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 β-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. 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 β-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. Divalent metal ions such as ferrous iron, Fe2+, Mn2+, Co2+, and Zn2+ are vital nutrients for living cells that participate as metabolic cofactors in a variety of biochemical processes involving electron transfers, including respiration and photosynthesis. The natural resistance-associated macrophage protein (Nramp) 1The abbreviations used are: Nrampnatural resistance-associated proteinBlamβ-lactamase (secreted form)Catchloramphenicol acetyl transferaseLBLuria-BertaniPBSphosphate-buffered salineTMDtransmembrane domainMntHproton-dependent manganese transporter.1The abbreviations used are: Nrampnatural resistance-associated proteinBlamβ-lactamase (secreted form)Catchloramphenicol acetyl transferaseLBLuria-BertaniPBSphosphate-buffered salineTMDtransmembrane domainMntHproton-dependent manganese transporter. belongs to a highly conserved family of integral membrane proteins found in a large spectrum of organisms, including mammals (2Vidal S.M. Malo D. Vogan K. Skamene E. Gros P. Cell. 1993; 73: 469-485Google Scholar, 3Gruenheid S. Cellier M. Vidal S. Gros P. Genomics. 1995; 25: 514-525Google Scholar), plants (4Belouchi A. Cellier M. Kwan T. Saini H.S. Leroux G. Gros P. Plant Mol. Biol. 1995; 29: 1181-1196Google Scholar), yeast (5West A.H. Clark D.J. Martin J. Neupert W. Hartl F.U. Horwich A.L. J. Biol. Chem. 1992; 267: 24625-24633Google Scholar), and bacteria (6Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Google Scholar, 7Cellier M. Belouchi A. Gros P. Trends Genet. 1996; 12: 201-204Google Scholar). Eukaryotic Nramp proteins were implicated in pH-dependent transport of divalent metals, including Fe2+ and Mn2+ (8Supek F. Supekova L. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Google Scholar, 9Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Google Scholar). Mammalian Nramp2 facilitates transferrin-independent iron absorption in the intestine and transferrin-dependent iron uptake by peripheral tissues (e.g. bone marrow, erythrocytes, and kidney), whereas Nramp1 is devoted to host resistance, acting at the level of the membrane of the phagosome in macrophages and neutrophils (10Forbes J.R. Gros P. Trends Microbiol. 2001; 9: 397-403Google Scholar). Likewise, three yeast homologs contribute to manganese and iron homeostasis by facilitating manganese acquisition either at the plasma membrane or from intracellular vesicles and iron mobilization from the vacuole (11Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Google Scholar). The phenotypes associated with Nramp1 and Nramp2 knockout in mice, innate susceptibility to various intracellular pathogens and microcytic anemia, respectively, demonstrate non-redundant roles of mammalian Nramp proteins in divalent metal homeostasis. Deregulation of their expression may also perturb normal metabolism due to the cytotoxic effects of excessive metal accumulation (12Muckenthaler M. Roy C.N. Custodio A.O. Minana B. deGraaf J. Montross L.K. Andrews N.C. Hentze M.W. Nat. Genet. 2003; 34: 102-107Google Scholar). natural resistance-associated protein β-lactamase (secreted form) chloramphenicol acetyl transferase Luria-Bertani phosphate-buffered saline transmembrane domain proton-dependent manganese transporter. natural resistance-associated protein β-lactamase (secreted form) chloramphenicol acetyl transferase Luria-Bertani phosphate-buffered saline transmembrane domain proton-dependent manganese transporter. Bacterial homologs of eukaryotic Nramp were subsequently characterized in Gram-positive and -negative species as proton-dependent manganese transporters and denominated MntH proteins. Detailed sequence analyses revealed the existence of three phylogenetic groups of MntH proteins that showed distinct evolutionary patterns, and these groups were designated MntH A, B, and C (13Cellier M.F. Bergevin I. Boyer E. Richer E. Trends Genet. 2001; 17: 365-370Google Scholar). One of three phylogenetic groups of bacterial homologs (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google Scholar) is distributed among Gram-positive and -negative bacteria and shows congruency between function, phylogenetic and taxonomic relationships, and amino acid substitution rate pattern, consistent with an early evolutionary origin (14Richer E. Courville P. Bergevin I. Cellier M. J. Mol. Evol. 2003; 57: 363-376Google Scholar). Several MntH A proteins were functionally characterized. In Gram-positive species, Bacillus subtilis MntH A is necessary for growth in minimal medium not supplemented with manganese, and mntH gene expression is regulated by the manganese-dependent repressor MntR (15Que Q. Helmann J.D. Mol. Microbiol. 2000; 35: 1454-1468Google Scholar). Mycobacterial MntH A was characterized by heterologous expression in Xenopus oocytes (16Agranoff D. Monahan I.M. Mangan J.A. Butcher P.D. Krishna S. J. Exp. Med. 1999; 190: 717-724Google Scholar) and yeast (17Reeve I. Hummel D. Nelson N. Voss J. Hummell D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8608-8613Google Scholar). MntH A proteins were also studied in Gram-negative species; Escherichia coli and Salmonella typhimurium MntH A proteins were characterized in our laboratory as proton-dependent transporters of divalent metals with strong preference for Mn2+ (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google Scholar, 18Boyer E. Bergevin I. Malo D. Gros P. Cellier M.F. Infect. Immun. 