The NRAMP family of divalent metal transporters
The natural resistance associated macrophage proteins (NRAMP) family of divalent metal transporters participates in plant intracellular Fe distribution. The presence of NRAMP transporters in both Strategy I and II plants suggests a common function in metal homeostasis. In Arabidopsis, three NRAMP proteins (NRAMP1, NRAMP3 and NRAMP4) revert yeast fet3Δfet4Δ growth in Fe-limited conditions, and exhibit increase mRNA levels upon Fe starvation (Curie et al. 2000; Thomine et al. 2000). Increased resistance to toxic Fe levels of NRAMP1 overexpressing plants and a preferential root localization suggest that Arabidopsis NRAMP1 functions in intracellular root Fe distribution (Curie et al. 2000). Studies on single and double knockout mutants demonstrate that NRAMP3 and NRAMP4 play a redundant role in Fe mobilization from vacuoles in Arabidopsis seeds (Lanquar et al. 2005; Thomine et al. 2003) (Fig. 1 and Table 1). The nramp3nramp4 double mutant exhibits germination arrest under low Fe nutrition, which is fully rescued by exogenous Fe supply (Lanquar et al. 2005). The in vivo imaging of individual metals in plant organelles has shown that nramp3nramp4 mutant seeds fail to retrieve Fe from vacuolar globoids during germination, which is crucial to support Arabidopsis early development previous to Fe acquisition from the soil (Lanquar et al. 2005). Finally, increased Cd hypersensitivity when overexpressed and suppression of growth defects associated with yeast cells deleted for the Mn transporter suggest that Arabidopsis NRAMP proteins also participate in Mn and Cd transport (Thomine et al. 2000).
More recently, a new vacuolar iron transporter 1 (VIT1), which does not belong to the NRAMP family, has been characterized in Arabidopsis plants (Kim et al. 2006). VIT1 complements the sensitivity of ccc1Δ yeast mutants to elevated levels of Fe, which strongly suggests that VIT1 functions in vacuolar Fe storage (Li et al. 2001; Kim et al. 2006). VIT1 localizes to the Arabidopsis vacuolar membrane, and it is highly expressed in the developing embryo and seed. Synchrotron X-ray fluorescence microtomography data have demonstrated that Fe localizes to the provascular strands of wild-type seeds, a distribution completely abolished in vit1-1 mutants (Kim et al. 2006). Furthermore, vit1-1 plants grow poorly in Fe-limiting soils, which is indicative of a VIT1 function in seedling development (Kim et al. 2006).
Copper-transporting P-type ATPases
The heavy metal P-type ATPase (HMA) family, also know as P1B-ATPases, utilizes ATP hydrolysis to efflux positively charged metals from the cytoplasm (Fig. 2). The Arabidopsis genome encodes eight predicted HMA proteins that are classified in two groups: HMA1 to HMA4, which have been implicated in divalent cation transport, including Zn2+, Cd2+ and Cu2+; and HMA5 to HMA8, which presumably function in transport of monovalent Cu+ ions (Fig. 1 and Table 2). HMA5 to HMA8 exhibit two particular characteristics: a CPC ion transduction motif in the sixth predicted TMD, and MxCxxC Cu+-binding motifs at the amino-terminal domain of the protein (Axelsen & Palmgren 2001; Williams & Mills 2005) (Fig. 2).
