Membrane topology of the NixA nickel transporter of Helicobacter pylori: Two nickel transport-specific motifs within transmembrane helices II and III

NixA, the high-affinity cytoplasmic membrane nickel transport protein of He licobacter pylori, imports Ni2+ into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonizati on, NixA fractionates with the cytoplasmic membrane, and protein cross-link ing studies suggest that NixA functions as a monomer. A preliminary topolog ical model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters . To test the proposed topology of NigA and relate critical residues to spe cific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusion s were created along the entire length of the protein. Expression of report er fusions was confirmed by Western blotting with beta-galactosidase- and a lkaline phosphatase-specific antisera. The activities of reporter fusions n ear to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni2+ transport localiz ed these motifs entirely within transmembrane domains II. and III. Fusion a ctivities localized six additional Asp and Glu residues which reduced Ni2transport by >90% when mutated within or immediately adjacent to transmembr ane domains II, V, VI, and VII. All fusions strongly support a model of Nix A in which the amino and carboxy termini are located in the cytoplasm and t he protein possesses eight transmembrane domains.