2.A.55 The Metal Ion (Mn2+-iron) Transporter (Nramp) Family
Homologues of this family are found in various yeasts, plants, animals, archaea, and Gram-negative and Gram-positive bacteria termed ''natural resistance-associated'' macrophage protein (NRAMP) proteins because one of the animal homologues plays a role in resistance to intracellular bacterial pathogens such as Salmonella enterica serovar Typhimurium, Leishmania donovani and Mycobacterium bovis. The natural history of SLC11 genes in vertebrates has been discussed by Neves et al. (2011). Proposed to be a distant member of the DMT Superfamily (TC# 2.A.7), several human pathologies may result from defects in Nramp-dependent Fe2+ or Mn2+ transport, including iron overload, neurodegenerative diseases and innate susceptibility to infectious diseases (Cellier 2012). Nramp-driven transport of iron across membranes is selective for ferrous ions although iron is mostly present as ferric ions in the growth media and in engulfed bacteria (Peracino et al. 2022).
Humans and rodents possess two distinct NRAMPs. The broad specificity NRAMP2 (DMT1), which transports a range of divalent metal cations, transports Fe2+ and H+ with a 1:1 stoichiometry and apparent affinities of 6 μm and about 1 μm, respectively. Variable H+:Fe2+ stoichiometry has also been reported. The order of substrate preference for NRAMP2 is Fe2+> Zn2+> Mn2+> Co2+> Ca2+> Cu2+> Ni2+> Pb2+. Many of these ions can inhibit iron absorption. Mutation of Nramp2 in rodents leads to defective endosomal iron export within the ferritin cycle, impaired intestinal iron absorption and microcytic anemia. Symptoms of Mn2+ deficiency are also seen. It is found in apical membranes of intestinal epithelial cells but also in late endosomes and lysosomes.
In contrast to the widely expressed NRAMP2, NRAMP1 is expressed primarily in macrophages and monocytes and appears to have a preference for Mn2+ rather than Fe2+. NRAMP1 (TC# 2.A.55.2.3) has been reported to function by metal:H+ antiport (Techau et al., 2007). It is hypothesized that a deficiency for Mn2+ or some other metal prevents the generation of reactive oxygenic and nitrogenic compounds that are used by macrophage to combat pathogens. This hypothesis is supported by studies on the bacterial NRAMP homologues which exhibit extremely high selectivity for Mn2+ over Fe2+, Zn2+ and other divalent cations. Regulation of these transporters in bacteria can occur through Fur, OxyR, and most commonly a DtxR homolog, MntR.
The Smf1 protein of Saccharomyces cerevisiae appears to catalyze high-affinity (Km = 0.3 μm) Mn2+ uptake while the closely related Smf2 protein may catalyze low affinity (Km = 60 μm) Mn2+ uptake in the same organism. Both proteins also mediate H+-dependent Fe2+ uptake. These proteins are of 575 and 549 amino acyl residues in length and are predicted to have 8-12 transmembrane α-helical spanners. The E. coli homologue of 412 aas exhibits 11 putative and confirmed TMSs with the N-terminus in and the C-terminus out. The yeast proteins may be localized to the vacuole and/or the plasma membrane of the yeast cell. Indirect and some direct experiments suggest that they may be able to transport several heavy metals including Mn2+, Cu2+, Cd2+ and Co2+. A third yeast protein, Smf3p, appears to be exclusively intracellular, possibly in the Golgi. Nramp2 (Slc11A2) of Homo sapiens (TC #2.A.55.2.1) has a 12 TMS topology with intracellular N- and C-termini. Two-fold structural symmetry in the arrangement of membrane helices for TM1-5 and TM6-10 (conserved Slc2 hydrophobic core) is suggested (Czachorowski et al., 2009). The NRAMP family genes in tea plant (Camellia sinensis) have been identified (Li et al. 2021).
The Nramp family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. Bozzi et al. 2016 determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Cysteine accessibility scanning results Allowed identification of the metal-permeation pathway in the alternate outward-open conformation. Two natural anemia-causing glycine-to-arginine mutations impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state (Bozzi et al. 2016).