Arabidopsis responsive-to-antagonist 1 (RAN1, or HMA7) was isolated in a genetic screen for seedlings that evoked the ethylene triple response in the presence of an ethylene antagonist (Hirayama et al. 1999). Given that Cu coordination is required for ethylene binding to the ETR1 ethylene receptor (Rodriguez et al. 1999), RAN1 has been proposed to participate in the biogenesis of functional ethylene receptors by supplying its metal cofactor at the endoplasmic reticulum (Hirayama et al. 1999; Woeste & Kieber 2000; Chen et al. 2002) (Fig. 1). Loss-of-function alleles of RAN1 affect multiple processes including cell elongation, which are partially alleviated by Cu supply (Woeste & Kieber 2000). Therefore, RAN1 will function in Cu delivery to the ethylene receptor, and presumably to other Cu proteins within the secretory pathway. On the contrary, several lines of evidence suggest that Arabidopsis HMA5, the closest RAN1 relative in the P-type ATPase family, functions in root Cu detoxification (Andres-Colas et al. 2006). Firstly, HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu treatment. Secondly, T-DNA insertion mutants are more sensitive to excess Cu, but not to increases in other metals, as shown by growth defect and root growth arrest. And thirdly, when grown under Cu excess, HMA5-defective plants accumulate Cu in roots to a greater extent than wild-type plants (Andres-Colas et al. 2006). While further studies on cellular and tissue HMA5 expression are needed, these data suggest that HMA5 can be involved in Cu efflux at specific root cells (Fig. 1). If this is the case, HMA5 overexpression in plants can be a strategy for improving Cu detoxification under Cu excess (see Copper phytoremediation).
Cu transport into the chloroplasts depends on the HMA proteins P-type ATPase of Arabidopsis 1 (PAA1, or HMA6) and PAA2 (or HMA8). Both genes were isolated in a screen for mutants that exhibited a high-chlorophyll-fluorescence phenotype, which is a consequence of reduced photosynthetic electron transport activity (Shikanai et al. 2003; Abdel-Ghany et al. 2005b). Both PAA1 and PAA2 proteins contain amino-terminal cleavable transit sequences that, when fused to GFP, target this reporter protein to the chloroplast (Abdel-Ghany et al. 2005b). Interestingly, while full-length fusion of PAA1 to GFP localizes to the chloroplast periphery, an amino-terminal portion of PAA2 protein including the first four TMDs localizes to the thylakoid membranes (Abdel-Ghany et al. 2005b). Analyses of chloroplast Cu proteins in wild-type paa1 and paa2 mutants demonstrate that both ATPases are required for Cu delivery to the thylakoidal protein plastocyanin (PC), while only the paa1 mutant shows a decrease in Cu delivery to the stromal Cu/ZnSOD (Abdel-Ghany et al. 2005b) (Fig. 1). Consistent with these data, PAA1 is expressed in both roots and shoots, while PAA2 is only detected in shoots (Abdel-Ghany et al. 2005b). A double paa1paa2 mutant results in seedling lethality, a more severe phenotype than that observed for plants defective for both PC genes (Weigel et al. 2003; Abdel-Ghany et al. 2005b), suggesting that PAA proteins play additional roles such as Cu delivery to Cu/ZnSOD.
Cu import into chloroplasts is not completely abolished in paa1 mutants. An additional HMA family member, HMA1, localized in the chloroplast envelope, has been recently implicated in Cu transport into the chloroplasts (Seigneurin-Berny et al. 2006). hma1 mutants exhibit diminished chloroplast Cu/ZnSOD activity, but normal PC content (Seigneurin-Berny et al. 2006), suggesting that Cu from HMA1 would be preferentially delivered to Cu/ZnSOD (Fig. 1). Furthermore, HMA1 may have specific functions in plants grown under adverse light conditions (Seigneurin-Berny et al. 2006). It should be stressed that the HMA1 protein does not contain the canonical MxCxxM amino-terminal Cu+-binding motifs, but instead, it is histidine rich at the amino terminus, suggesting that, rather than Cu+, it may transport Cu2+.
Once inside the cell, the limited solubility and high reactivity of Cu+ requires the participation of specialized cellular factors named Cu chaperones. These factors are Cu+-binding soluble proteins that mediate intracellular Cu delivery to specific target apoproteins to form biological active Cu proteins (O'Halloran & Culotta 2000; Huffman & O'Halloran, 2001). Several independent Cu chaperones have been described in S. cerevisiae: Antioxidant 1 (Atx1) shuttles Cu to a P-type ATPase located within a post-Golgi compartment for translocation into the secretory pathway (Pufahl et al. 1997); Cu chaperone for Cu/ZnSOD (Ccs1) is required for Cu insertion into the active site of cytosolic Cu/ZnSOD (reviewed by Culotta, Yang & O'Halloran T 2006), and Cox17 is involved in Cu trafficking towards mitochondrial cytochrome c oxidase (COX) (Cobine, Pierrel & Winge 2006). Cu chaperones seem to be conserved in most eukaryotes, but unique metallochaperone characteristics have emerged in plants, probably aimed to fit plant-specific requirements in their Cu delivery pathways (Fig. 3).
