Ammonium/methylammonium transport (Amt) proteins facilitate diffusion of NH ₃ bidirectionally

Abstract

The ammonium͞methylammonium transport (Amt) proteins of enteric bacteria and their homologues, the methylammonium͞ ammonium permeases of Saccharomyces cerevisiae, are required for fast growth at very low concentrations of the uncharged species NH3. For example, they are essential at low ammonium (NH4 ؉ ؉ NH3) concentrations under acidic conditions. Based on growth studies in batch culture, the Amt protein of Salmonella typhimurium (AmtB) cannot concentrate either NH3 or NH4 ؉ and this organism appears to have no means of doing so. We now show that S. typhimurium releases ammonium into the medium when grown on the alternative nitrogen source arginine and that outward diffusion of ammonium is enhanced by the activity of AmtB. The latter result indicates that AmtB acts bidirectionally. We also confirm a prediction that the AmtB protein would be required at pH 7.0 in ammonium-limited continuous culture, i.e., when the concentration of NH3 is <50 nM. Together with our previous studies, current results are in accord with the view that Amt and methylammonium͞ammonium permease proteins increase the rate of diffusion of the uncharged species NH3 across the cytoplasmic membrane. These proteins are examples of protein facilitators for a gas. Members of the ammonium͞methylammonium transport (Amt) protein family are found in all three domains of life (1, 2). These cytoplasmic membrane proteins are involved in acquisition of ammonium, often the best nitrogen source for microbes. As a function of pH, ammonium (NH 3 ϩ NH 4 ϩ ) exists as a mixture of uncharged (gaseous) and protonated forms. [The pKa of ammonium is 9.25 (3).] Because the uncharged species, NH 3 , diffuses freely across phospholipid bilayers, there has been controversy about the need for a transport system(s) for ammonium and about the species being transported. Although many reports propose that Amt and methylammonium͞ammonium permease (MEP) proteins actively transport the charged species NH 4 ϩ (4, 5), there are several lines of evidence against this proposal (6, 7). First, apparent concentration of the ammonium analog, [ 14 C]methylammonium ( 14 CH 3 NH 2 ϩ 14 CH 3 NH 3 ϩ ), in an Amt͞MEP-dependent manner, was accounted for by metabolic trapping in enteric bacteria and by diffusion trapping into vacuoles and other acidic compartments in fungi. In each case accumulation depended on a subsequent energy-requiring reaction, one catalyzed by glutamine synthetase in bacteria or the V-type H ϩ -ATPase in fungi. Hence neither example provides evidence for active transport of methylammonium. Second, growth studies in Salmonella typhimurium indicated that the Amt protein of this organism (AmtB) did not concentrate ammonium and that the organism had no means of doing so. Third, in batch culture, amt mutants of enteric bacteria and mep mutants of Saccharomyces cerevisiae had a profound growth defect at low ammonium concentrations only at acid pH, conditions under which the concentration of the uncharged species NH 3 is very low. This finding was in accord with the view th...

Ammonium Toxicity in Bacteria

“…A possible explanation of this observation is an energyindependent transport by ammonium channels, which facilitates diffusion, rather than by permeases, which concentrate ammonium inside the cell by an energydependent transport mechanism. This kind of transport mechanism was first suggested by Soupene and coworkers for ammonium transporter AmtB from Salmonella typhimurium [32,33] and was further supported by the recently published structure of E. coli AmtB [17] as well as by transport data [14].…”

Amt/MEP/Rh proteins conduct ammonia

“…Based on the predominantly nonpolar nature of the pore of AmtB, both structural studies have concluded that conduction of the charged form appears energetically highly unfavorable and that NH 3 must be the permeating species as suggested by the Kustu group for some time [37,39]. Stopped-flow experiments with purified AmtB reconstituted into proteoliposomes [19] and a recent in vivo functional characterization of E. coli AmtB are also consistent with this view [18].…”

“…In contrast to the prevailing view of interpreting cellular methylammonium accumulation as Amt/MEP-mediated secondary transport as reviewed by von Wire´n and Merrick [42], Kustu and collaborators [37,39,40] have attributed this accumulation to metabolic trapping by glutamine synthetase or to trapping in acidic vacuoles in the case of fungi. They proposed instead that Amt/MEP proteins simply mediate bi-directional NH 3 transport and this view is now strongly supported by the structural results.…”

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

“…From experimental evidence suggesting that AmtB and Mep proteins facilitate diffusion of NH 3 across the cytoplasmic membrane of bacteria and fungi respectively [40,41], Soupene and co-workers have also speculated that mammalian Rh proteins might be gas channels. However, based on the relatively restricted organ and tissue distribution of Rh proteins and the observation that mutants of Chlamydomonas reinhardtii lacking the paralogue Rh1 protein grow very slowly under high CO 2 conditions, these authors further suggested that mammalian Rh proteins might function as CO 2 rather than as NH 3 gas channels [42,43].…”