Divalent metal transport in the green microalga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> is mediated by a protein similar to prokaryotic Nramp homologues

Information about the molecular mechanisms of metal transport in algae is scarce, despite the significant status these organisms have in aquatic ecosystems. In the present study, we describe the cloning and functional characterization of a divalent metal transporter (named DMT1) in the green microalga Chlamydomonas reinhardtii Dangeard. The longest open reading frame of the cloned DMT1 cDNA encodes a protein of 513 amino acids with 11 putative transmembrane domains. The protein belongs to the Nramp family of divalent metal transporters and shows surprisingly higher similarity to some prokaryotic than to eukaryotic polypeptides. Especially the N-terminus, which is longer than of every other homologue considered in this study, displays – uniquely among selected eukaryotic Nramps – exclusively prokaryotic characteristics. Functional complementation experiments in yeast strains with impaired metal transport systems, revealed that C. reinhardtii DMT1 has a broad specificity, acting in the transport of several divalent metals (manganese, iron, cadmium, copper), but excluding zinc. Published online December 2004     

Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo

The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters.     

Importance of Conserved Acidic Residues in MntH,the Nramp homolog of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of ⁵⁴Mn²⁺ uptake with a V_max value of approximately 10% of wild-type and a slightly elevated K_m value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn²⁺. Other residues such as Glu-102 and Asp238 may play a role in the release of Mn²⁺ to the cytoplasm or may be involved in maintaining secondary structure.This revised version was published online in June 2005 with a corrected cover date.     

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity^r) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn²⁺ transport by MntH and SitABCD, as well as Zn²⁺ and Mn²⁺ transport by ZupT. In vitro uptake of ⁵⁴Mn²⁺ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.     

Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters.

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 TMS1 and 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.     

Cloning and characterization of the OsNramp family from Oryza sativa,a new family of membrane proteins possibly implicated in the transport of metal ions

The mammalian Nramp1 protein is an integral membrane protein expressed exclusively in macrophages, where it plays a critical role in the ability of these cells to destroy ingested microbes. The bactericidal mechanism of action of Nramp1 remains unknown. We report the identification and characterization of cDNA clones corresponding to three homologues of the mammalian Nramp1 gene from the genome of Oryza sativa, OsNramp1, OsNramp2, and OsNramp3. These three genes encode a novel group of highly similar hydrophobic polypeptides sharing between 64% and 75% sequence similarity, that show similar hydropathy profiles, and predicted secondary structure, including the same number, position, and sequence characteristics (including conserved charges) of transmembrane domains. Together, these define a highly conserved membrane associated hydrophobic core. The three plant proteins show a remarkable degree of sequence similarity with their mammalian counterpart (60% to 70% similarity), including primary and secondary structure elements previously described in ion transporters and channels. Expression studies in normal plant tissues indicate that while OsNramp1 is expressed primarily in roots, and OsNramp2 is primarily expressed in leaves, OsNramp3 is expressed in both tissues. The recent discovery that the yeast Nramp homologue SMF1 functions as a manganese transporter raises the exciting possibility that OsNramp encodes a family of metal ion transporters in plants.     