Figure 3. Subcellular distribution of copper by Arabidopsis metallochaperones. A speculative diagram showing subcellular distribution of copper in a generic plant cell is represented. Cu transporters and Cu proteins are represented in blue, while Cu chaperones are in orange, green and pink. Dotted lines indicate putative Cu delivery pathways; continuous lines represent interactions demonstrated by yeast two-hybrid, and discontinuous lines mean the lack of interaction unless the protein is processed. Question marks indicate unclear chaperones or steps. COPT, copper transporter; COX, cytochrome c oxidase; CCH, copper chaperone; ATX1, antioxidant 1; CCS, Cu chaperone for Cu/ZnSOD; SOD, superoxide dismutase; HMA, heavy metal P-type ATPase; RAN1, responsive-to-antagonist 1; PAA, P-type ATPase of Arabidopsis; PC, plastocyanin.
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The Arabidopsis CCH protein was the first Cu chaperone described in plants (Himelblau et al. 1998). Although constitutively expressed, CCH is up-regulated in leaves undergoing senescence (Himelblau et al. 1998). At its amino-terminal domain, CCH exhibits the conserved features of the ATX1-like metallochaperone family including a predicted overall βαββαβ fold structure and an MxCxxC Cu+-binding motif. However, CCH presents a plant-exclusive carboxy-terminal domain with special structural characteristics (Mira, Martinez-Garcia & Penarrubia 2001a; Mira et al. 2001b; Mira et al. 2004). This domain adopts an extended conformation in solution and forms well-ordered amyloid-like fibrils. Moreover, in the presence of anionic detergents, the carboxy-terminal domain displays altered electrophoretic mobility (Mira et al. 2004). Because CCH is present in phloem-enucleated sieve elements, a plant-specific role in Cu symplasmic transport through the plasmodesmata during senescence-associated nutrient mobilization has been proposed for this extra carboxy-terminal domain of CCH (Mira et al. 2001a) (Fig. 3). Low CCH expression in cells lacking functional plasmodesmata, such as pollen, is consistent with this putative function (Table 3). The Arabidopsis genome contains a gene encoding an additional Atx1 homolog (MIPS code: At1g66240), which lacks the CCH plant-specific carboxy-terminal extension. Recent yeast two-hybrid studies show interaction between Arabidopsis ATX1 metallochaperone, and both RAN1 and HMA5 P-type ATPases metal-binding domains (Andres-Colas et al. 2006 and our unpublished results) (Fig. 3). Interestingly, deletion of the CCH carboxy-terminal domain is required for the interaction between the metallochaperone and P-type ATPases metal binding domains (Andres-Colas et al. 2006, and our unpublished results). These results suggest either a regulatory role for the CCH carboxy-terminal extension, the processing of this domain prior to interaction with ATPases or the requirement for an intermediate regulatory protein. Copper delivery to other P-type ATPases, such as chloroplastic PAA1 and PAA2, has not been described so far in higher plants. Because Cu delivery to Cu-transporting ATPases for thylakoid import in chloroplast-related cyanobacteria involves Atx1-like metallochaperones (Banci et al. 2006), candidates for higher plant chloroplast Cu chaperones include CCH and ATX1, as well as other proteins with ATX1-like Cu+-binding motifs encoded by the Arabidopsis genome (Wintz & Vulpe 2002) (Fig. 3).
