(32) . For sequencing of the COPT1 cDNA from plasmid pKK31, the oligonucleotides 5`-GTTTTCAAGTTCTTAGATGC-3` for reading from the PGK promoter toward the insert and 5`-AGCGTAAAGGATGGGG-3` for reading from the PGK terminator toward the insert (26) were used. Further sequencing on both strands was performed by primer walking. COPT1-specific primers were synthesized with an oligonucleotide synthesizer from Applied Biosystems.

Growth of Plants

Plants of A. thaliana ecotypes Columbia and Landsberg erecta were grown in sand at 23 °C with a photoperiod of 16 h light/8 h dark for 6 weeks before harvesting for the extraction of RNA and genomic DNA. Plants were watered with a nutrient solution of defined composition (33) .

Northern and Southern Analysis

Total RNA was extracted from A. thaliana organs entirely as described by Verwoerd et al.(34) . For Northern analysis, 10 µg of total RNA was denaturated with glyoxal according to Sambrook et al.(27) and electrophoresed on a 1.5% (w/v) agarose gel. HindIII-digested DNA was end labeled with polynucleotide kinase in the presence of [-P]ATP as in Sambrook et al.(27) and subjected to the same glyoxal blot. The RNA was fixed on a Hybond-N membrane (Amersham) by exposure to UV light for 3 min. The membrane was prehybridized for 30 min at 65 °C in 0.25 M sodium-phosphate buffer (pH 7.2), 1% (w/v) bovine serum albumin, 1 mM EDTA, 7% (w/v) SDS. Hybridization was performed for 16 h at 65 °C in the same solution plus the complete COPT1 cDNA (NotI fragment of 0.8 kb from pKK31) labeled by the random primer method with [alpha-P]dCTP using the Quick Prime Kit (Pharmacia Biotech Inc.) according to the supplier. Afterward, the membrane was washed two to three times at 58 °C for 15 min in 0.05 M sodium-phosphate buffer (pH 7.2), 0.5% (w/v) SDS and autoradiographed using X-OMAT films from Kodak. Genomic DNA was extracted from A. thaliana plants by the method of Mettler (35) and additionally purified by CsCl centrifugation according to Sambrook et al.(27) . DNA (2 µg) was digested with the appropriate restriction endonuclease and electrophoresed on a 0.7% (w/v) agarose gel. The P end-labeled HindIII-digested DNA served as length standard. Prehybridization, hybridization, and washing conditions were the same as described above.

Hydrophobicity Analysis

The hydropathy profiles of the proteins studied were obtained by applying the method of Kyte and Doolittle (36) with the program SOAP in PCGENE (IntelliGenetics, Inc., Mountain View, CA) using a window size of 11 residues. Potential transmembrane helices were predicted employing the method of Rost et al.(37) . (^2)


RESULTS

Complementation of the High Affinity Copper Uptake Mutant ctr1-3 from Yeast

The S. cerevisiae ctr1-3 mutant strain 83 is defective in high affinity copper uptake and high affinity ferrous iron uptake(6) . As a result, it is unable to grow on a nonfermentable carbon source and under conditions of iron or copper starvation(6, 7) . To identify plant copper transporter cDNAs, strain 83 was transformed with an episomal plasmid containing a cDNA library derived from Arabidopsis seedlings under the control of the PGK promoter(26) . Primary transformants (110,000 colonies) were selected on uracil-free medium, the cells were washed from the plates, and an aliquot was plated on medium containing glycerol as the sole carbon source. A cDNA with a size of 0.75 kb (pKK31) was able to complement the yeast mutation. To confirm that no reversion of the original mutation had occurred, plasmid pKK31 was isolated and retransformed into the copper transport mutant. Mutant cells transformed with pKK31 regained the ability to grow on the nonfermentable carbon source glycerol (Fig. 1A). The restriction of growth of ctr1-3 cells on plates either containing the copper chelator BCS to lower free copper concentration or the ferrous iron chelator ferrozine to decrease the free iron concentration was also complemented by the presence of pKK31 (Fig. 1B).


