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

Courville, Pascal; Chaloupka, Roman; Veyrier, Frédéric ORCID: https://orcid.org/0000-0002-8574-0547 et Cellier, Mathieu ORCID: https://orcid.org/0000-0002-6084-434X (2004). Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog Journal of Biological Chemistry , vol. 279 , nº 5. pp. 3318-3326. DOI: 10.1074/jbc.M309913200.

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Résumé

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.

Type de document:	Article
Mots-clés libres:	-
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	15 nov. 2024 20:15
Dernière modification:	15 nov. 2024 20:15
URI:	https://espace.inrs.ca/id/eprint/14521

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