Info

aTMD: transmembrane domain; aa: amino acids. Features in parentheses are not fully conserved. ^Possible localization. Pm: plasma membrane; Pl: plastid; Chl: chloroplast.

Copper is thought to be incorporated in two steps, where Cu(II) would first be reduced to Cu(I) through the action of a Cu(II)-chelate reductase activity and Cu(I) would subsequently be captured by plant roots in a transporter-mediated process. This mechanism is operative for iron uptake in dicotyledons (28) and compelling data suggest that the same strategy could be utilized as well for copper uptake. Effectively, copper deficiency has been shown to induce Fe(III)-chelate reductase activity, which reduces both Fe(III) and Cu(II), in roots of pea seedlings (29). In addition, Arabidopsis frdl mutants, which carry a recessive mutation in the FRO2 gene, encoding an Fe(III)-chelate reductase (30), lose the ability to reduce Cu(II) chelates (31).

However, some other authors defend that this strategy is not functional in plants. In this sense, it has been reported that Cu(II) but not Cu(I) was efficiently absorbed by maize roots (32). Moreover, copper accumulation was not reduced in Arabidopsis frdl plants grown on plates (31), suggesting that copper reduction is not essential for the uptake of this metal.

Regarding copper transport across the plasma membrane, three families of heavy-metal transporters have been identified so far (see Table 2):

1. The ZIP (ZRT/IRT-related protein) family defines a group of transition metal transporters with broad specificity.

2. The Nramp (natural resistance-associated macrophage protein) family is also a group of heavy-metal transporters with wide spectrum.

3. The CTR (copper transporters) family is integrated by homologs from the yeast high-affinity plasma membrane copper transporters.

The first Arabidopsis ZIP members (ZIP1, ZIP2, and ZIP3) were isolated by their ability to transport zinc, but not iron, in yeast cells (33). Interestingly, metal-uptake competition experiments indicate that ZIP2 seems to prefer Cu and Cd over other transition metals. A fourth member, ZIP4, was identified by sequence homology, although no metal-transport competence has been detected in yeast. However, the fact that it contains a putative chloroplast target signal suggests a role in metal transport to this compartment in plant cells (33).

In a screen for plant cDNAs that encoded for iron transporters, Eide et al. (34) identified the Arabidopsis IRT1 gene by functional complementation of yeast iron-uptake mutants. Later, IRT1 was also shown to transport Mn and Zn, but not Cu (35). IRT1 is thought to be an Fe(II) transporter that takes up Fe from soil in a manner that is coupled to the Fe(III)-chelate reductase (1).

In higher plants, 10 Nramp-related genes have been identified to date (36-38,40,41). Attending to sequence comparison analyses, they are divided into two major classes (see Table 2). Members from the two subgroups have been demonstrated to transport Fe, Mn, and Cd in yeast cells (36,37). It has been suggested that distinct members of the family perform their function at different subcellular compartments and tissues (37). Therefore, all of these transporters would act coordinately to regulate the pool of cytoplasmic metals in the whole plant.

Ethylene insensitive 2 (EIN2) represents a special Arabidopsis member of this Nramp group, involved in the ethylene transduction pathway (42). Ethylene is a plant hormone that influences many aspects of plant growth and development, some of them with outstanding economical impact on agriculture, which traditionally made this gaseous hormone a main focus of attention in plant biology (43). Mechanisms of ethylene action are being elucidated thanks to molecular genetic approaches using the ethylene-evoked triple-response phenotype of Arabidopsis seedlings (44). EIN2 encodes a protein with a hydrophobic Nramp-like amino terminal domain and an interesting exclusive hydro-philic carboxy terminal domain involved in the expression of genes that respond to oxidative stress and hormones such as jasmonates and ethylene, therefore integrating several important plant signal transduction pathways (38). The EIN2 Nramp-like domain is evolutionary distant from the two other groups of plant Nramp genes (36). In fact, neither EIN2 nor truncated versions of the protein displayed metal-transporting capacity when expressed in heterologous systems (38). In contrast, the

Fig. 2. Sequence comparison of the five members family of Arabidopsis copper transporters (COPT) belonging to the CTR family. At the upper part, bold lines indicate predicted transmembrane domains (TMD) and asterisks display the position of a putative copper-binding motif in COPT1 and COPT2.