Figure 1: Functional complementation of the ctr1-3 mutant strain 83 of yeast by Arabidopsis COPT1. S. cerevisiae strains CM3262 and 83 (ctr1-3) untransformed or transformed with the vector pFL61 or plasmid pKK31 (COPT1 cDNA under the control of the PGK promoter) are shown. A, cells were streaked on a YG plate containing glycerol as a test for respiratory competence. Incubation lasted for 7 days at 30 °C. B, strains were grown overnight in SDC and, cell numbers were determined by spectrophotometry at 600 nm. Each culture was adjusted to an A of 1 and then serially 10-fold diluted in water (lanes 1-5). 20-µl aliquots of each strain and dilution were spotted onto YNBFC plates containing 1 mM ferrozine, 160 µM BCS, 900 µM CuSO(4), or 0 µM CuSO(4) (no addition of CuSO(4)) and incubated for 14 days, respectively, at 30 °C prior to photography.



Copper is toxic at elevated levels, which may relate to its ability to catalyze the formation of the extremely reactive hydroxyl radicals (38) . To further support the conclusion that the isolated Arabidopsis cDNA promotes copper uptake, strains 83 (pFL61) and 83 (pKK31) were examined for the effects of copper-mediated growth inhibition. The ctr1-3 strain harboring plasmid pKK31 was more sensitive to toxicity on a solid plate containing 900 µM CuSO(4) than the same strain transformed with the vector pFL61 only (Fig. 1B), indicating an efficient delivery of copper to the yeast cells. This putative metal uptake system that apparently mediates a copper transport has been designated COPT1.

Sequence Analysis of the Arabidopsis COPT1 cDNA

The COPT1 cDNA is 747 base pairs in length and contains one continuous open reading frame, which encodes a putative protein of 169 amino acid residues (Fig. 2). The 5`-proximal ATG triplet has been assumed to be the start codon since according to the ``first-AUG-rule'' it serves as the initiator codon to be used in the translation of about 95% of the eukaryotic mRNAs(39) . This assignment is supported by the nucleotide context around the start codon; AAACCATGG has a purine at position -3 from the initiator codon, which is another conserved feature of eukaryotic mRNAs(39) . In front of the start codon an in-frame stop codon is present in position -21 to -19, supporting the conclusion that the open reading frame is of full-length.


Figure 2: Nucleotide and predicted amino acid sequence of the A. thaliana COPT1 cDNA. The sequence presented corresponds to the COPT1 cDNA isolated in this study (from pKK31) via the functional complementation of the high affinity copper transporter mutant ctr1-3 of yeast. The EMBL sequence data bank accession number of the COPT1 cDNA sequence is Z49859.



A comparison of the nucleotide sequence with those in the EMBL nucleotide sequence data base and the Swiss-PROT sequence data base revealed a significant homology to only two proteins, a putative protein encoded by the open reading frame of YHR185c, recently identified by sequencing of yeast chromosome VIII(30) , and the QP protein from the protozoan parasite Theileria parva(40) . The function of these two proteins is unknown(30, 40) ; however, the yeast protein was tentatively named Copper Transporter 2 (CTR2) for reasons that will be elaborated in the following section. A sequence alignment of the predicted COPT1 protein of A. thaliana with the CTR2 protein from yeast (23.4% identity, 38.3% similarity) and the QP protein of T. parva (21.9% identity, 40.3% similarity) is shown in Fig. 3A. Whereas COPT1 and CTR2 are similar in length, the QP protein is much longer with 480 amino acid residues, of which only the carboxyl-terminal 180 residues display homology to COPT1 (Fig. 3A). Another region of sequence homology is covered by the first 44 amino-terminal residues of COPT1, which are enriched in methionine (25%) and histidine (16%) residues (Fig. 3B). A similar amino acid sequence is present in the amino terminus of the P-type ATPase CopB of Enterococcus hirae (34.8% identity, 56.5% similarity) (41) and in two proteins of Pseudomonas syringae, CopB (32.6% identity, 52.2% similarity) and CopA (26.1% identity, 58.7% similarity)(42) . These bacterial proteins are all involved in the handling of copper. The COPT1 protein of A. thaliana has no detectable homology to the high affinity copper transporter CTR1 from yeast.


Figure 3: Alignment of the predicted amino acid sequence of A. thaliana COPT1 protein in (A) with S. cerevisiae CTR2 and T. parva QP proteins and in (B) with putative copper binding sites in E. hirae CopB and the P. syringae CopB and CopA proteins. Eh, E. hirae; Ps, P. syringae. Numbers indicate amino acid positions. Identical amino acids are marked by asterisks, and similar amino acids are indicated by colons above the aligned sequences. The dots mark artificial sequence gaps introduced to improve the homologies between the proteins.



Analysis of the hydropathy profile of the amino acid sequence of the COPT1 protein using the method of Kyte and Doolittle (36) demonstrates that the A. thaliana protein is highly hydrophobic (Fig. 4). Thus, COPT1 is most likely an integral membrane protein. Employing the method of Rost et al.(37) , under the assumption that a stretch of 16 amino acid residues is sufficiently long to span the lipid bilayer(43) , revealed the presence of three potential transmembrane helices in the COPT1 protein (Fig. 4). The potential transmembrane helices extend in COPT1 from residues 63 to 84, 104 to 122, and 127 to 144 (Fig. 4). The distribution of hydrophilic and hydrophobic amino acids along the polypeptide chains is also very similar when comparing the CTR1 and QP protein with COPT1 (Fig. 4), which further supports their relatedness.


Figure 4: Hydropathy profiles of the amino acid sequences of the COPT1 protein from A. thaliana, the CTR2 protein from S. cerevisiae, and the QP protein from T. parva. The method of Kyte and Doolittle (36) was employed using a window size of 11 successive residues (hydrophilic, negative values; hydrophobic, positive values). In each case, the predicted three membrane-spanning alpha-helices are indicated by cross-bars (1-3) extending in COPT1 approximately from residues 63 to 84, 104 to 122, and 127 to 144; in CTR2 from residues 81 to 101, 135 to 151, and 152 to 168; and in QP from 371 to 391, 405 to 429, and 433 to 456.



Disruption of the CTR2 Gene from Yeast

Our results suggest that the COPT1 protein is a plant metal transporter, which presumably transports copper. To further test this hypothesis, we constructed a disruption mutation in the CTR2 gene, which encodes a yeast homologue of COPT1 (Fig. 3A). This disruption allele, designated ctr2::HIS3, was obtained via the insertion of the HIS3 gene into a BglII restriction site located in codon 120 of CTR2. The disruption allele was transformed into a haploid ctr1-3 and a haploid wild type strain. Homologous recombinants of both recipient strains that contained the ctr2::HIS3 allele were obtained and examined for the correct integration at the wild type CTR2 gene locus via PCR analysis (data not shown). The CTR2 gene is not essential for cell viability, even in a ctr1-3 mutant background.