The generalized transport reaction catalyzed by Nramp family proteins is:
Me2+ (out) H+ (out) ⇌ Me2+ (in) H+ (in)
High-affinity Me2+ (Fe2+, Mn2+, Zn2+, Cu2+, Cd2+, Ni2+, Co2+) uptake transporter, Smf1p or ESP1p of 575 aas and 11 TMSs. Important for oxidative stress protection. Its activity is regulated by the Tre1 protein alone, and it's degradation is dependent on the Bsd2, Rsp5, Tre1 and Tre2 proteins (Stimpson et al. 2006).
Eukaryotes, bacteria, archaea
Smf1p of Saccharomyces cerevisiae
Low-affinity Me2+ (Mn2+, Cu2+) uptake transporter, Smf2p. Essential for manganese uptake.
Eukaryotes, bacteria, archaea
Smf2p of Saccharomyces cerevisiae
Manganese transporter, Pdt1, of 584 aas and 11 TMSs. It also transports cadmium (Cd2+ and iron (Fe2+) (Ahmad et al. 2022).
Pdt1 of Schizosaccharomyces pombe
Plasma membrane NRAMP divalent cation (Fe2+ and Cd2+ demonstrated) uptake system of 571 aas and 11 TMSs. Cd2+ down regluates expression (Wei et al. 2015).
NRAMP of Exophiala pisciphila
NRAMP Mn2+ uptake porter, DmtA, of 575 aas and 11 TMSs. DmtA is physiologically important for the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow and export (Fejes et al. 2020).
DmtA of Aspergillus niger
Divalent heavy metal (Fe2+, Zn2+, Mn2+, Cu2+, Cd2+, Co2+, Ni2+ and Pb2+) ion:H+ symporter, Nramp2 or divalent metal transporter, DMT1 = SLC11A2 (Garrick et al. 2003). A 12 TMS topology with intracellular N- and C-termini is established. Two-fold structural symmetry in the arrangement of membrane helices for TMSs 1-5 and TMSs 6-10 (conserved Slc11 hydrophobic core) is suggested (Czachorowski et al., 2009). A conserved motif in a central flexible region of TMS1 (DPGN) binds the metal ion (Wang et al. 2011). It is upregulated by iron deficiency and downregulated by iron loading (Nam et al. 2013). NRAMP2 also serves as the Sindbis alpha virus receptor (Rose et al. 2011). DMT1 interacts with the iron chaparone protein, PCBP2 (Q15366), in an iron-dependent fashion, and may be essential for iron uptake (Lane and Richardson 2014). Mutations cause a syndrome of congenital microcytic hypochromic anemia, poorly responsive to oral iron treatment, with liver iron overload associated paradoxically with normal to moderately elevated serum ferritin levels (Beaumont et al. 2006). Nigral iron accumulation and activation of NMDA receptors contribute to the neurodegeneration of dopamine neurons in Parkinson's disease, and activation of NMDA receptors participates in iron metabolism in the hippocampus (Xu et al. 2018). NMDA receptor inhibitors MK-801 and AP5 protect nigrostriatal projection systems and reduce nigral iron levels. NMDA treatment increased the expression of DMT1 and decreased the expression of the iron exporter ferroportin 1 (Fpn1) (TC# 2.A.100.1.4) (Xu et al. 2018). DMT1 cannot be a direct donor of catalytic copper because it does not have the cytosol domain present in Ctr1, which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes (Ilyechova et al. 2019). Slc11a2 activity is essential for intestinal non-heme iron absorption after birth, and is also required for normal hemoglobin production during the development of erythroid precursors (Gunshin et al. 2005). May also take artemisinin (Girardi et al. 2020). Optimal conditions for Western blotting for this and other proteins requires that the sample not be boiled (Tsuji 2020). DMT1 polymorphisms affect blood lead levels of occupationally exposed individuals (Mani et al. 2021).