Nramp-like domain is necessary for regulating the EIN2 carboxy-terminal domain hormone responses. Thus, the EIN2 Nramp domain has been suggested to be involved in sensing divalent metal ions, rather than transporting them across membranes. Concomitantly, the EIN2 carboxy-ter-minal domain would transduce the signal to downstream elements, maybe interacting with components of other hormone transduction cascades. If true, a metal ion is predicted to act as a second messenger in the ethylene signaling pathway (38).

Finally, members of a widespread family of eukaryotic CTR transporters have been identified in yeast, worms, flies, mammals, and plants. All of the members belonging to this family contain three predicted transmembrane segments and some of them posses an N-terminal putative metal-binding domain (Fig. 2). The Arabidopsis COPT1 gene was originally identified by its ability to suppress the phenotype of yeast mutants defective in high-affinity copper uptake (39). COPT1 encodes a polypeptide of 170 amino acids with 49% similarity to its yeast counterpart CTR1 (1). The 44 amino-terminal residues of COPT1 are enriched in methionine and histidine residues and display similarity with bacterial copper binding motifs (44). This COPT1 amino terminal domain is predicted to face the extracytoplasmic region (1), suggesting copper binding previous to its transport across plasma membrane. Complementation experiments with truncated forms of the protein indicate that the COPT1 amino-terminal domain is necessary for high-affinity copper uptake in yeast (Mira and Penarrubia, unpublished results).

Sequence analysis of the complete Arabidopsis genome (45) reveals a five-member family of CTR-related genes, temptatively named COPT1-5 (Fig. 2 and Table 2). The hydrophobicity profiles of the predicted proteins is in accordance with the three transmembrane spanning domains (TMD) postulated for the CTR family. The alignment of the polypeptides reveals that whereas the TMDs are highly conserved within the COPT family, the amino- and carboxy-terminal protein regions are variable in sequence and length. COPT2 is closely related to COPT1 (72% identity) and also has a histidine and methionine-rich amino-terminal box, whereas COPT3-5 are more distantly related to COPT1 and do not posses any recognizable copper binding motif (Fig. 2).

Although Northern blot showed that COPT1 mRNA was undetectable in roots (39), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) data indicate that COPT1 is indeed expressed in these nutrient-uptake organs. COPT2 is also capable of rescuing yeast high-affinity copper-uptake mutants and expressed in roots. Both COPT1 and COPT2 mRNA levels are downregulated by copper treatment in leaves, suggesting a role of these genes in copper transport in plants (Sancenon and Penarrubia, unpublished results).

Although we have tried to summarize disperse data available about metal transporters, further phenotypic characterization of genetically modified plants that silence or overexpress these genes turn out to be essential for assessing the role of the three transporter families in plant copper homeo-stasis.

3.2.2. The Transport at the Endomembrane System

The RAN1 (Responsive to Antagonist 1) gene from Arabidopsis thaliana has been recently cloned and it encodes a homolog of P-type ATPases from yeast (Ccc2) and humans (Menkes and Wilson proteins) located at post-Golgi vesicles (Fig. 1). RAN1 presents two putative copper-binding boxes of the GMTCXXC type and complements the deficiency in copper transport to the secretory pathway of the yeast mutant Accc2 (46). However, RAN1 lacks the leucine repeats taht act as signals for the copper-mediated traffic of the protein between Golgi and plasma membranes in the human homolog (46,47). It would be interesting to check RAN1 subcellular localization under different copper status and its tissue distribution during development in order to gain further insight into its specific functionality in plants.

This copper homeostasis-related gene was isolated in a screen for plants with an unusual response to the ethylene antagonist trans-cyclooctene (46), further underscoring the critical role of metals in the ethylene signaling pathway. Among the members of this pathway, perception takes place through a small family of receptors. One of these receptors, ETR1, has been shown to form disulfide-linked homodimers (48) that require a Cu atom for high-affinity ethylene binding (9) (Fig. 1). ETR1 encodes a polypeptide resembling the sensor module of the two-component regulatory systems widely used by different living organisms. However, ETR1 combines the sensor domain for hormone binding and a membrane-localized regulatory histidine kinase signaling domain (reviewed in ref. 49). Ethylene-depleted receptors act as negative regulators of the transduction pathway. Upon hormone-binding, the receptor is inactivated and the resulting phenotypic effects are known as the ethylene response. Thus, mutations in the hormone binding domain provoke dominant insensitivity to ethylene because the receptor cannot be inactivated, whereas disruptions in the signaling domain originate a constitutive ethylene response (50,51).

Two types of RAN1 defective plants have been described. In the first group (composed of ran1-1 and ran1-2), the mutations cause a slight impairment in the receptor structure in such a way that modifies the response to the ethylene antagonist trans-cyclooctene. Interestingly, no other pheno-typic effects have been observed (46). There is no molecular explanation for this behavior yet, because the mechanisms of the trans-cyclooctene action and the receptor structure still remain unsolved. The second class is composed of ran1 loss-of-function plants and include ran1-3, a strong allele mutated in a glycine residue conserved within the rest of homologs (52), ran1-4, caused by a transfer-DNA insertion (53) and the transgenic CaMV 35S::RAN1 plants that cause cosupression of RAN1 (46). All of these plants appear to have a constitutive ethylene response showing a phenotype similar to the mutants with multiple disruption of ethylene receptors (51). It has been suggested that this fact would indicate the requirement of RAN1 to pump copper into the post-Golgi compartment where it would be added to ethylene aporeceptors as they move toward the plasma membrane. In RAN1-defective en Ol

Fig. 3. Sequence comparison of Atx1-like metallochaperones from different species: Homo sapiens, Canis familiaris, Ovis aries, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, and Arabidopsis thaliana. In the upper part, the predicted secondary structure for the Saccha-romyces chaperone is indicated. p-Sheets are represented by arrows, a-helices are indicated by rectangles, and loops are indicated by lines. In the bottom part, asterisks show identical amino acids and dots indicate conservative substitutions. A schematic representation of the a-helix conformation predicted for the central part of the C-domain (from residue 90 to 108) denominated Hello motif.

Fig. 3. Sequence comparison of Atx1-like metallochaperones from different species: Homo sapiens, Canis familiaris, Ovis aries, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, and Arabidopsis thaliana. In the upper part, the predicted secondary structure for the Saccha-romyces chaperone is indicated. p-Sheets are represented by arrows, a-helices are indicated by rectangles, and loops are indicated by lines. In the bottom part, asterisks show identical amino acids and dots indicate conservative substitutions. A schematic representation of the a-helix conformation predicted for the central part of the C-domain (from residue 90 to 108) denominated Hello motif.

plants, the absence of copper delivery to the secretory pathway would avoid metal incorporation to cuproproteins passing through the endomembrane system, including ethylene receptors. These anomalous receptors would cause the permanent activation of the ethylene signaling pathway and the observed constitutive response. Moreover, ethylene-independent effects, such as a rosette-lethal phe-notype originated by a defect in cell expansion, have also been observed in the ran1-3 mutant under an ethylene-insensitive background (52). This phenotype could be caused by the absence of the metal in other copper-requiring enzymes that incorporate this cofactor in a post-Golgi compartment by a RANl-dependent mechanism. In this sense, it would be interesting to check the activity of apoplastic cuproenzymes, such as laccase, diamino oxidase, and ascorbate oxidase, in these mutants. Furthermore, genetic analysis and the response of the ran1-3 to ethylene inhibitors indicate that, in addition to forming completely functional ethylene-binding sites, copper is also required for the receptor signaling function (52). The molecular meaning of this observation will require the structural characterization of the ethylene receptor, as well as the reorganizations that take place upon hormone binding. These studies will open new perspectives into the knowledge of how copper influences ethylene action and, to a greater extent, they are promoting a growing and preponderant interest of plant biologists about how plant cells deal with copper.

