Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH₃ across the Cytoplasmic Membrane

Abstract

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH 4 ؉ ؉ NH 3 ). Transport and accumulation of the ammonium analog [14 C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH 4 ؉ . In bacteria, however, ATP-dependent conversion of methylammonium to ␥-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A ⌬vph1 mutant of S. cerevisiae and a ⌬vma1 mutant, which lack vacuolar H ؉ -ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH 3 NH 3 ؉ in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH 3 NH 2 . By inference, their natural substrate is NH 3 , a gas. We postulate that MEP proteins facilitate diffusion of NH 3 across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO 2 .Methylammonium and ammonium permeases MEP1, MEP2, and MEP3 of Saccharomyces cerevisiae (35) and the ammonium and methylammonium transport B (AmtB) protein of enteric bacteria (64) are members of a unique family of cytoplasmic membrane transporters that are specific for ammonium (48). (We use ammonium to designate both the charged and uncharged species.) The MEP/Amt family (nomenclature, TC 2.49) occurs ubiquitously in bacteria, archaea, and eukarya (19,36). Beginning with the pioneering studies of Hackette et al. (16), the activity of MEP/Amt proteins has been assessed by studying transport and accumulation of the ammonium analog methylammonium, which can be 14 C labeled. Based on studies with methylammonium it has been proposed that members of the MEP/Amt family transport the charged species NH 4 ϩ across the cytoplasmic membrane and concentrate it in an energy-dependent manner (19, 65).We showed previously that enteric bacteria convert methylammonium to ␥-N-methylglutamine in the ATP-dependent reaction catalyzed by glutamine synthetase and hence that methylammonium cannot be used to assess energy-dependent concentrative uptake (62). The same metabolic conversion occurs in other proteobacteria, including methylotrophic pseudomonads (2, 1...

Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

“…Our results support previous speculation (17) that Rh proteins may facilitate diffusion of NH 3 . However, it was also proposed recently that the Rh1 protein paralog of the green alga Chla- mydomonas reinhardtii might be regarded as a gas channel for CO 2 , because mutants lacking this protein grow only very slowly when cultured in high CO 2 conditions (48).…”

“…Some studies suggested that RhAG-mediated uptake of MA in oocytes was an electroneutral NH 4 ϩ ͞H ϩ exchange (13), whereas electrophysiological studies in voltage-clamped RhBG-and RhCGexpressing oocytes suggested that transport is (14,15) or is not (16) electrogenic. It was also speculated that Rh and Mep͞Amt proteins might be facilitators for NH 3 gas transport (17).…”

Lack of the Rhesus protein Rh1 impairs growth of the green alga Chlamydomonas reinhardtii at high CO 2

“…Although inconsistent, all of the studies cited above concluded that Rh proteins, like their only known paralogues, the ammonium͞methylammonium transport (Amt) proteins [also called methylammonium permeases (Mep) in Saccharomyces cerevisiae], were active transport systems for the ion NH 4 ϩ . Disruption of an Rh gene in the slime mold Dictyostelium discoideum yielded no phenotype (16).Contrary to views of others, we have proposed that Amt͞Mep proteins are bidirectional channels for the uncharged species NH 3 and constitute an example of biological gas channels (12,(17)(18)(19)(20). There is now considerable genetic and physiological evidence to support this view in several microorganisms.…”

Amt/MEP/Rh proteins conduct ammonia

“…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.…”