2002; 70: 6032-6042Google Scholar). MntH proteins B and C have also been studied recently; one MntH C protein was reported to contribute to Staphylococcus aureus virulence (19Horsburgh M.J. Wharton S.J. Cox A.G. Ingham E. Peacock S. Foster S.J. Mol. Microbiol. 2002; 44: 1269-1286Google Scholar), and several mntH genes from groups B and C were functionally expressed in E. coli and shown to confer sensitivity to divalent metals (14Richer E. Courville P. Bergevin I. Cellier M. J. Mol. Evol. 2003; 57: 363-376Google Scholar). Therefore, despite significant distance between currently known Nramp homologs, their sequence preserved structural features that correspond to a conserved function in proton-dependent divalent metal uptake. Based on their evolutionary features, the prokaryotic genes of mntH group A most likely represent precursors of eukaryotic Nramp genes (13Cellier M.F. Bergevin I. Boyer E. Richer E. Trends Genet. 2001; 17: 365-370Google Scholar, 14Richer E. Courville P. Bergevin I. Cellier M. J. Mol. Evol. 2003; 57: 363-376Google Scholar); the corresponding proteins should thus exhibit similar structures because they perform similar functions (20Saier M.H. Winkelmann, G. Microbial Transport Systems. Wiley-VCH, Weinheim, Germany2001: Scholar). of the transmembrane topology is a to study the structural and functional of a membrane transporter M. Microbiol. Mol. Biol. 2000; Scholar), and a combination of in silico and is to a model D. D. J. T. C.N. G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar). in the of a functional sequence analysis of eukaryotic Nramp homologs a transmembrane topology that was on several conserved features as the of a of amino acid sequence for the of and the strong for loops to and the of a in a loop and a conserved transport topology placed the terminus by a conserved of transmembrane domains and either one or highly the C terminus or The topology been in of The of yeast been by a A at either of the was by consistent with the of in the membrane of the yeast The data indicated that the in yeast proteins is from the the the C terminus (11Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Google Scholar). the and C of the mammalian protein Nramp1 were on the cytoplasmic of the membrane using either a the of the protein or by a at the C terminus S.M. E. P. S. Gros P. J. 1996; Scholar). The group using the that the loop between the and was a A V. G. N. Gros P. J. Biol. Chem. 2000; Scholar). a study indicated that a corresponding to Nramp2 a from the to in B. D. J. J. Biol. 1993; Scholar). have been one study that Nramp1 a transmembrane topology and C on that with either the extra loop between and or the C terminus or not respectively, of iron with J. Biol. 2001; Scholar). bacterial homologs, a study of the of the of the MntH A protein a transmembrane topology from that (17Reeve I. Hummel D. Nelson N. Voss J. Hummell D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8608-8613Google Scholar). These data the of the of in prokaryotic MntH proteins and of the of prokaryotic MntH and eukaryotic Nramp and transmembrane In the we the transmembrane topology of the E. coli Nramp ortholog MntH A which an structural and functional model of eukaryotic Nramp using genetic a combination of in silico predictions, reporter gene and including proteins for which metal uptake and metal-dependent proton transport were Bacterial and mntH E. coli cells were used in the were at and in Luria-Bertani supplemented with the or The medium (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google Scholar) was used for the assay and using the mntH E. coli and of the used in represent the of the represent the The represent the of the The represent the in a and used in and or or S. P. Mol. Microbiol. 1995; H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google was used for most and with H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google was used to the and with and not and with fusion was used for most was used to the in a were used to a topology of the MntH A and were using the of and J. J. Mol. Biol. for an and by a of and of (Blam) was in Scholar). of the mntH gene were by with and and with III using the to the The fusion at the amino acid was by with and with to the The were with and with the secreted by with and of the Scholar). The was in E. coli and were The were on three respectively, and and and and were The minimal of for the were by of a and on of and supplemented with were from and with to the of the corresponding to were using from and of were by targeted fusion the amino and The were with and and to the with the and and to the gene by and and a The at the and were using and and a was used at The were for to chloramphenicol using a to of medium and for were to the constructs with for and of a were on chloramphenicol and for the for which was Constructs and were in and respectively, using and and of were by to Scholar) the amino and The were in using and The were with and to a The proteins were in and minimal were as using a of as an of The was also used to a fusion The of the constructs was by a for at and which was in to a of and in the of of a The level of of the was