Yeast Ccs1 contains three conserved and functionally distinct domains: the amino-terminal domain bears striking homology to the Atx1 metallochaperone; the central domain with sequence homology to its target protein Cu/ZnSOD, which physically interacts with SOD; and the carboxy-terminal domain that carries a conserved CxC motif that plays a crucial role in copper transfer to Cu/ZnSOD (Casareno, Waggoner & Gitlin 1998; Schmidt et al. 1999). Furthermore, Ccs1 oxidizes an intrasubunit disulphide in Cu/ZnSOD in an oxygen-dependent process necessary for dimerization into the catalytically active form (reviewed by Culotta et al. 2006). The Arabidopsis genome contains only one gene with significant homology to CCS metallochaperone (Table 2), but three Cu/ZnSOD isoforms that localize to cytosol, chloroplast and peroxisome. This raises the question of how plants specifically deliver Cu to three different subcellularly located SODs with just one chaperone. Recent results show that Arabidopsis CCS rescues the growth defects associated with yeast ccs1Δ mutants, and localizes to chloroplasts in plant cells (Abdel-Ghany et al. 2005a). However, ccs T-DNA insertion lines show a decrease in all three Cu/ZnSOD activities (Chu et al. 2005). Furthermore, a CCS truncated form lacking the 66 amino-terminal amino acids, which target the protein to the chloroplasts, only rescues cytosolic and peroxisomal Cu/ZnSOD activities (Chu et al. 2005). These results suggest that CCS dual targeting to the chloroplasts and the cytosol would allow proper Cu delivery to different SODs (Fig. 3). It has been demonstrated that mammalian and Caenorhabditis elegans Cu/ZnSOD can acquire Cu by a not yet completely understood CCS-independent mechanism (Carroll et al. 2004; Jensen & Culotta 2005). This pathway for Cu delivery would explain the basal Cu/ZnSOD activities and the lack of a more severe phenotype associated with Arabidopsis ccs knockout mutants.
Studies in yeast postulated that Cox17 is the Cu chaperone implicated in Cu trafficking to the mitochondrial COX (reviewed by Cobine et al. 2006). However, a Cox17 protein tethered to the mitochondrial inner membrane resulted in complementation of the respiratory defect of yeast cox17Δ mutants (Maxfield, Heaton & Winge 2004). Most mitochondrial Cu is located within the matrix in a soluble and accessible non-proteinaceous form, suggesting that this pool can be the source of Cu for Cox17 (Cobine et al. 2004). Within the inner membrane space, Cox17 delivers Cu to two mitochondrial inner membrane proteins, Sco1 and Cox11, which are thought to be Cu donors for the COX CuA and CuB sites, respectively (reviewed by Cobine et al. 2006). Although two Cox17 homologs have been identified in Arabidopsis by complementation of the respiratory deficiency of yeast cox17Δ mutants (Balandin & Castresana 2002; Wintz & Vulpe 2002) (Fig. 3 and Table 2), further studies are necessary to determine their role within the plant.
The presence of putative Cu2+ transporters in plants (e.g. HMA1) suggests that additional mechanisms may exist for proper Cu delivery. In this sense, the Arabidopsis CUTA protein, which binds Cu2+ and localizes to the intermembrane chloroplastic space, has been proposed as a candidate for a Cu2+ metallochaperone (Burkhead et al. 2003).
Once incorporated into the root, Fe and Cu are loaded into the xylem sap by a not yet characterized system. It has been proposed that organic acids, especially citrate, are the main metal-chelators in xylem (Rauser 1999; Curie & Briat 2003; Hell & Stephan 2003) (Fig. 1). A key component of Fe xylem loading is FRD3, a member of the multidrug and toxin efflux (MATE) family of small molecule transporters. Arabidopsis plants defective in FRD3 exhibit constitutive Fe deficiency responses independently of Fe supply and are chlorotic, in spite of accumulating elevated Fe levels (Rogers & Guerinot 2002). The explanation for these apparently contradictory results is that in frd3 mutants, Fe accumulates within the xylem, while the Fe content in leaf protoplasts is half the level of wild type (Green & Rogers 2004). Interestingly, it has been recently shown that FRD3 is capable of efflux citrate when expressed in Xenopus oocytes (E.E. Rogers, personal communication) (Fig. 1).
