A Functional Study Identifying Critical Residues Involving Metal Transport Activity and Selectivity in Natural Resistance-Associated Macrophage Protein 3 in Arabidopsis thaliana

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1δ, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1δ. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.

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Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

10.1101/402412 ◽

2018 ◽

Cited By ~ 3

Author(s):

Aaron T. Bozzi ◽

Lukas B. Bane ◽

Christina M. Zimanyi ◽

Rachelle Gaudet

Keyword(s):

Metal Binding ◽

Voltage Dependence ◽

Mechanistic Model ◽

Salt Bridge ◽

Metal Transport ◽

Natural Resistance ◽

Metal Type ◽

Proton Symport ◽

Macrophage Protein ◽

Stimulation Of

AbstractSecondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate “coupling”. We untangle the “proton-metal coupling” behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: ΔpH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.

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Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis

Membranes ◽

10.3390/membranes10100255 ◽

2020 ◽

Vol 10 (10) ◽

pp. 255

Author(s):

Matthias Gantner ◽

Theodoros Laftsoglou ◽

Honglin Rong ◽

Vincent L. G. Postis ◽

Lars J. C. Jeuken

Keyword(s):

Transition Metals ◽

Enterococcus Faecalis ◽

Fluorescent Dyes ◽

Metal Transport ◽

Natural Resistance ◽

Experimental Conditions ◽

Metal Transporters ◽

Supported Membrane ◽

Essential Trace Elements ◽

Macrophage Protein

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.

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Association between rheumatoid arthritis and genetic variants of natural resistance-associated macrophage protein 1 gene: A meta-analysis

International Journal of Rheumatic Diseases ◽

10.1111/1756-185x.13366 ◽

2018 ◽

Vol 21 (9) ◽

pp. 1651-1658 ◽

Cited By ~ 4

Author(s):

Hong Wang ◽

Fei-Fei Yuan ◽

Zi-Wei Dai ◽

Bin Wang ◽

Dong-Qing Ye

Keyword(s):

Rheumatoid Arthritis ◽

Genetic Variants ◽

Meta Analysis ◽

Natural Resistance ◽

Macrophage Protein

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Natural resistance-associated macrophage protein 1 gene polymorphisms and response to vaccine against or challenge with Salmonella enteritidis in young chicks

Poultry Science ◽

10.1093/ps/82.2.259 ◽

2003 ◽

Vol 82 (2) ◽

pp. 259-266 ◽

Cited By ~ 43

Author(s):

W Liu ◽

MG Kaiser ◽

SJ Lamont

Keyword(s):

Gene Polymorphisms ◽

Salmonella Enteritidis ◽

Natural Resistance ◽

Macrophage Protein ◽

Young Chicks

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Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import

The Journal of General Physiology ◽

10.1085/jgp.201912428 ◽

2019 ◽

Vol 151 (12) ◽

pp. 1413-1429 ◽

Cited By ~ 7

Author(s):

Aaron T. Bozzi ◽

Lukas B. Bane ◽

Christina M. Zimanyi ◽

Rachelle Gaudet

Keyword(s):

Proton Transport ◽

Metal Uptake ◽

Deinococcus Radiodurans ◽

Salt Bridge ◽

Metal Transport ◽

Natural Resistance ◽

Transport Pathway ◽

Directional Bias ◽

Proton Cotransport ◽

Stimulation Of

Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by “coupling” proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp’s structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.

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