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.
Thomas
G H
GH
Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, UK.
Mullins
J G
JG
Merrick
M
M
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