Put the metal to the petal: metal uptake and transport throughout plants

Compared to other organisms, plants have expanded families of transporters that are involved in the uptake and efflux of metals. Fortunately, in many cases, the examination of double mutants has been sufficient to overcome the challenge of studying functionally redundant gene families. Plants that lack two heavy-metal-transporting P-type ATPase family members (HMA2 and HMA4) reveal a function for these transporters in Zn translocation from roots to shoots. Likewise, the phenotype of plants that lack two natural resistance associated macrophage protein (NRAMP) homologs (NRAMP3 and NRAMP4) implicate these metal uptake proteins in the mobilization of vacuolar Fe stores during seed germination. Most families of metal transporters are ubiquitous but the Yellow Stripe1-Like (YSL) family is plant specific and YSL family members have been implicated in the transport of metals that are complexed with a plant specific chelator called nicotianamine (NA).     

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene. In order to facilitate further studies on this important gene, we now report the nearly complete structure of the bovine NRAMP 1 gene, including sizes and positions of 13 introns relative to the bovine NRAMP 1 gene coding sequence and the DNA sequence of intron-exon junctions. Comparison of the bovine, murine and human NRAMP 1 gene structures revealed a high degree of conservation in intron placement, though the lengths of several introns were less-well conserved. In general, the greatest divergence in intron lengths occurred in regions of the NRAMP 1 gene displaying the lowest coding sequence conservation. In addition, mutations near intron-exon junctions could account for 25 of the 75 total amino acid differences between murine and bovine NRAMP 1. Using information gained through this study, it was possible to rapidly identify a novel polymorphism within the bovine NRAMP 1 gene intron X. This polymorphism was shown by direct DNA sequence analysis to consist of insertion of three guanine nucleotides at positions 37,40 and 98 relative to the intron X start point. Initial scans of several cattle breeds suggest that the two intron X alleles identified here are stable and widespread in the Bos taurus population.     

Polymorphism in the rhesus macaque (Macaca mulatta) NRAMP1 gene: lack of an allelic association to tuberculosis susceptibility

Although previous tuberculosis (TB) research has suggested that underlying genetic factors influence a host's response and ability to survive Mycobacterium infection, only recently has a gene been identified, the 'natural resistance-associated macrophage protein 1' (NRAMP1) gene, which provides a degree of natural resistance to infection by some Mycobacterium species. To date, however, the role that NRAMP1 may play in resistance to Mycobacterium infection has only been examined in mouse and man. Here, we present data generated at NRAMP1 among a group of rhesus macaques (Macaca mulatta) that were euthanized because of an outbreak of Mycobacterium tuberculosis during quarantine. Data were also generated on unrelated (and healthy) rhesus macaques in order to better determine the frequency and degree of genetic polymorphism within Macaca at the NRAMP1 locus. These data represent the first study designed to examine the role that NRAMP1 may play in TB susceptibility among rhesus macaques.     

The NRAMP family of metal-ion transporters

The family of NRAMP metal ion transporters functions in diverse organisms from bacteria to human. NRAMP1 functions in metal transport across the phagosomal membrane of macrophages, and defective NRAMP1 causes sensitivity to several intracellular pathogens. DCT1 (NRAMP2) transport metal ions at the plasma membrane of cells of both the duodenum and in peripheral tissues, and defective DCT1 cause anemia. The driving force for the metal-ion transport is proton gradient (protonmotive force). In DCT1 the stoichiometry between metal ion and proton varied at different conditions due to a mechanistic proton slip. Though the metal ion transport by Smf1p, the yeast homolog of DCT1, is also a protonmotive force, a slippage of sodium ions was observed. The mechanism of the above phenomena could be explained by a combination between transporter and channel mechanisms.     

Association study of the NRAMP1 gene promoter polymorphism and early-onset type 1 diabetes

Natural resistance-associated macrophage protein 1 (NRAMP1) has an important role in regulating macrophage functions that affect innate resistance as well as immune responses. We analyzed the microsatellite polymorphism in the promoter region of the human NRAMP1 gene in 206 type 1 diabetes patients and 200 normal children to determine whether this polymorphism might be associated with type 1 diabetes in the Japanese population. The frequency of allele 2 (180 bp) of the promoter microsatellite polymorphism of the NRAMP1gene was slightly lower in the early-onset population (2-10 years of age) of type 1 diabetes patients than in controls, although the difference did not reach statistical significance. The association study of the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene, located near the NRAMP1 gene, and type 1 diabetes showed that the CTLA-4 gene significantly contributed to the development of type 1 diabetes, whereas NRAMP1 had an additional effect on the onset of type 1 diabetes in the young population.     

Cloning, sequencing and linkage mapping of the NRAMP1 gene of sheep and deer

The ovine NRAMP1 and cervine NRAMP1 cDNAs were cloned by RT PCR of RNA derived from macrophage enriched leukocyte preparations. The complete coding and 3' regions were sequenced. Both sheep and deer NRAMP1 proteins are 548 amino acids long. There are 77 and 73 amino acid differences, respectively, compared to the mouse Nramp1 sequence. Dinucleotide repeats were found in both the ovine and cervine 3' non-coding sequence. Amplification of these regions in individual sheep and deer showed them to be polymorphic micro-satellites. They have polymorphism information content values of 0·76 and 0·84 in sheep and deer, respectively. Using these microsatellites, the ovine NRAMP1 gene was mapped in a linkage group on ovine chromosome 2q and cervine NRAMP1 was mapped in a linkage group syntenic with human chromosome 2, mouse chromosome 1 and sheep chromosome 2.     