from the and membrane were for fusion A. M. in and Scholar). The were in and and at for were by using a The proteins were on a membrane from at for with and for at in a were with the for for in and for with the secondary and in were revealed using the from The used were an an and and of were by the of E. coli using a A. J. B. S. Scholar). In of E. coli for with of were and in and The were and for in The were and in of membrane and of proteins were on and was as were using the protein assay by MntH A metal uptake was by a disk assay of metal were a at and The were in the to a of of bacteria was to of and supplemented with the of to a disk with of and for at or (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google Scholar). and divalent metal-dependent intracellular accumulation of was by the fluorescence of pH-dependent H. M. H. Microbiol. 2002; Scholar, G. Nature. Scholar). cells of E. coli mntH were with as was by from H. M. H. Microbiol. 2002; Scholar) using the and with the were of for for and for by of for for and for was L.K. M. S. Microbiol. Scholar) using and the was using using and to which was by transport cells of E. coli mntH were with MntH were in the of at in which was supplemented with fluorescence to the expression of MntH proteins. were by in and in the to an of was measured on a fluorescence fluorescence was by a at and respectively, the was at was for and intracellular of to the was by the of as H. M. H. Microbiol. 2002; Scholar). or or were to the the of the A topology J. B. G. 2000; Scholar) was to prokaryotic MntH a transmembrane similar to eukaryotic using that in S. 2001; 17: Scholar, T. T. In Biol. 2002; Scholar, K. A. G. J. Mol. Biol. 2003; Scholar) were is a using amino acid derived from characterized membrane proteins and structural and to topology and a of and of Scholar). that a model by structural of a membrane protein A. B. G. J. Mol. Biol. 2001; Scholar). the of our we a transmembrane topology using the evolutionary B. P. Sci. 1996; Scholar) in a of MntH A proteins from Gram-positive and -negative species and the In an E. coli MntH A was using the amino acid of and and a of that transmembrane J. J. Mol. Biol. Scholar). The results of these were consistent with with eukaryotic Nramp M. Belouchi A. Gros P. Trends Genet. 1996; 12: 201-204Google Scholar) and a topology with the terminus by 11 and the C terminus we used as of the periplasmic and cytoplasmic of the the secreted form of and been used as an to the D. D. J. T. C.N. G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar) to the topology of membrane proteins Scholar, S. H. M.A. J. Scholar); reporter is in the to to or in a to the periplasmic M. Microbiol. Mol. Biol. 2000; Scholar). The cytoplasmic reporter been used in several to data with either or A. E. 1996; 35: Scholar, J. E. J. 2002; Scholar, T. E. T. A. J. Scholar); to chloramphenicol on the cytoplasmic of the been reported to as an that a protein that is on the the of data also results B. D. J. J. Biol. 1993; Scholar, M. Microbiol. Mol. Biol. 2000; Scholar, J. Sci. 2002; Scholar). were the analysis to that strong to periplasmic The of was on the and targeted to the of loops level sequence The results with are in III and A and B. were to the of the is indicated in and were in membrane by analysis using an constructs in a significant of the form of to secreted were in the membrane and and and In one a of fusion protein with level in with that the level of not with the level of expression of fusion proteins S. H. M.A. J. Scholar, Mol. Microbiol. 1995; Scholar, J. Biol. Chem. 1993; Scholar). the that of and in their expression both were as with that the fusion were a in exposure of the in the in a level of Three indicated a that is in with the that placed the loop between and 11 on the periplasmic the cytoplasmic of the membrane of EcoliA fusion to (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google in a The three targeted strong of them functional as their expression sensitivity to Fe2+ data and the in their and functional which to the expression using the The targeted was with and no secreted was either strong to expression of the at functional was with the of the in a not EcoliA data the of either the or a similar as both constructs to type MntH The data that the loop 6/7 and the C terminus are consistent with the and with most fusion the cytoplasmic exposure of the loop between and 11 that and in III represent a targeted fusion of was fusion in the loops 2/3, 6/7, and were used as and on the and an the was used as not confer to chloramphenicol the not was expressed and for cells which the of the The of an was found to the protein expression level and the of the protein was in analysis of membrane using an metal uptake and not confer to chloramphenicol These results confirmed that the EcoliA C terminus is of targeted assay of metal of in a to cytoplasmic strong chloramphenicol and The constructs of was in membrane of of the of these was in for was confirmed by metabolic and of level in a of and protein with data The deduced cytoplasmic exposure of is consistent with the and results of the and These three the that is to the cytoplasmic of on the J. Sci. 2002; Scholar, Mol. Microbiol. 