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.
Courville
Pascal
P
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7.
Chaloupka
Roman
R
Veyrier
Frédéric
F
Cellier
Mathieu F M
MF
D000595:Amino Acid Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002104:Cadmium / Q000737:chemistry; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D019143:Evolution, Molecular; D017930:Genes, Reporter; D007477:Ions; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors; D001618:beta-Lactamases / Q000378:metabolism; D000099173:Natural Resistance-associated Macrophage Protein 1
D002104:Cadmium; D027682:Cation Transport Proteins; D015500:Chloramphenicol O-Acetyltransferase; D007477:Ions; D011522:Protons; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases; D000099173:Natural Resistance-associated Macrophage Protein 1