To examine a possible role of the CTR2 gene in copper uptake, the CTR2 disruption mutant was compared with the parental wild type strain for the ability to grow on the nonfermentable carbon source glycerol, as a test for respiration competence, and on a minimal medium containing the ferrous iron chelator ferrozine, a test for high affinity ferrous iron uptake. On these media, the high affinity copper transport mutant ctr1-3 fails to grow(6) . As shown in Fig. 5, a mutation of CTR2 did not result in impaired growth on glycerol or iron-starved medium. We therefore tested the growth response of the ctr2::HIS3 single and the ctr1-3 ctr2::HIS3 double mutant on copper-starved medium containing the copper chelator BCS and on copper-rich medium. Whereas the ctr2::HIS3 mutant again did not show a growth impairment on copper-starved medium, the double mutant ctr1-3 ctr2::HIS3 was slightly stronger growth inhibited in the presence of 80 µM BCS than the ctr1-3 single mutant (Fig. 5B). However, both, the ctr2::HIS3 single and the ctr1-3 ctr2::HIS3 double mutant strain were more resistant to copper toxicity than the respective parental strains (800 µM CuSO(4) in Fig. 5B). Under the same conditions, a mutation only in CTR1 does not result in an increased resistance to copper when compared to a wild type strain (Fig. 5B). This was to be expected, since copper excess has been shown to strongly repress CTR1 gene expression(7) . The growth responses of the CTR2 disruption mutant on copper-deprived and copper-rich media may suggest that CTR2 allows a low affinity copper uptake. This notion is supported by the observation that overexpression of CTR2 (pKK52) does not result in the complementation of the high affinity copper transport mutant ctr1-3 on solid medium containing as sole carbon source glycerol or containing ferrozine to lower the free iron concentration (data not shown). However, cells of the ctr1-3 ctr2::HIS3 mutant strain KK4 overexpressing CTR2 from plasmid pKK52 showed a slightly enhanced growth in presence of the copper chelator BCS (Fig. 5C). Overexpression of CTR2 resulted in increased sensitivity to toxicity on a plate containing 700 µM CuSO(4) (Fig. 5C), which is consistent with mediation of an efficient copper transport into the cell via CTR2 when high external copper concentrations are present.


Figure 5: Phenotypes resulting from various CTR2 genotypes. Culture plates either contained YG medium with glycerol or YNBFC medium. YNBFC was with 1 mM ferrozine, BCS (40 or 80 µM), CuSO(4) (700 or 800 µM), or no copper addition (0 µM CuSO(4)). The S. cerevisiae strains included the parental strain CM3262 (CTR1 CTR2), strain KK3 (ctr2::HIS3), strain 83 (ctr1-3), and strain KK4 (ctr1-3 ctr2::HIS3). A, cells streaked on YNBFC plates containing ferrozine to assess high affinity ferrous iron uptake. B, strains were grown in YPD overnight, adjusted to A of 1, and serially diluted as described in Fig. 1. C, strains either transformed with vector pFL61 or plasmid pKK52 (CTR2 under control of PGK promoter) were grown for 48 h in SDC. Serial dilutions were performed as described in Fig. 1. All plates were incubated for 14 days except for the YG and YNBFC plates (no copper addition), which were incubated only for 7 days at 30 °C prior to photography.



Consistent with a possible role of CTR2 in copper uptake is the highly hydrophobic nature of this protein (Fig. 4), suggesting that CTR2 is an integral membrane protein. Inspection of the CTR2 amino acid sequence revealed the presence of three potential transmembrane helices, like for the plant homologue COPT1 (Fig. 4). The predicted transmembrane helices extend in CTR2 from residues 81 to 101, 135 to 151, and 152 to 168 (Fig. 4).

Northern and Southern Blot Analyses of Arabidopsis COPT1

A COPT1 mRNA species of approximately 0.9 kb was found in total RNA extracted from different organs of A. thaliana (Fig. 6B). The transcript was of equal size in flowers, stems, and leaves; however, it was not detectable in roots. The transcript was most abundant in leaves.


Figure 6: Expression of COPT1 in different organs of Arabidopsis and Southern blot analysis of COPT1. A, Arabidopsis genomic DNA (2 µg per track) extracted from ecotypes Columbia (C) and Landsberg erecta (L) was digested with DraI (lane 1), SacI (lane 2), EcoRV (lane 3), PstI (lane 4), and HindIII (lane 5). The blot was probed with a P-labeled NotI fragment from pKK31 covering the entire COPT1 cDNA (Fig. 2). Note, the COPT1 cDNA does not contain SacI, EcoRV, PstI, or HindIII sites, but a single DraI site in position 728 is present (Fig. 2). B, Northern blot analysis of total RNA (10 µg per track) extracted from A. thaliana flowers (lane 1), stems (lane 2), leaves (lane 3), and roots (lane 4). The same COPT1 probe as in A has been used. Length standard (kb) in A and B was HindIII-digested and P-labeled DNA.



