title: Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.
abstract: Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.
authors: Institute for Biochemistry, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany;
authors: Tobias
authors: T
authors: Wacker
authors: Department of Biochemistry, University of Zürich, 8057 Zürich, Switzerland;Institute of Complex Systems-Cellular Biophysics Research Center Jülich, 52428 Jülich, Germany; and.
authors: Juan J
authors: JJ
authors: Garcia-Celma
authors: Institute for Biochemistry, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany;
authors: Philipp
authors: P
authors: Lewe
authors: Institute for Biochemistry, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany;BIOSS Centre for Biological Signalling Studies, 79104 Freiburg, Germany andrade@bio.chemie.uni-freiburg.de.
authors: Susana L A
authors: SL
authors: Andrade
mesh_terms: D064751:Ammonium Compounds / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport
chemical_list: D064751:Ammonium Compounds; D002352:Carrier Proteins
keywords: Amt/Rh family; ammonium transport proteins; cation transport