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 id entified experimentally as high-affinity ammonium transporters. We have det ermined 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 Amt B-LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning heli ces, with the N-terminus on the exterior face of the membrane and the C-ter minus on the interior. A small subset, including E. coli AmtB, probably hav e an additional twelfth membrane-spanning region at the N-terminus. Additio n 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 [C-14] -methylammonium uptake. This C-terminal region, along with four membrane-sp anning helices, contains multiple residues that are conserved within the Me p/Amt protein family. Structural modelling of the E. coli AmtB protein sugg ests a number of secondary structural features that might contribute to fun ction, including a putative ammonium binding site on the periplasmic face o f the membrane at residue Asp-182. The implications of these results are di scussed in relation to the structure and function of the related human Rhes us proteins.