Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH(3) 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 (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 γ-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 Δvph1 mutant of S. cerevisiae and a Δvma1 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).

酿酒酵母（Saccharomyces cerevisiae）的甲基铵与铵（MEP）通透酶，属于一类仅能转运铵（NH₄⁺ + NH₃）的广泛分布细胞质膜蛋白家族。针对铵类似物[¹⁴C]甲基铵——一种弱碱——的转运与积累现象，曾有研究者提出假说，认为该家族成员可通过能量依赖方式浓缩铵离子NH₄⁺。但在细菌中，谷氨酰胺合成酶（glutamine synthetase）可将甲基铵以ATP依赖的方式催化转化为γ-N-甲基谷氨酰胺，这使得该类似物无法用于评估细胞质膜的浓缩转运过程。我们已证实甲基铵不会在酿酒酵母中发生代谢，同时发现其在丝状真菌粗糙脉孢菌（Neurospora crassa）中也几乎不被代谢。然而，甲基铵的积累依赖于液泡的能量依赖性酸化。酿酒酵母的Δvph1突变体与Δvma1突变体均缺乏液泡型H⁺-ATP酶（vacuolar H(+)-ATPase）活性，在甲基铵积累方面存在显著（五倍及以上）缺陷，而其初始转运速率几乎未受影响。粗糙脉孢菌的vma-1突变体则可将甲基铵大量代谢为甲基谷氨酰胺。因此，与细菌类似，真菌中甲基铵后续的能量依赖性利用过程，同样使其无法用于评估细胞质膜的主动转运。质子梯度可将带电形式CH₃NH₃⁺隔离于酸性液泡中，这一现象表明MEP蛋白的底物为不带电形式CH₃NH₂。由此推断，其天然底物为气体NH₃。我们推测MEP蛋白可促进NH₃跨细胞质膜扩散，并进一步提出假说：与MEP蛋白同属一个结构域家族的人类Rh蛋白（Rhesus proteins），可促进CO₂的扩散。