Recent progress in structure–function analyses of Nramp proton-dependent metal-ion transportersThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 960-978 ◽  
Author(s):  
P. Courville ◽  
R. Chaloupka ◽  
M.F.M. Cellier
Keyword(s):  
Structure Function ◽  
Metal Ion ◽  
Hypochromic Anemia ◽  
Experimental Models ◽  
Natural Resistance ◽  
Bacterial Cells ◽  
Divalent Metal Ions ◽  
Recycling Endosomes ◽  
Transmembrane Segments ◽  
Macrophage Protein

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me2+, including Mn2+, Fe2+, Co2+, and Cd2+). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me2+ from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure–function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me2+ and proton H+ transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1and 6 as crucial structural segments for Me2+ and H+ symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me2+ homeostasis in human health and diseases.

scholarly journals Mycobacterium tuberculosis Expresses a Novel Ph-Dependent Divalent Cation Transporter Belonging to the Nramp Family

1999 ◽  
Vol 190 (5) ◽  
pp. 717-724 ◽  
Author(s):  
Daniel Agranoff ◽  
Irene M. Monahan ◽  
Joseph A. Mangan ◽  
Philip D. Butcher ◽  
Sanjeev Krishna
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metal Ion ◽  
Divalent Cations ◽  
Natural Resistance ◽  
Xenopus Laevis Oocytes ◽  
Mrna Levels ◽  
Susceptibility To Infection ◽  
Reverse Transcription Pcr ◽  
Acidic Extracellular Ph ◽  
Macrophage Protein

Mammalian natural resistance–associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe2+, Mn2+, Zn2+, and Cu2+. Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces ∼20-fold increases in 65Zn2+ and 55Fe2+ uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated 65Zn2+ and 55Fe2+ transport is abolished by an excess of Mn2+ and Cu2+, confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe2+ and Cu2+ between <1 and 5 μM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5–70 μM Cu2+. M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter

2001 ◽  
Vol 354 (3) ◽  
pp. 511-519 ◽  
Author(s):  
Tapasree GOSWAMI ◽  
Arin BHATTACHARJEE ◽  
Paul BABAL ◽  
Susan SEARLE ◽  
Elizabeth MOORE ◽  
...  
Keyword(s):  
Metal Ions ◽  
Metal Ion ◽  
Proton Gradient ◽  
Natural Resistance ◽  
Bivalent Cation ◽  
Bivalent Cations ◽  
Carbonyl Cyanide ◽  
Endosomal Membranes ◽  
Data Points ◽  
Macrophage Protein

In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1G169; P = 3.22×10-6) and human NRAMP1 (P = 3.87×10-5) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH5.5, Nramp1G169 (P = 1.34×10-13) and NRAMP1 (P = 1.09×10-6) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130±57 nA were evoked by 100µM Zn2+ at pH7.5, and 139±47 nA by 100µM Fe2+ at pH7.0, in Nramp1G169 oocytes; currents of 254±49 nA and 242±26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614nM for Nramp1G169 and approx. 562nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

scholarly journals Manganese is a Physiologically Relevant TORC1 Activator in Yeast and Mammals.

2021 ◽  
Author(s):  
Raffaele Nicastro ◽  
Helene Gaillard ◽  
Laura Zarzuela ◽  
Elisabet Fernandez-Garcia ◽  
Mercedes Tome ◽  
...  
Keyword(s):  
Metal Ion ◽  
Human Diseases ◽  
Natural Resistance ◽  
Homeostatic Process ◽  
Downstream Effectors ◽  
Mtorc1 Signaling ◽  
The Family ◽  
Macrophage Protein

The essential biometal manganese (Mn) functions as a cofactor for several enzymatic activities that are critical for the prevention of human diseases. Whether intracellular Mn levels may also modulate signaling events has so far remained largely unexplored. The target of rapamycin complex 1 (TORC1, mTORC1 in mammals) is a conserved protein kinase complex that requires metal co-factors to phosphorylate its downstream effectors as part of a central, homeostatic process that coordinates cell growth and metabolism in response to nutrient and/or growth factor availability. Using genetic and biochemical approaches, we show here that TORC1 activity is exquisitely sensitive to stimulation by Mn both in vivo and in vitro. Mn-mediated control of TORC1 depends on Smf1 and Smf2, two members of the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters, the turnover of which is subjected to feedback control by TORC1 activity. Notably, increased Mn levels and consequent activation of TORC1 cause retrograde dysregulation and antagonize the rapamycin-induced gene expression and autophagy programs in yeast. Because Mn also activates mTORC1 signaling in aminoacid starved human cells, our data indicate that intracellular Mn levels may constitute an evolutionary conserved physiological cue that modulates eukaryotic TORC1/mTORC1 signaling. Our findings therefore reveal a hitherto elusive connection between intracellular Mn levels, mTORC1 activity, and human diseases.

scholarly journals The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel

2004 ◽  
Vol 385 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Daniel AGRANOFF ◽  
Lauren COLLINS ◽  
David KEHRES ◽  
Tom HARRISON ◽  
Michael MAGUIRE ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Metal Ion ◽  
Ph Dependence ◽  
Opportunistic Pathogen ◽  
Membrane Topology ◽  
Natural Resistance ◽  
Xenopus Laevis Oocytes ◽  
Bivalent Cation ◽  
Gradient Based ◽  
Macrophage Protein

