Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium.
P-type ATPases are a family of cation transport enzymes present in all species from bacteria to mammals whose members mediate membrane flux of all common biologically relevant cations. More than 50 members of this family of transporters have been sequenced; extensive structural data are available, and several members have been analyzed by site-directed mutagenesis. Nonetheless, there is no current consensus regarding their membrane topology. In this work, the Salmonella typhimurium Mg2+ transporting P-type ATPase encoded by the MgtB locus has been used as a model for P-type ATPases. Unlike other prokaryotic P-type ATPases, the MgtB protein is similar in length, amino acid sequence, and hydropathy profile to known eukaryotic P-type ATPases. The membrane topology of MgtB was analyzed by several epitope insertions in MgtB and from the activity of 35 protein fusions between MgtB and the reporter enzymes BlaM (beta-lactamase) and LacZ (beta-galactosidase). The epitope insertions within MgtB all retained function as assessed by cation uptake assays and were regulated normally by the level of Mg2+ within the growth medium. The epitope insertion and fusion protein data are completely incompatible with the numerous previously proposed models for P-type ATPases predicting 7, 8, 9, or 12 transmembrane segments. Rather, they indicate that MgtB contains 10 transmembrane segments with both amino and carboxyl termini residing within the cytosol. By extension, we suggest that all eukaryotic P-type ATPases contain 10 transmembrane segments with both termini within the cytosol.
Smith
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Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965.
Tao
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Maguire
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D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009532:Nickel; D001616:beta-Galactosidase; D001618:beta-Lactamases; D000251:Adenosine Triphosphatases; C083836:MgtB protein, Salmonella typhimurium; D008274:Magnesium