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine‐based motif

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP‐fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild‐type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N‐terminal dileucine‐based motif. The NRAMP3 or NRAMP4 N‐terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4‐dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N‐terminal dileucine‐based motif as well as AP4.      

Reciprocal inhibition of Cd and Pb sulfocomplexes for uptake in Caco-2 cells

Cadmium-lead interactions for uptake were studied in the TC7 clone of human enterocytic-like Caco-2 cells as a function of inorganic metal speciation. We have previously shown that Cd uptake in these cells involves both the free cation Cd2+ and chlorocomplex (CdCln(2-n)) species. Here we show 1.9 times higher uptake levels for 109CdCln(2-n) compared to 210PbCln(2-n). Reciprocal inhibitions of chlorocomplexes were observed with a much higher inhibitory effect of Cd compared to Pb. Replacing Cl- by NO3- increased both the level of aquo ion 109Cd2+ and 109Cd accumulation. In contrast, higher levels of 210Pb2+ did not favor 210Pb uptake. For both metals, higher uptake data were recorded in the presence of SO4(2-), leading to sulfocomplex formation, compared with Cl-. Reciprocal inhibitions were minimal at high-cation levels but were significant and comparable in the presence of sulfo-complexes. We conclude that, in addition to Cd2+ (but not Pb2+), sulfocomplexes of both metals would preferentially be taken up compared to chlorocomplexes. NRAMP2 is not involved in Pb2+ uptake, and the NRAMP2-mediated Cd2+ uptake is insensitive to Pb. Uptake of Pb chlorocomplexes could involve specific mechanisms but of very low affinity, whereas uptake of Pb sulfocomplexes occurs with high affinity.     

Genotype and allele frequencies of divalent metal transporter 1 polymorphism in Turkish population

Divalent metal transporter 1 (DMT1 also known as DCT1, NRAMP2 or SCL11A2) is a membrane-bound divalent metal transporter which is conserved from prokaryotes to higher eukaryotes. It has been postulated to play important roles in intestinal iron absorption at the brush border of duodenal enterocytes, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. DMT1 gene which contains at least four isoforms (1A/+IRE, 1A/-IRE, 2/+IRE and 2/-IRE) is located on chromosome 12q13 in human. DMT1 mediates the transport of a wide range of metals, including the essential metals Fe2+, Zn2+, Mn2+, Cu2+, Co2+, Ni2+ and toxic metals such as Cd2+ and Pb2+. The intention of this study is to determine that IVS4+44C/A single nucleotide polymorphism in DMT1 gene of Turkish population. For this purpose blood samples from 192 female and 192 male volunteers were analyzed. DMT1 gene was amplified with the polymerase chain reaction-restriction fragment length polymorphism technique and 351 bp oligonucleotide was produced. The amplified oligonucleotides were cut with MnlI restriction enzyme according to their polymorphic characteristics. Digested and undigested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator. The genotype frequencies of DMT1 IVS4+44C/A polymorphism were determined as 47.9% for CC, 40.1% for AC and 12.0% for AA genotypes. The frequency of the C allele was found to as 68.0% and of the A allele as 32.0%. The genotype frequencies were consistent with Hardy-Weinberg equilibrium (χ2=2.394; Exact P=0.128).     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.     

Identification of Polymorphisms and Sequence Variants in the Human Homologue of the Mouse Natural Resistance–Associated Macrophage Protein Gene

The most common mycobacterial disease in humans is tuberculosis, and there is evidence for genetic factors in susceptibility to tuberculosis. In the mouse, the Bcg gene controls macrophage priming for activation and is a major gene for susceptibility to infection with mycobacteria. A candidate gene for Bcg was identified by positional cloning and was designated “natural resistance-associated macrophage protein gene” (Nramp1), and the human homologue (NRAMP1) has recently been cloned. Here we report on (1) the physical mapping of NRAMP1 close to VIL in chromosome region 2q35 by PCR analysis of somatic cell hybrids and YAC cloning and (2) the identification of nine sequence variants in NRAMP1. Of the four variants in the coding region, there were two missense mutations and two silent substitutions. The missense mutations were a conservative alanine-to-valine substitution at codon 318 in exon 9 and an aspartic acid–to-asparagine substitution at codon 543 in the predicted cytoplasmic tail of the NRAMP1 protein. A microsatellite was located in the immediate 5' region of the gene, three variants were in introns, and one variant was located in the 3' UTR. The allele frequencies of each of the nine variants were determined in DNA samples of 60 Caucasians and 20 Asians. In addition, we have physically linked two highly polymorphic microsatellite markers, D2S104 and D2S173, to NRAMP1 on a 1.5-Mb YAC contig. These molecular markers will be useful to assess the role of NRAMP1 in susceptibility to tuberculosis and other macrophage-mediated diseases.     

Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells

NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal transporter 1) is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates active proton-coupled transport. Recently, it has been shown that the microcytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical membrane iron transporter in intestinal epithelial cells and the endosomal iron transporter in transferrin cycle endosomes of other cells. To investigate the cellular functions of NRAMP2, we generated a polyclonal antibody against the N-terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2 N-terminal antibody recognized a 90-116-kDa membrane-associated protein, and this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F. Subcellular fractionation revealed that NRAMP2 co-sedimented with the late endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not with the transferrin receptor in early endosomes. The intracellular localization of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in vesicular structures and were colocalized with LAMP-2, but not with EEA1 (early endosome antigen 1) or the transferrin receptor. These results indicated that NRAMP2 is localized to the late endosomes and lysosomes, where NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in the transferrin cycle.     

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.