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Journal of Bacteriology, December 2006, p. 8343-8351, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01383-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis{triangledown}

Ellen B. Duffy and Blanca Barquera*

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

Received 30 August 2006/ Accepted 25 September 2006

The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the 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 the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm 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 flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at 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, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies 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.

* Corresponding author. Mailing address: Rm. 2239, Biotechnology Center, 110 Eighth St., Troy, NY 12180. Phone: (518) 276-3861. Fax: (518) 276-2851. E-mail: barqub{at}rpi.edu.

{triangledown} Published ahead of print on 13 October 2006.

Journal of Bacteriology, December 2006, p. 8343-8351, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01383-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Juarez, O., Nilges, M. J., Gillespie, P., Cotton, J., Barquera, B. (2008). Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae. J. Biol. Chem. 283: 33162-33167 [Abstract] [Full Text]  
  • Lin, P.-C., Turk, K., Hase, C. C., Fritz, G., Steuber, J. (2007). Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production in Vibrio cholerae. J. Bacteriol. 189: 3902-3908 [Abstract] [Full Text]  


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