3.3. Cytosolic Distribution

According to the Saccharomyces model, it is conceivable that a small family of cytosolic metallochaperones could also operate in plants (Fig. 1). In this sense, cDNA sequences with homology to yeast CCS, which code for putative chaperones that deliver copper to the Cu/Zn-SOD (superoxide dismutase) cytosolic isoform, have been found in the databases from both Arabidopsis (GenBank accession no. AF179371) and tomato (GenBank accession no. AF117707). Tomato CCS, obtained as recombinant protein, has already been structurally characterized (54).

On the other hand, no information is available about how plants transport copper to their organela (Fig. 1). With regard to the yeast chaperone Cox17, which drives copper to the mitochondrial cytochrome oxidase, no plant homologs have been found yet in spite of the fact that the Arabidopsis genome has been already released (45) and sequencing programs are in progress for other higher plants. Moreover, plant-specific organelae (i.e., the chloroplasts) should have their own mechanism to guarantee copper delivery to cuproproteins such as plastocyanin and the plastidic isoform of the Cu/Zn-SOD. In these cases, new approaches would be necessary to uncover these plant exclusive processes.

Special attention will be paid next to the copper routing toward the secretory pathway, because the corresponding Arabidopsis copper chaperone has been the first metallochaperone described in plants and our main focus of interest over the past several years.

3.3.1. The Copper Chaperone CCH

The cDNA clone encoding a homolog of the yeast ATX1 (55) was identified in the Arabidopsis expressed sequence tag collection and named CCH for copper chaperone (56) (Fig. 1). Although CCH has an amino-terminal part (N-domain) that exhibits a 36% identity to Atx1, it also has an extra carboxy-terminal region (C-domain) composed of 48 amino acids that is absent in all other nonplant homologs identified to date (Fig. 3).

When compared to the rest of Atx1-like proteins, the CCH N-domain presents two conserved regions. The first region includes the copper-binding motif MXCXXC located at the L1 connection within the structural module pappap, characteristic of both metallochaperones and copper ATPases (57,58) (Fig. 3). The cysteines in the Atx1 MTCSGC box for copper binding are conserved in the CCH-MSCQGC box. However, threonine is replaced by serine, a conservative substitution, in the Arabidopsis motif (Fig. 3). The second region is composed of basic residues where the conservation of the Arabidopsis lysine necessary for the antioxidant role of the Atx1 and its human homolog Hah1

is noticeable (59,60) (Fig. 3). In Atxl, these basic residues are at the protein surface and probably participate in electrostatic interactions with acidic residues in the cytosolic exposed surface of Ccc2 (57,61).

3.3.1.1. Characterization of the Plant-Exclusive CCH C-Domain

The CCH C-domain has an unusual amino acid composition with no significant identity with any other sequences in the databases. It is composed of 44% charged residues alternating with noncharged ones, mostly prolines, alanines and hydrophobic-p-branched amino acids, generating a singular (i, i + 1) periodicity. Secondary-structure predictions indicate that the central part of this domain could be arranged as an a-helix with a spatial distribution of basic amino acids on one side and acidic amino acids on the other (Fig. 3). This central part has been named Hello motif for helix with lateral opposite charge. From the structural point of view, circular-dicroism (CD) spectroscopy data indicate that the C-domain could adopt an a-helical conformation in the presence of trifluorethanol, but an extended conformation is acquired in aqueous solution, which is further stabilized by lowering the temperature or in the presence of submicellar concentrations of sodium dodecyl sulfate (SDS). The extended conformation that we refer to here could be obtained as a result of the contribution of two or more different types of secondary structures, like type-II polyproline helix and/or antiparallel p-sheet (62). Although this complex structural behavior deserves further characterization, it could reflect the conformational flexibility of the C-domain in response to environmental changes.

