PMID:10931328
| Citation |
Thomas, GH, Mullins, JG and Merrick, M (2000) Membrane topology of the Mep/Amt family of ammonium transporters. Mol. Microbiol. 37:331-44 |
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| Abstract |
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. |
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| Keywords |
Alkaline Phosphatase/genetics; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biological Transport; Blotting, Western; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cation Transport Proteins; Cell Membrane/physiology; Escherichia coli/chemistry; Escherichia coli/metabolism; Escherichia coli Proteins; Evolution, Molecular; Genes, Reporter; Humans; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Methylamines/metabolism; Models, Biological; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Sequence Homology, Amino Acid; beta-Galactosidase/genetics |
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