A Southern blot of genomic DNA from the A. thaliana ecotypes Columbia and Landsberg erecta, which was digested with DraI, SacI, EcoRV, PstI, and HindIII and probed with the entire COPT1 cDNA (Fig. 2), is shown in Fig. 6A. Washing under a mild stringency revealed only one hybridizing band in each of the tracks (Fig. 6A). This result is in agreement with the assumption that no other closely related gene is cross-hybridizing under these conditions, suggesting that COPT1 is a single copy gene. Digestion of the genomic DNA with HindIII revealed the presence of a polymorphism when comparing the two ecotypes (Fig. 6A, lane 5).


DISCUSSION

Very little is known about the molecular basis of copper transport across plant membranes. Since the functional complementation of yeast mutants for the isolation of plant cDNAs from an expression library has been used successfully to identify plant transporter cDNAs (16, 17, 18, 19, 20, 21, 22) , we started to dissect the plant copper transport system by complementation of yeast mutants.

We have isolated a full-length cDNA of a putative copper transporter (COPT1) from A. thaliana by functional complementation of the high affinity copper transport mutant ctr1-3 from yeast. The cDNA encodes a polypeptide of 169 amino acid residues, which, interestingly, shows no detectable sequence homology to the copper transporter CTR1 from yeast. The distribution of hydrophilic and hydrophobic amino acid residues along the polypeptide chain suggests the presence of three potential transmembrane helices (Fig. 4); thus, COPT1 is most likely an integral membrane protein. The presence of only three potential membrane-spanning domains does not contradict a possible role of COPT1 in metal transport since also for the high affinity copper transporter from yeast, CTR1, only three transmembrane domains have been predicted (6) . In addition, there are other metal transporters known from the cytoplasmic membrane of prokaryotes that also have only three transmembrane segments, for example magnesium and mercury transport systems(44, 45) . The COPT1 protein resides presumably in the cytoplasmic membrane since the complementation of the ctr1-3 mutant on copper-limited plates (Fig. 1B) would be otherwise difficult to interpret. The ctr1-3 mutant is well characterized and has been clearly demonstrated to suffer from a copper uptake defect across the cytoplasmic membrane(6, 7) , which is obviously overcome by heterologous expression of COPT1 (Fig. 1B). However, the COPT1 protein lacks a predictable amino-terminal leader sequence and may thus utilize one of the transmembrane domains to initiate insertion into the membrane. The CTR1 protein is lacking a leader sequence as well(6) .

A copper transporter would be predicted to first interact with copper before subsequent translocation of the copper ion across the membrane. The methionine- and histidine-rich amino terminus composed of the first 44 amino acid residues of the COPT1 protein might be such a copper binding domain since it displays considerable sequence homology to similar motifs present in the bacterial copper binding proteins CopA and CopB of P. syringae(42) and CopB of E. hirae(41) (Fig. 3B). These bacterial proteins have been demonstrated experimentally to bind copper(46, 47) . The CopB-ATPase of E. hirae is a cytoplasmic membrane protein and functions as an efflux pump of copper(46) . The Cop proteins of P. syringae mediate the sequestration of copper outside the cytoplasm; CopB is an outer membrane protein, and CopA is localized in the periplasm as part of a copper resistance mechanism(47) . Since this methionine- and histidine-rich domain is the only common denominator among these bacterial copper binding proteins, which are otherwise completely unrelated in amino acid sequence, it has been proposed that this motif represents a copper binding domain(41, 42, 46, 47) . The presence of this putative copper binding domain at the amino terminus of COPT1 is consistent with a direct role in copper transport and points to its possible ability to interact directly with copper.