DMT1 (SLC11A2) of Homo sapiens
NRAMP2 (NRAMPB; SLC11a2) of 552 aas and 10 TMSs. Inovoled in iron hoemeostasis and infectivity together with NRAMP1 (TC#2.A.55.2.9). Present in the contractile vacuole which regulates osmolarity and possible stores iron (Peracino et al. 2013). Nramp1 and NrampB (Nramp2) are in membranes of macropinosomes and in phagosomes or the contractile vacuole network, respectively (Peracino et al. 2022).
NRAMP2 of Dictyostelium discoideum
NRAMP2 of 596 aas and 12 TMSs. NRAMP2 also serves as the Sindbis alpha virus receptor (Rose et al. 2011).
NRAMP2 of Drosophila melanogaster
VO2+ (vanidate) NRAMP uptake system in vacuoles of vanadocytes (587 aas; Ueki et al. 2011).
Vanidate transporter of Ascidia sydneiensis samea (Vanadium-rich ascidian)
Enterocyte iron uptake system, NRAMP or DMT1 of 558 aas and 13 TMSs. Inhibited by lead and cadmium ions competitively (Kwong et al. 2010). The close (85% identity) homologue from Scophthalmus maximus (Turbot) (Psetta maxima) has been characterized (Chen et al. 2007).
DMT1 of Oncorhynchus mykiss (Rainbow trout) (Salmo gairdneri)
NRAMP3 iron/cadmium transporter of 512 aas (Wei et al. 2009).
NRAMP3 of Noccaea caerulescens
Divalent cation and aluminum transporter, Smf3. Mediates aluminum-induced dopamine neuron degeneration (VanDuyn et al. 2013).
Smf3 of Caenorhabditis elegans
Fe2+/Mn2+ transporter, Smf1 of 562 aas and 12 TMSs (Au et al. 2009).
Smf1 of Caenorhabditis elegans
DMT1, SLC11A2 or NRAMP2, isoform 1, of 564 aas and 12 TMSs. It is an electrogenic Mn2+ transporter that is expressed at high levels in the brush-border membranes of enterocytes (Bai et al. 2008).
DMT1 of Gallus gallus (chicken)
NRAMP4 of 512 aas and 12 TMSs, a vacuolar metal transporter involved in intracellular metal homeostasis. It can transport iron (Fe), manganese (Mn) and cadmium (Cd), and regulates metal accumulation under Fe starvation. It acts redundantly with NRAMP3 (which is 85% identical to it) to mobilize vacuolar Fe and provide sufficient Fe during seed germination (Lanquar et al. 2005). In association with NRAMP3, it is required for optimal growth and photosynthesis under Mn deficiency. It exports Mn from vacuoles in leaf mesophyll cells, making Mn available for functional photosystem II in chloroplasts (Lanquar et al. 2010). Its ortholog in Thlaspi japonicum has been characterized (Mizuno et al. 2005). Expression of particular transmembrane transporters (e.g., members of the ZIP (ZNT1) and NRAMP (NRAMP4) families) leads to metal tolerance and accumulation in plants (Fasani et al. 2021).
NRAMP4 of Arabidopsis thaliana
NRAMP5 of 538 aas and 12 TMSs. Transports Cd2+. Mutations lead to low Cd2+ accumulation in plants and seeds (Cao et al. 2019).
NRAMP5 of Oryza sativa subsp. japonica (Rice)
Me2+ (Fe2+, Cd2+, Co2+):H+ symporter, DCT1 (Nramp2) (Splice variant isoforms serve different functions). The stoichiometry between metal ion and proton in the symport process catalyzed by DCT1 varies under different conditions due to mechanistic proton slip. A single reciprocal mutation, I144F, in TMS2 of DCT1 abolished the metal ion transport activity, increased the slip currents, and generated sodium slip currents (Nevo, 2007). A double mutation, adding F227I in TMS4 to I144F restored the uptake activity of DCT1 and reduced the slip currents. Thus, these regions are important in coupling metal ion and proton symport (Nevo, 2007). NRAMP2 also serves as the Sindbis alpha virus receptor (Rose et al. 2011). Three isoforms are expressed differentially in different cell types and are developmentally regulated (Ding et al. 2013).
