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J. Biol. Chem., Vol. 279, Issue 5, 3318-3326, January 30, 2004

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Determination of Transmembrane Topology of the Escherichia coli Natural Resistance-associated Macrophage Protein (Nramp) Ortholog^*

Pascal Courville, Roman Chaloupka¶, Frédéric Veyrier, and Mathieu F. M. Cellier||

From the Institut National de la Recherche Scientifique-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7 and the Institute of Physics, Charles University in Prague, Ke Karlovu 5, 121 16 Prague 2, Czech Republic

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 -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.

Received for publication, September 8, 2003 , and in revised form, November 6, 2003.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-78014-MI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a table and graph depicting impairment of EcoliA transport activity (Supplemental Data 1) and expression of Cat fusion proteins shown by SDS-PAGE (Supplemental Data 2).

^¶ Supported by short term NATO Science Fellowship Grant CZ 3/2003.

^|| Scholar of the Fond pour la Recherche en Santé du Québec and to whom correspondence should be addressed: INRS-Institut Armand-Frappier, 531, Bd. des Prairies, Laval, Québec, Canada H7V 1B7. Tel.: 450-687-5010 (ext. 4681); Fax: 450-686-5301; E-mail: mathieu.cellier{at}inrs-iaf.uquebec.ca.

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