The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese,But Not Iron

The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn²⁺ and Cd²⁺, but not for Fe²⁺ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice.Transport of mineral elements from soil to different organs and tissues of plants requires different types of transporters (Hall and Williams, 2003; Nevo and Nelson, 2006; Yokosho et al., 2009; Olsen and Palmgren, 2014; Sasaki et al., 2016), which include the zinc-regulated transporters, iron-regulated transporter-like protein family; the natural resistance-associated macrophage protein (Nramp) family of transporters; the multidrug and toxic compound extrusion protein transporters; the heavy metal ATPase transporters; the oligopeptide transporters family; the ATP-binding cassette family of transporters; and the cation-diffusion facilitator family of transporters. Among them, Nramp represents a transporter family for metal ion in all organisms including bacteria, animals, and plants (Curie et al., 2000; Nevo and Nelson, 2006). Some members of this family in plants have been functionally characterized, especially in model plants such as Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). In Arabidopsis, there are six members of Nramp transporter proteins. AtNramp1 is localized to the plasma membrane of root cells and functions as a high-affinity transporter for Mn uptake (Cailliatte et al., 2010). Both AtNramp3 and AtNramp4 are localized to the tonoplast and play redundant roles in Fe, exporting from the vacuole during seed germination and in Mn homeostasis at the adult stage (Thomine et al., 2000; Lanquar et al., 2005, 2010). AtNramp6 is targeted to a vesicular-shaped endomembrane compartment and is implicated in the distribution or availability of Cd within cells (Cailliatte et al., 2009). However, the function of AtNramp2 and AtNramp5 has not been characterized.On the other hand, there are seven members of Nramp transporter family in the rice genome, of which four have been functionally characterized. They all are localized to the plasma membrane but show different roles. OsNramp1 shows transport activity for Fe and Cd in yeast and is proposed to be involved in Cd accumulation (Takahashi et al., 2011). OsNramp3 is localized at the vascular tissues of nodes and plays an important role in distribution of Mn, but not Fe and Cd (Yamaji et al., 2013). On the other hand, OsNrat1 (OsNramp4) transports trivalent Al ion (Xia et al., 2010) and is required for high Al tolerance in rice roots. Finally, OsNramp5 functions as a major transporter responsible for root Mn and Cd uptake (Ishimaru et al., 2012; Ishikawa et al., 2012; Sasaki et al., 2012). However, the function of OsNramp2, OsNramp6, and OsNramp7 is unknown.In addition to Nramp members characterized in rice and Arabidopsis, some members in other plant species have also been characterized. For example, a soybean (Glycine max) Nramp transporter, GmDMT1 is implicated in the ferrous iron transport (Kaiser et al., 2003). Nramp1 isolated from Noccaea caerulescens, a Zn/Cd hyperaccumulator, is involved in the influx of Cd across the endodermal plasma membrane and plays a key role in Cd flux into the stele and root-to-shoot Cd transport (Milner et al., 2014). In Malus baccata, Nramp1 is capable of mediating the distribution of ions as well as transport of Fe, Mn, and Cd (Xiao et al., 2008). Besides, Nramp1 and Nramp3 in tomato (Solanum lycopersicum) have also been suggested to be involved in Mn transport (Bereczky et al., 2003). When NcNramp3 and NcNramp4 from Noccaea caerulescens were expressed in yeast, NcNramp3 transported Fe, Mn, and Cd, while NcNramp4 also transported Zn in addition to Fe, Mn, and Cd (Oomen et al., 2009). However, Nramp4 isolated from Thlaspi japonicum, a Ni hyperaccumulator, showed transport activity for Ni but not for Zn, Cd, or Mn in yeast (Mizuno et al., 2005). These findings indicate that Nramp members have a diverse role in metal transport in plants.Barley (Hordeum vulgare) is the fourth most important cereal crop in the world; however, less progress has been made in understating of molecular mechanisms on mineral element transport in barley due to its large genome size. For example, no Nramp members in barley have been functionally characterized so far. In this study, we first isolated barley Nramp member, HvNramp5, which is a close homolog of rice OsNramp5. Detailed functional analysis revealed that HvNramp5 is involved in the uptake of both Mn and Cd, but not of Fe in barley roots. Furthermore, we found that different from OsNramp5, HvNramp5 showed a distinct pattern in the gene expression, cellular localization, and transport substrate.     

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea

Background

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.

Methods

We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.

Results

Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.

General Significance

This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.     

Managing the manganese: molecular mechanisms of manganese transport and homeostasis

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.     

Natural history of SLC11 genes in vertebrates: tales from the fish world

Background  

The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members.     

Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog

The natural resistance-associated macrophage protein (Nramp) defines a conserved family of secondary metal transporters. Molecular evolutionary analysis of the Nramp family revealed the early duplication of an ancestral eukaryotic Nramp gene, which was likely derived from a bacterial ortholog and characterized as a proton-dependent manganese transporter MntH (Makui, H., Roig, E., Cole, S. T., Helmann, J. D., Gros, P., and Cellier, M. F. (2000) Mol. Microbiol. 35, 1065-1078). Escherichia coli MntH represents a model of choice to study structure function relationship in the Nramp protein family. Here, we report E. coli MntH transmembrane topology using a combination of in silico predictions, genetic fusion with cytoplasmic and periplasmic reporters, and MntH functional assays. Constructs of the secreted form of beta-lactamase (Blam) revealed extra loops between transmembrane domains 1/2, 5/6, 7/8, and 9/10, and placed the C terminus periplasmically; chloramphenicol acetyltransferase constructs indicated cytoplasmic loops 2/3, 6/7, 8/9, and 10/11. Two intra loops for which no data were produced (N terminus, intra loop 4/5) both display composition bias supporting their deduced localization. The extra loops 5/6 and 6/7 and periplasmic exposure of the C terminus were confirmed by targeted reporter insertion. Three of them preserved MntH function as measured by a disk assay of divalent metal uptake and a fluorescence assay of divalent metal-dependent proton transport, whereas a truncated form lacking transmembrane domain 11 was inactive. These results demonstrate that EcoliA is a type III integral membrane protein with 11 transmembrane domains transporting both divalent metal ions and protons.     

