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Membrane topology of the Mep/Amt family of ammonium transporters
Author(s) -
Thomas Gavin H.,
Mullins Jonathan G. L.,
Merrick Mike
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01994.x
Subject(s) - periplasmic space , biology , biochemistry , c terminus , escherichia coli , membrane protein , membrane topology , protein family , transport protein , n terminus , membrane transport protein , function (biology) , membrane , peptide sequence , microbiology and biotechnology , gene , amino acid
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 α‐peptide to the C‐terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [ 14 C]‐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.