Cryptococcus neoformans is an important human opportunistic pathogen and a facultative intracellular parasite, particularly in HIV-infected individuals. Little is known about metal ion transport in this organism. C. neoformans encodes a single member of the Nramp (natural resistance-associated macrophage protein) family of bivalent cation transporters, known as Cramp, which we have cloned and expressed in Xenopus laevis oocytes and Spodoptera frugiperda Sf 21 insect cells. Cramp induces saturable transport of a broad range of bivalent transition series cations, including Mn2+, Fe2+, Co2+ and Ni2+. Maximal cation transport occurs at pH 5.5–6.0, consistent with the proton gradient-based energetics of other Nramp orthologues. Mn2+ transport is diminished in the presence of 140 mM Na+, compatible with a Na+ slippage mechanism proposed for the Saccharomyces cerevisiae Nramp orthologue Smf1p. Cramp resembles Smf1p with respect to predicted membrane topology, substrate specificity and pH dependence, but differs in terms of its apparent affinity for Mn2+ and negligible inhibition by Zn2+. Cramp is the first Nramp orthologue from a fungal pathogen to be functionally characterized. Insights afforded by these findings will allow the formulation of new hypotheses regarding the role of metal ions in the pathophysiology of cryptococcosis.

scholarly journals Bioinformatics and Transcriptional Study of the Nramp Gene in the Extreme Acidophile Acidithiobacillus ferrooxidans Strain DC

Minerals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 544 ◽  
Author(s):  
Bo Miao ◽  
Li Shen ◽  
Xueduan Liu ◽  
Weimin Zeng ◽  
Xueling Wu
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Metal Ion ◽  
Cytoplasmic Membrane ◽  
Ferrous Ion ◽  
Natural Resistance ◽  
Online Tools ◽  
The Family ◽  
Bioinformatics Software ◽  
Macrophage Protein ◽  
High Concentrations

The family of Nramp (natural resistance-associated macrophage protein) metal ion transporter functions in diverse organisms from bacteria to humans. Acidithiobacillus ferrooxidans (At. ferrooxidans) is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous ion (600 mM). The AFE_2126 protein of At. ferrooxidans of the Dachang Copper Mine (DC) was analyzed by bioinformatics software or online tools, showing that it was highly homologous to the Nramp family, and its subcellular localization was predicted to locate in the cytoplasmic membrane. Transcriptional study revealed that AFE_2126 was expressed by Fe2+-limiting conditions in At. ferrooxidans DC. It can be concluded that the AFE_2126 protein may function in ferrous ion transport into the cells. Based on the ΔpH of the cytoplasmic membrane between the periplasm (pH 3.5) and the cytoplasm (pH 6.5), it can be concluded that Fe2+ is transported in the direction identical to that of the H+ gradient. This study indirectly confirmed that the function of Nramp in At. ferrooxidans DC can transport divalent iron ions.

scholarly journals The Iron Transport Protein NRAMP2 Is an Integral Membrane Glycoprotein That Colocalizes with Transferrin in Recycling Endosomes

1999 ◽  
Vol 189 (5) ◽  
pp. 831-841 ◽  
Author(s):  
Samantha Gruenheid ◽  
François Canonne-Hergaux ◽  
Susan Gauthier ◽  
David J. Hackam ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Cell Types ◽  
Integral Membrane Protein ◽  
Tissue Expression ◽  
Natural Resistance ◽  
Macrophage Cell ◽  
Recycling Endosomes ◽  
Endosomal Membrane ◽  
Lower Extent ◽  
Macrophage Protein ◽  
Insight Into

The natural resistance associated macrophage protein (Nramp) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90–100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe2+ across the endosomal membrane and into the cytoplasm.

scholarly journals Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

2021 ◽  
Vol 89 (1-2) ◽  
pp. 103-116
Author(s):  
Julie Baker Phillips ◽  
David H. Ardell
Keyword(s):  
Structure Function ◽  
Metal Ion ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Heterologous Gene ◽  
Multigene Families ◽  
Ion Binding ◽  
Trna Genes ◽  
Structural Factors ◽  
Trna Structure

AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes

2001 ◽  
Vol 204 (6) ◽  
pp. 1053-1061 ◽  
Author(s):  
A. Sacher ◽  
A. Cohen ◽  
N. Nelson
Keyword(s):  
Xenopus Laevis ◽  
Metal Ions ◽  
Metal Ion ◽  
Divalent Cations ◽  
Ion Uptake ◽  
Xenopus Laevis Oocytes ◽  
Ion Transporters ◽  
Divalent Metal Ions ◽  
Transport Pathway ◽  
Metal Ion Uptake

Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life. Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells. Little is known about the transport mechanism of metal ions by eukaryotic cells. Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters. Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a ‘slippage’ phenomenon of different monovalent cations unique to each transporter. Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties. We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to ‘slip’ through the DCT1 transporter. A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells. 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials. In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials. Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered. Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential. A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed. The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.

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