1995; Scholar) and on fusion data with EcoliA of and not confer sensitivity to manganese and sensitivity to the metals and of the similar level sensitivity to the metals and not in is to in the assay of metal sensitivity of metal transport secondary to III and bacterial sensitivity to metals such as and may in assay because of their (14Richer E. Courville P. Bergevin I. Cellier M. J. Mol. Evol. 2003; 57: 363-376Google Scholar). Therefore, to demonstrate the functional between MntH proteins and and and a truncated form of MntH to that sensitivity to and we studied a of MntH transporters the proton to transport of eukaryotic Nramp2 showed that intracellular divalent metal intracellular H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Google Scholar). demonstrate the EcoliA MntH transport function, we divalent metal-dependent intracellular to MntH expression using a pH-dependent protein H. M. H. Microbiol. 2002; Scholar, G. Nature. Scholar). The data in that expression of EcoliA MntH an intracellular the of not the of that a divalent metal for MntH transport (1Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Google Scholar) is for proton MntH similar with on the of A and B, and and In expression of the not the intracellular the growth sensitivity to with is not likely due to proton-dependent of the the showed intracellular data These results demonstrate that a MntH transport function and the topology our a analysis the to the of both and the of not been reported for of Nramp family. In we used functional to study the of Nramp proteins using the E. coli MntH A ortholog as a The results that EcoliA is a type III integral transmembrane protein with the terminus cytoplasmic by 11 and the C terminus on the periplasmic of the membrane The periplasmic of the C terminus of E. coli MntH A was with the of the and that were both functional in bacterial sensitivity to Fe2+ and Mn2+ and divalent metal-dependent the to whereas not confer chloramphenicol The of the EcoliA terminus was revealed by fusion constructs and that strong to in a that membrane was due to the is by the sequence the likely these demonstrate that the and of the protein and are as their in known transmembrane a likely structural 2001; Scholar), by their in Nramp and exposure of the extra loop 6/7 was also with the targeted that in a to that of the fusion which that the of transmembrane in the constructs and were expressed at significant with level of a the The MntH strong to that the model in is because preserved MntH transport of both divalent metals and which is a of Nramp of both proton transport and metal sensitivity and in cells the and that the transmembrane topology deduced from these results a functional MntH A The of the 11 EcoliA was derived from the analysis of the fusion or constructs that were in of extra loops or and in of intra loops or the constructs revealed extra loops between and and and and and and the constructs indicated cytoplasmic exposure of the loops 2/3, 6/7, and 10/11. The intra loops for which no data were produced (N terminus, intra loop 4/5) both display a significant and whereas the extra loop a of The amino acid composition bias of these thus their deduced D. L. D. J. Chem. Sci. 2002; Scholar). The for which no fusion was produced is one of the most in the E. coli MntH A is of in MntH B and most MntH A proteins as as in type eukaryotic and most MntH C a conserved or Nramp2 was as an of and B. D. J. J. Biol. 1993; Scholar), that the on a of the transmembrane that to 2001; Scholar). a natural or an that a in the of is to both Nramp1 (2Vidal S.M. Malo D. Vogan K. Skamene E. Gros P. Cell. 1993; 73: 469-485Google Scholar) and Nramp2 function M.A. M.A. Andrews N.C. Proc. Natl. Acad. Sci. U. S. A. Scholar). In both the strong of EcoliA and and found in the of transmembrane and and strong for with and consistent with the topology the existence of EcoliA The targeted in the extra loop 5/6 confirmed the with the targeted fusion fusion was expressed at a level and a level of was expressed at a level the and MntH A functional of the the was also associated with EcoliA metal sensitivity and proton data not These results are consistent with level expression of and that the of is to The sensitivity of the to with the results with in which similar and in is a that is conserved among MntH A the is either a or a to the both the of and the of conserved structural may the sensitivity of to the and that were the for three that produced an and is by the that these fusion are of a that the to the cytoplasmic of the Therefore, the results the model derived from the and The 11 topology of EcoliA that of yeast proteins or homologs, which have the model in study of the E. coli MntH A protein is consistent with the and data that were eukaryotic The that is most conserved in Nramp proteins and strong structural and functional to both divalent metal transport and proton The are consistent with the that eukaryotic Nramp transporters derived from an ancestral mntH A level sequence relationship with MntH proteins from groups A and B display similar that MntH B proteins a transmembrane topology similar to on their to eukaryotic MntH C proteins should display a topology similar to the model including MntH proteins found in bacteria to and the C terminus In the model in study for structural and functional of both bacterial MntH and eukaryotic Nramp transporters of divalent metals and protons. and for their as as M. A. H. and F. or that were used in with

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