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.

Formate is a major metabolite in the anaerobic fermentation of glucose by many enterobacteria. It is translocated across cellular membranes by the pentameric ion channel/transporter FocA that, together with the nitrite channel NirC, forms the formate/nitrite transporter (FNT) family of membrane transport proteins. Here we have carried out an electrophysiological analysis of FocA from Salmonella typhimurium to characterize the channel properties and assess its specificity toward formate and other possible permeating ions. Single-channel currents for formate, hypophosphite and nitrite revealed two mechanistically distinct modes of gating that reflect different types of structural rearrangements in the transport channel of each FocA protomer. Moreover, FocA did not conduct cations or divalent anions, but the chloride anion was identified as further transported species, along with acetate, lactate and pyruvate. Formate, acetate and lactate are major end products of anaerobic mixed-acid fermentation, the pathway where FocA is predominantly required, so that this channel is ideally adapted to act as a multifunctional export protein to prevent their intracellular accumulation. Because of the high degree of conservation in the residues forming the transport channel among FNT family members, the flexibility in conducting multiple molecules is most likely a general feature of these proteins.

Amt-1 from Archaeoglobus fulgidus (AfAmt-1) belongs to the Amt/Rh family of ammonium/ammonia transporting membrane proteins. The transport mode and the precise microscopic permeation mechanism utilized by these proteins are intensely debated. Open questions concern the identity of the transported substrate (ammonia and/or ammonium) and whether the transport is passive or active. To address these questions, we studied the overall thermodynamics of the different transport modes as a function of the environmental conditions. Then, we investigated the thermodynamics of the underlying microscopic transport mechanisms with free energy calculations within a continuum electrostatics model. The formalism developed for this purpose is of general utility in the calculation of binding free energies for ligands with multiple protonation forms or other binding forms. The results of our calculations are compared to the available experimental and theoretical data on Amt/Rh proteins and discussed in light of the current knowledge on the physiological conditions experienced by microorganisms and plants. We found that microscopic models of electroneutral and electrogenic transport modes are in principle thermodynamically viable. However, only the electrogenic variants have a net thermodynamic driving force under the physiological conditions experienced by microorganisms and plants. Thus, the transport mechanism of AfAmt-1 is most likely electrogenic.

PII-like proteins, such as GlnK, found in a wide variety of organisms from prokaryotes to plants constitute a family of cytoplasmic signaling proteins that play a central regulatory role in the assimilation of nitrogen for biosyntheses. They specifically bind and are modulated by effector molecules such as adenosine triphosphate, adenosine diphosphate and 2-oxoglutarate. Their highly conserved, trimeric structure suggests that cooperativity in effector binding might be the basis for the ability to integrate and respond to a wide range of concentrations, but to date no direct quantification of this cooperative behavior has been presented. The hyperthermophilic archaeon Archaeoglobus fulgidus contains three GlnK proteins, functionally associated with ammonium transport proteins (Amt). We have characterized GlnK2 and its interaction with effectors by high-resolution X-ray crystallography and isothermal titration calorimetry. Binding of adenosine nucleotides resulted in distinct, cooperative behavior for ATP and ADP. While 2-oxoglutarate has been shown to interact with other GlnK proteins, GlnK2 was completely insensitive to this key indicator of a low level of intracellular nitrogen. These findings point to different regulation and modulation patterns and add to our understanding of the flexibility and versatility of the GlnK family of signaling proteins.