The interpretation that COPT1 might be a plant metal transporter capable of transporting copper is based on the following observations by heterologous expression in the well characterized high affinity copper transporter mutant ctr1-3 of yeast. (i) Expression of COPT1 allows the yeast strain 83 (ctr1-3) to grow on the nonfermentable carbon source glycerol (Fig. 1A). The ctr1-3 mutant is unable to grow on a nonfermentable carbon source due to a respiratory defect, which is a consequence of the high affinity copper uptake defect(6) . Regaining the ability to grow on glycerol plates in presence of COPT1 is consistent with increased copper uptake via the plant membrane protein. (ii) Expression of COPT1 restores the growth defect of the ctr1-3 mutant on iron-starved plates (Fig. 1B). Since the high affinity ferrous iron transport is strictly copper dependent, the FET3 oxidase, which is a component of the high affinity ferrous iron transporter system, contains copper in the active site(8, 10) ; the ctr1-3 mutant suffers from both a copper and an iron uptake defect. Regained growth on iron-deprived plates in presence of COPT1 is again consistent with enhanced copper transport across the cytoplasmic membrane of the ctr1-3 mutant. (iii) Expression of COPT1 allows yeast strain 83 (ctr1-3) to grow more efficiently on copper-starved plates (Fig. 1B). As pointed out by Dancis et al.(7) , the growth arrest of CTR1 mutants in low copper-containing medium does not seem to be related to iron deficiency but to a copper deficiency, since it is not rescued by iron addition. The most plausible explanation for this phenotype is that A. thaliana COPT1, heterologously expressed in the ctr1-3 mutant, promotes enhanced copper uptake and not iron uptake. (iv) Expression of COPT1 inhibits growth of the ctr1-3 mutant on copper-rich medium (Fig. 1B, 900 µM CuSO(4)). Thus, the COPT1 protein mediates the efficient delivery of copper to the cells, even at high external copper concentrations, and thereby resulting in copper toxicity. The same visible phenotype has been described upon overexpression of the high affinity copper transporter CTR1 from yeast on copper-rich plates(7) . The toxic nature of copper for yeast cells at elevated concentrations is well known(48) . We should point out that we cannot currently exclude the possibility that COPT1 might transport, in addition to copper, other metals.

The COPT1 protein of A. thaliana shows a low but significant homology (23.4% identity, 38.3% similarity) to the predicted protein of an open reading frame on yeast chromosome VIII (Fig. 3A). We have named this gene CTR2 and hypothesize that it encodes a metal uptake system of low affinity for copper. In fact, Dancis et al.(6) observed that the CTR1 mutant still displays a measurable copper uptake activity, although strongly reduced when compared to the wild type. However, high levels of exogenous copper (500 µM) completely suppressed the ferrous iron uptake defect in the CTR1 mutant(6) , which is indicative for the presence of a low affinity copper uptake system in the yeast cytoplasmic membrane. The conclusion that CTR2 might mediate a low affinity copper uptake into yeast cells is based on the following results. (i) Overexpression of CTR2 results in increased sensitivity to copper toxicity on copper-rich plates (Fig. 5C), which is consistent with enhanced copper uptake via CTR2. (ii) The disruption mutant ctr2::HIS3, on the other hand, displays increased resistance to copper toxicity (Fig. 5B), indicative for a strongly reduced copper uptake. (iii) The disruption mutant ctr2::HIS3 does not show a growth defect on a nonfermentable carbon source nor on an iron- or copper-starved medium (Fig. 5, A and B). However, the double mutant ctr1-3 ctr2::HIS3 is slightly stronger growth inhibited in presence of the copper chelator BCS than the single mutant ctr1-3 (Fig. 5B). Thus, CTR2 seems to make only a minor contribution to the delivery of copper into cells under conditions of a copper starvation when the high affinity copper transporter CTR1 is still functional. (iv) Overexpression of CTR2 results in a slight growth promotion in a ctr1-3 ctr2::HIS3 mutant background (Fig. 5C) but not in a complementation of the ctr1-3 mutant on medium containing a nonfermentable carbon source or the iron chelator ferrozine. This observation strongly supports the hypothesis that CTR2 may encode a metal uptake system of a low affinity for copper, transport capacity of which is not high enough to fully complement all the growth defects of the ctr1-3 mutant, especially when only a low external copper concentration is available. The CTR2 protein is predicted, like its plant homologue COPT1, to contain three potential transmembrane domains (Fig. 4). Interestingly, the CTR2 protein does not exhibit a histidine- and methionine-rich amino-terminal domain like COPT1. Assuming that CTR2 directly binds copper, a novel metal binding motif must be involved. As has been discussed already by Dancis et al.(6) , different copper binding motifs have to be expected dependent on the strength of the binding to copper, which not only can be permanent but transient, in case of a copper transporter, as well. It wouldn't be surprising if a high affinity copper transporter would comprise a completely different copper binding domain than a low affinity copper transporter. An alternative explanation could be that CTR2 actually transports other metals instead of or besides copper, which would explain the lack of a defined copper binding motif and the inability to fully complement the ctr1-3 mutant.