DCT1 of Rattus norvegicus
NRAMP1 or DMT1 of 513 aas and 11 or 12 putative TMSs. It shows higher similarity to prokaryotic than eukaryotic homologues. The N-terminus displays exclusively prokaryotic characteristics. Functional complementation experiments revealed that DMT1 has a broad specificity, transporting several divalent metals (manganese, iron, cadmium and copper), but not zinc (Rosakis and Köster 2005).
NARMP1 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
NRAMP of 555 aas and 12 TMSs (Chen et al. 2004). This protein is 93% identical to 2.A.55.2.23.
NRAMP of the red sea bream (Pagrus major)
Ethylene-insensitive protein 2-like isoform X2 of 1302 aas with 12 N-terminal TMSs and a long hydrophilic C-terminal domain. There are 11 nramp genes in the tea genome (Li et al. 2021), and some are expressed in roots, while others are expressed in the leaves and stems. This protein is 52% identical to TC# 2.A.55.2.8 throughout its length, and is therefore probably orthologous to this Arabidopsis protein.
NRAMP metal ion transporter of Camellia sinensis (tea)
Natural resistance-associated macrophage protein, MsNRAMP of 554 aas and 12 TMSs. Chesapeake Bay striped bass (Morone saxatilis) is experiencing an epizootic of mycobacteriosis that threatens the health of this species. Burge et al. 2004 characterized an Nramp gene in this species and show that induction follows Mycobacterium exposure. MsNramp contains all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TMSs 7 and 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TMSs 6 and 7. MsNramp was present in all tissues assayed. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells (Burge et al. 2004).
MsNRAMP of Morone saxatilis
Slc11a-a or Slc11α of 581 aas and 12 TMSs (Sibthorpe et al. 2004).
Slc11a-a of Takifugu rubripes (pufferfish)
NRAMP2 Fe2+, Mn2+, H+ cation transporter of 684 aas and 11 TMSs
NRAMP porter of Plasmodium falciparum
Macrophage and intestinal divalent cation (Mn2+ > Fe2+):H+ antiporter (catalyzes divalent cation efflux and regulates cation homeostasis), NRAMP1, scavenger receptor-BI (SR-BI) or Slc11a2. (Techau et al., 2007). The C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains are both necessary for HDL signaling. Direct binding of cholesterol to the C-terminal transmembrane domain has been demonstrated (Assanasen et al. 2005). Thus, HDL signaling requires cholesterol binding and efflux as well as the C-terminal domains of SR-BI; thus, SR-BI serves as a cholesterol sensor on the plasma membrane. The G212V mutation leads to microcytic anemia and liver iron overload (Bardou-Jacquet et al., 2011). Multiple targeting motifs direct NRAMP1 into lysosomes (Cheng and Wang, 2012). The structure and topology has been studied revealing a symmetric but inversely oriented arrangement (Li et al., 2012). Structural modeling and molecular dynamics simulations of the caprine and bovine orthologs led to mechanistic predictions (Triantaphyllopoulos et al. 2019). Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 to increase phagolysosomal iron availability (Banerjee and Datta 2020). It may also transport, or play a role in transport, of α- and β-carotene as well as cyptoxanthine, lutein, lycopene (Reboul 2013).
SLC11A1 of Homo sapiens
The major root plasma membrane high affinity Me2+(Fe2+, Co2+. Mn2+, Cd2+) uptake transporter, NRAMP-1 (also called NRAMP1 and NRAMP-6) is stimulated by Mn2+ deficiency) (Cailliatte et al., 2010). In silico analyses of plant NRAMPs have been performed using NRAMP1 as the query sequence (Vatansever et al. 2016). Cd2+ stress induces Ca2+ signals in Arabidopsis roots. The calcium-dependent protein kinases, CPK21 and CPK23, interact with the Cd transporter, NRAMP6/NRAMP1, through a variety of protein interactions. CPK21/23 phosphorylate NRAMP6 primarily at Ser489 and Thr505 to inhibit the Cd2+ transport activity of NRAMP6, thereby improving the Cd tolerance of plants (Zhang et al. 2022). Two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23, positively regulate the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, as well as higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution in the plasma membrane and enhance plant tolerance to Mn toxicity. Hence, the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provides insight into a mechanism of tolerance of plants to Mn toxicity (Zhang et al. 2023).