A functional-phylogenetic classification system for transmembrane solute transporters.

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.     

Emerging mechanisms for heavy metal transport in plants

Heavy metal ions such as Cu(2+), Zn(2+), Mn(2+), Fe(2+), Ni(2+) and Co(2+) are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd(2+), Hg(2+) and Pb(2+), can become extremely toxic. Thus mechanisms must exist to satisfy the requirements of cellular metabolism but also to protect cells from toxic effects. The mechanisms deployed in the acquisition of essential heavy metal micronutrients have not been clearly defined although a number of genes have now been identified which encode potential transporters. This review concentrates on three classes of membrane transporters that have been implicated in the transport of heavy metals in a variety of organisms and could serve such a role in plants: the heavy metal (CPx-type) ATPases, the natural resistance-associated macrophage protein (Nramp) family and members of the cation diffusion facilitator (CDF) family. We aim to give an overview of the main features of these transporters in plants in terms of structure, function and regulation drawing on information from studies in a wide variety of organisms.     

Metal transporters that contribute copper to metallochaperones in Saccharomyces cerevisiae

Copper metallochaperones represent a new family of soluble, low-molecular-weight proteins that function to deliver copper to specific sites within a cell. How the metallochaperones acquire their copper, however, is not known. In this study, we have conducted a survey of known metal ion transporters in bakers' yeast, Saccharomyces cerevisiae, to identify those that contribute copper to pathways involving the metallochaperones Atxlp and Lys7p. The results indicatethat, in addition to the well known Ctr1p and Ctr3p high-affinity copper transporters, the metallochaperones can acquire their copper through pathways involving the relatively non-specific divalent metal ion transporter Fet4p and the putative low-affinitycopper transporter Ctr2p. We have examined the localization of Ctr2p using an epitope tagged version of the protein and find that Ctr2p does not localize to the cell surface but may operate at the level of the vacuole to mobilize intracellular copper. Inaddition to Ctrlp, Ctr2p, Ctr3p and Fet4p, other metal transport systems can act as upstream donors of copper for the metallochaperones when copper availability in the medium is increased. Although the nature of these auxiliary systems is unknown, they do not appear to involve the yeast members of the Nramp family of divalent transporters, or uptake mechanisms that involve endocytosis. Since vastly different metal transporters located at either the cell surface or intracellular sites can all contribute copper to metallochaperones, it is unlikely that the metallochaperones directly interact with the metal transporters to obtain the metal.     

Point mutations change specificity and kinetics of metal uptake by ZupT from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.     

Switching substrate specificity of AMT/MEP/ Rh proteins

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH₄⁺, NH₃ + H⁺, NH₄⁺ + H⁺ or NH₃, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH₃ transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH₃ across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH₃ transport through the central subunit pore is suggested.     

Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

To overcome the metal restriction imposed by the host’s nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore‐mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad‐spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram‐negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB–OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal‐loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa.     

Genetic susceptibility to intracellular infections: Nramp1, macrophage function and divalent cations transport

Nramp1 is one of the few host resistance genes that have been characterized at the molecular level. Nramp1 is an integral membrane protein expressed in the lysosomal compartment of macrophages and is recruited to the membrane of bacterial phagosomes where it affects intracellular microbial replication. Nramp1 is part of a very large gene family conserved from bacteria and man that codes for transporters of divalent cations transporters. We propose that Nramp1 affects the intraphagosomal microbial replication by modulating divalent cations content in this organelle. Both mammalian and bacterial transporters may compete for the same substrate in the phagosomal space.