The enhanced demand of phloem-mediated transport of metals during reproductive phases, especially under mineral deficiency, poses the question of the identity of the associated ligands. Studies with the castor bean Ricinus communis implicate iron transport protein (ITP), a 96-amino acid protein with high similarity to the stress-related family of late embryogenesis abundant proteins, and the non-proteinogenic amino acid nicotianamine (NA) in phloem Fe and maybe Cu distribution (Kruger et al. 2002; Hell & Stephan 2003). ITP binds Fe3+in vivo in the phloem, but it also complexes Cu2+ and other metals in vitro (Kruger et al. 2002) (Fig. 1). NA is a ubiquitous metal-chelator in all plants, which is synthesized by nicotianamine synthase (NAS) from S-adenosyl-L-methionine. The first evidence for a role of NA in internal metal transport came from Cu and Fe-related phenotypes associated with the NA synthesis-defective chloronerva tomato mutant, which shows interveinal chlorosis (Ling et al. 1996; Mori 1999). In Strategy II plants, NA is a substrate for nicotianamine aminotransferase (NAAT), acting as intermediate in the phytosiderophore biosynthesis pathway. Given that NAAT is absent in Strategy I plants, NA has been greatly reduced in tobacco plants by expressing a barley NAAT (Takahashi et al. 2003). NA-defective tobacco plants exhibit phenotypes that point to the essentiality of NA for metal transport in veins and interveinal areas, and for reproductive growth and fertility (Takahashi et al. 2003) (Fig. 1). Because NA is mostly complexed with Fe2+ in the phloem (von Wiren et al. 1999), while transported as an Fe3+-ITP complex, it has been proposed that NA can act as a shuttle by chelating Fe2+ from ITP-bound Fe3+ during loading and unloading of Fe in the phloem (Kruger et al. 2002). If this is the case, a reduction system, perhaps including an FRO family member, would also be necessary, as well as the participation of specific Fe2+-NA transporters.
The primary uptake of Fe3+-phytosiderophore complexes in Strategy II plants is mediated by a member of the oligopeptide transporter (OPT) superfamily known in maize as yellow stripe 1 (YS1) (Curie et al. 2001). Yeast complementation assays and transport studies with Xenopus oocytes demonstrate that YS1 encodes a proton-coupled transporter for phytosiderophores and NA broad-range metal chelates (Schaaf et al. 2004). Despite the fact that Strategy I plants do not synthesize and secrete phytosiderophores, they express multiple yellow stripe-like (YSL) genes (Table 1), which have been proposed to function in transport of Fe-NA complexes. Arabidopsis YSL1 loss-of-function mutants contain lower levels of Fe-NA in their seeds and display a transient defect in germination that can be rescued by Fe supply (Le Jean et al. 2005). An exacerbated phenotype is observed for ysl1ysl3 double knockout mutants, which exhibit interveinal chlorosis in leaves caused by decreased Fe levels, and reduced fertility as a consequence of defective anther and embryo development (Waters et al. 2006). These phenotypes are at least partially rescued by addition of exogenous Fe and ectopic expression of YSL3. Interestingly, ysl1ysl3 double mutants are less efficient in mobilizing metals, especially Cu, from senescent leaves. These results, and YSL1/YSL3 expression in the vasculature of shoots and reproductive organs, suggest a function in metal delivery from vascular tissues, as well as in Fe-NA delivery to seeds (Waters et al. 2006) (Fig. 1). Localization of Arabidopsis YSL2 to root endodermis and pericycle cells facing the meta-xylem tubes has suggested that it also participates in lateral movement of Fe and/or Cu within the veins (DiDonato et al. 2004; Schaaf et al. 2005). However, further studies are necessary to elucidate YSL2 substrate specificity, because contradictory results have been reported for YSL2-mediated transport in yeast (DiDonato et al. 2004; Schaaf et al. 2005).
The assumption that the members of the OPT family transport only peptides is being challenged by the surprising discovery that some OPTs may also be capable of divalent metal ion transport. AtOPT2 and AtOPT3 are differentially expressed under metal deficiencies, and heterologous expression of AtOPT3 in yeast suggests that it can transport Cu2+, Mn2+ and Fe2+ (Wintz et al. 2003).