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J. Bacteriol. doi:10.1128/JB.01383-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Membrane topology mapping of the Na⁺-pumping NADH:quinone oxidoreductase from Vibrio cholerae by PhoA/GFP fusion analysis

Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute

^* To whom correspondence should be addressed. Email: barqub{at}rpi.edu.

	Abstract

The membrane topology of the six subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae was determined by a combination of topology prediction algorithms and construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green-fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of topology prediction algorithms did not predict any transmembrane helices for NqrA. Lack of PhoA activity when fused to the C-terminus of NqrA and the observed fluorescence of the GFP C-terminal fusion confirms that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for FMN, resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site, residue T225, on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, FAD, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topology of the subunits of Na⁺-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.

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