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

Abstract

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.

FIG. 1.

Epifluorescent micrograph of GFP fusions. (A) E. coli pBAD-GFP. (B) Wild-type V. cholerae pBAD-GFP. (C) GFP fusion to the complete nqrA gene (GANC) in E. coli Top10 cells. (D) GANC in wild-type V. cholerae. (E) GFP fusion to nqrD from the N terminus to amino acid 125 (GDN125) in V. cholerae Δnqr. (F) GFP fusion to the complete nqrD gene plus a linker (GDNC+L) in V. cholerae Δnqr. The scale bar in panel B applies to all panels.

FIG. 2.

Membrane topology model of NqrC. The FMN binding site at residue T225 is shown. C-terminal fusion sites are indicated in the figure using the codes shown in Table 1.

FIG. 3.

Membrane topology model of NqrB. Conserved negatively charged residues located in the membrane spans are represented as black squares. The FMN binding site at residue T236 and a predicted quinone binding site at residue G141 are shown. C-terminal fusion sites are indicated in the figure using the codes shown in Table 1.

FIG. 4.

Coomassie blue-stained and UV-illuminated SDS-PAGE gel of membrane fractions of the alkaline phosphatase fusion to the complete nqrB gene (ABNC) and the alkaline phosphatase fusion to the complete nqrC gene (ACNC) in V. cholerae Δnqr. The fluorescent bands indicate the presence of the covalently bound FMNs. Thirty micrograms of membrane protein was used in each lane.

FIG. 5.

Membrane topology model of NqrD. Conserved negatively charged residues located in the membrane spans are represented as black squares. C-terminal fusion sites are indicated in the figure using the codes shown in Table 1.

FIG. 6.

Membrane topology model of NqrE. Conserved negatively charged residues located in the membrane spans are represented as squares. C-terminal fusion sites are indicated in the figure using the codes shown in Table 1.

FIG. 7.

Membrane topology model of NqrF. The binding sites for NADH, the 2Fe-2S center, and FAD are indicated. The residues of the weak hydrophobic region predicted to be a possible second transmembrane helix are shown as white squares. C-terminal fusion sites are indicated in the figure using the codes shown in Table 1.

FIG. 8.

Scheme illustrating the relationship between redox cofactor sidedness and the directionality of ion translocation in primary pumps. (A) H⁺-pumping NADH:quinone oxidoreductase (complex I). (B) Na⁺-pumping NADH:quinone oxidoreductase (Na⁺-NQR). (C) Cytochrome c (cyt C) oxidase.