PMID:14607838

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Citation

Courville, P, Chaloupka, R, Veyrier, F and Cellier, MF (2004) Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog. J. Biol. Chem. 279:3318-26

Abstract

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.

Links

PubMed Online version:10.1074/jbc.M309913200

Keywords

Amino Acid Sequence; Biological Transport; Blotting, Western; Cadmium/chemistry; Cation Transport Proteins/chemistry; Cation Transport Proteins/metabolism; Cell Membrane/metabolism; Chloramphenicol O-Acetyltransferase/metabolism; Cytoplasm/metabolism; Escherichia coli/metabolism; Evolution, Molecular; Genes, Reporter; Ions; Models, Biological; Molecular Sequence Data; Plasmids/metabolism; Protein Conformation; Protein Structure, Tertiary; Protons; Recombinant Fusion Proteins/metabolism; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Structure-Activity Relationship; Time Factors; beta-Lactamases/metabolism

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Methods:Determining_intracellular_pH

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See also

References

pHluorin paper[1]


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  1. Miesenböck, G et al. (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394 192-5 PubMed