The Southern data (Fig. 6A) indicate that COPT1 is encoded by a single gene in the A. thaliana genome. The observed polymorphism between ecotypes Columbia and Landsberg erecta can now be exploited to locate the position of the COPT1 gene in the Arabidopsis genome(49) . It will be very interesting to see if there are already mutants known that map to this position. In this context, recent work by Murphy and Taiz (50) should be mentioned. These authors described 59 putative copper-sensitive Arabidopsis mutants. From the Northern blot, a single COPT1 messenger of 0.9 kb was detected in leaves, flowers, and stems. However, no COPT1 transcript could be detected in roots. Thus, it is at the moment questionable if COPT1 is directly involved in copper uptake from soil via the roots or rather in the distribution within the plant, and here especially in the green parts.

In conclusion, we believe that the A. thaliana COPT1 protein is a copper transporter that presumably resides in the cytoplasmic membrane. The yeast homologue, CTR2, is most likely a metal transporter as well and might well function as a low affinity copper uptake system. We are currently trying to test this hypothesis by performing transport measurements with radioactive copper by heterologous expression of the COPT1 cDNA in yeast cells. Detailed studies will be needed to clearly define the substrate specificities and the kinetic characteristics of these novel transport proteins. It could well be possible that these transporters are capable of transporting copper and iron in addition to other metals. Functional complementation in yeast was used in the work described here to identify a putative copper transporter from plants. Via this approach, we may identify further components of plant copper transport systems. The intracellular transport of copper within yeast cells has just started to be dissected, and mutants are already available(51) . The availability of plant copper transporter genes together with the possibility of constructing transgenic plants where the expression is either down- or up-regulated will provide us with novel insights into the inorganic nutrition and partitioning of copper in higher plants.


FOOTNOTES

*
This work was supported in part by grants from the Belgian Program on Interuniversity Poles of Attraction (Prime Minister's Office, Science Policy Programming, no. 38). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z49859[GenBank].

§
Funded by the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed: Institut für Biochemie und Molekulare Physiologie, Universität Potsdam, Maulbeerallee 2a, D-14469 Potsdam, Germany. Tel.: 331-9771906; Fax: 331-9771948.

Funded by the Belgian Science Office.

**
Funded by the European Environmental Research Organization.

(^1)
The abbreviations used are: BCS, bathocuproinedisulfonic acid; PCR, polymerase chain reaction; kb, kilobase(s); MES, 4-morpholineethanesulfonic acid.

(^2)
This method is available via internet (PredictProtein@EMBLHeidelberg.DE).


ACKNOWLEDGEMENTS

We thank Dr. Michèle Minet and Dr. François Lacroute (CNRS, Gif-sur-Yvette, France) for providing an A. thaliana cDNA library and plasmid pFL39. Yeast strains CM3262 and 83 were kindly provided by Dr. Richard Klausner (NICHD, National Institutes of Health). We also thank Dr. Jan Demolder (University of Gent) for very helpful advice concerning the yeast work. We are indebted to Dr. Martin Steup (University of Potsdam) for critically reading the manuscript. Help by Karel Spruyt with the photos and Martine De Cock with preparing the manuscript is gratefully acknowledged.


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