Membrane topology of the Mep/Amt family of ammonium transporters.
The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high-affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB-PhoA fusion proteins with a complementary set of AmtB-LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning helices, with the N-terminus on the exterior face of the membrane and the C-terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane-spanning region at the N-terminus. Addition of PhoA or LacZ alpha-peptide to the C-terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]-methylammonium uptake. This C-terminal region, along with four membrane-spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp-182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.
Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, UK.
G H
GH
Thomas
J G
JG
Mullins
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Merrick
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000235:genetics
C111096:AmtB protein, E coli; D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008744:Methylamines; D011993:Recombinant Fusion Proteins; C027451:methylamine; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase