Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH3 across the cytoplasmic membrane

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH4+ + NH3). Transport and accumulation of the ammonium analog [C-14] methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH4+. In bacteria, however, ATP-dependent conversion of methylammonium to gamma -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 Delta vph1 mutant of S. cerevisiae and a Delta 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 methyl-glutamine. 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 CH3NH3+ in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH3NH2. By inference, their natural substrate is NH3, a gas. We postulate that MEP proteins facilitate diffusion of NH3 across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO2.