NRAMP-1 of Arabidopsis thaliana (Q9SAH8)
Nramp aluminum transporter 1, Nrat1; specific for trivalent Al ion in rice (Xia et al., 2010; Xia et al. 2011). There are 7 isoforms of NRAMPs in rice, and they transport a variety of metals, Zn2+, Mn2+, Fe2+, Cd2+, etc. (Mani and Sankaranarayanan 2018).
Nrat1 of Oryza sativa (Q6ZG85)
Mn2+ transporter, MntH (Hohle and O'Brian, 2009)
MntH of Bradyrhizobium japonicum (Q89K67)
Iron transporter, NRAMP isoform III (Lin et al., 2011).
NRAMP isoform III of Perkinsus marinus (D5FGJ2)
NRAMP1 (SLC11a1) of 533 aas and 11 TMSs. Regulates iron homeostasis and bacterial infection; present in phagosomes and macropinosomes (Peracino et al. 2013). Transports metal cations out of the phagolysosome, thereby depleting iron. Nramp1 overexpression protects cells from L. pneumophila infection (Peracino et al. 2006). Nramp1 and NrampB (Nramp2) are in membranes of macropinosomes and in phagosomes or the contractile vacuole network, respectively (Peracino et al. 2022).
NRAMP1 of Dictyostelium discoideum
Me2+ (Mn2+, Fe2+, Cd2+, Co2+, Zn2+, Ni2+):H+ symporter, MntH (Mn2+ · MntR and Fe2+ · Fur repressible). Specific resides in TMS1 and 6 line the pore and play a role in pH regulation (Courville et al., 2004; Haemig et al. 2010). Important for virulence in Salmonella (Karlinsey et al., 2010).
MntH (YfeP) of E. coli (P0A769)
YcsG (YcsH) of 404 aas and 11 TMSs. In addition to MntH, it is a Mn2+ uptake porter that is transcriptionally regulated by MntR and AhrC. Glucose controls manganese homeostasis through transcription factors regulating both MntH and YcsG (Ogura et al. 2023).
YcsG of Bacillus subtilis (P42964)
Manganese transport protein MntH (YdaR) of 425 aas and 11 TMSs. It is a H+-stimulated, divalent metal cation uptake system involved in manganese uptake. It can probably also transport cadmium, cobalt, copper and zinc, but not iron. It may be the predominant transporter of manganese during logarithmic phase growth. Its transcription is regulated by MntR and AhrC (Que and Helmann 2000; Ogura et al. 2023).
MntH of Bacillus subtilis
Putative metal ion transport protein
Putative metal ion transport protein of Streptomyces coelicolor
Uncharacterized permease of 406 aas
UP of Pseudomonas stutzeri
NRAMP homologue; putative manganese porter of 544 aas and 13 TMSs.
Mn2+ porter of Bradyrhizobium sp.
H+-stimulated, divalent metal cation uptake system, MntH of 436 aas and 11 TMSs. The x-ray structure has been determined, revealing the probable ion translocation pathway (Bozzi et al. 2016). Metal ion and proton may enter the transporter via the same external pathway to the ir binding sites, but they follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species (Bozzi et al. 2019). The results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Transmembrane helix 6b links proton- and metal-release pathways and drives conformational changes (Bozzi et al. 2019).
MntH of Deinococcus radiodurans
Uncharacterized protein of 465 aas and 11 TMSs (Hug et al. 2016).
UP of Candidatus Peribacter riflensis
NRAMP homologue of 483 aas and 11 TMSs in a 6 + 5 TMS arrangement.
NRAMP homologue of Saccharopolyspora erythraea