In order to know whether the presence of this extra domain is a common feature in plants, two homologs from soybean and rice have been sequenced and shown to contain C-terminal regions with similar sizes and amino acid composition (Mira and Penarrubia, unpublished results). To gain insight into the evolutionary appearance of the plant extra C-domains, it would be of interest to check its presence in other orthologs from different photosynthetic eukaryotes.

As a first attempt to address the role that the C-domains accomplishes in plant metallochaperones, functional studies were performed in yeast. Our results have shown that the CCH N-terminal domain retains the Atxl-like properties (e.g., the antioxidant function and the delivery of copper to the Ccc2 transporter in the secretory pathway) (55,59,60), indicating that these properties are not affected by the C-terminal part (62). As postulated for other multimodular proteins, the extra C-domain could have been added "in line" (63), a kind of module addition that would allow the structure of the N-terminal part of the protein to remain virtually identical. Moreover, by comparison with the Atx1 structure, recently determined by X-ray, no major structural reorganizations would be expected to take place at the C-domain of the CCH protein upon copper binding to the N-terminal part (57). As a consequence, this kind of assembly would explain the lack of interference with the original intracel-lular function played by this metallochaperone in single-cell organisms, while probably allowing a novel or more complex role related to pluricellularity in plants, as suggested for other extra domains (64). Nevertheless, because direct interaction between CCH and RAN1 has not been demonstrated yet, a role of this extra domain in protein-protein recognition cannot be discarded. However, Ccc2 and the Arabidopsis homolog RAN1 do not seem to differ enough to justify the existence of the extra CCH C-domain to interact with RAN1 (46).

3.3.1.2. Expression and Localization of CCH

The soybean CCH homolog was initially isolated by a search for upregulated genes during leaf senescence (65) and Arabidopsis CCH mRNA is induced during natural and ozone-accelerated leaf senescence (56,66). These facts indicate that CCH can be classified as a senescence-associated gene (SAG), a group of diverse genes that are mainly involved in nutrient salvage from decaying organs (67).

Leaf senescence involves an orderly loss of structures and metabolic functions characterized by macromolecule degradation and mobilization of the resulting nutrients to other parts of the plant (68,69). Metal ions released after metalloproteins break down are recycled from senescing leaves to growing organs (70-73). During these processes, the vascular system, in particular phloem, plays a key role in the translocation of resources. As detected in Western blots from Arabidopsis phloem exudates, CCH belongs to the pool of soluble polypeptides found in these transport tissues (74), suggesting its requirement for copper remobilization from decaying organs to demanding reproductive structures. In this context, an Arabidopsis metallothionein mRNA has been located to the vascular bundle (23). However, metallothioneins presumably act as chelators that are upregulated by copper (20). In contrast, CCH expression is specifically repressed by copper, and, at least in yeast, the CCH protein has the ability to bind and deliver copper to a specific target (56,62), thus indicating that the functions of metallothioneins and CCH are, in fact, different. Moreover, because CCH and its homologs exhibit antioxidant properties (55,59,62), it cannot be discarded that CCH could participate in avoiding the damage of the vascular system produced by oxidant toxic molecules originated during senescence and in keeping these structures functional for the progressive dismantling of decaying organs.

Immunolocalization experiments show that, despite its housekeeping character, CCH is specially abundant in specific phloem elements of green leaves and stems and that, during senescence, CCH levels increase and extend to surrounding cells (74). Perhaps, the high levels of CCH in the phloem are necessary to compete with other molecules for copper and to guarantee its delivery to the secretory pathway, mediating the loading of extracellular copper-requiring proteins along the way from senescent tissues to developing organs. Specially remarkable is the fact that CCH is mainly located at specific phloem cells named sieve elements. These cells suffer a selective degradation of internal structures during differentiation that results in the lost of their competence for transcription, translation and protein maturation, which makes them dependent on associated companion cells for much of their metabolic functions (75,76). Thus, CCH, after being synthesized in companion cells, must cross to the sieve elements through the cell-connecting structures called plasmodesmata. In this sense, CCH is the first metallochaperone shown to be transported from cell to cell, and it is tempting to speculate that the exclusive CCH C-domain of unknown function could be involved in this plant-exclusive transport process.

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