Soupene E, Ramirez R M, Kustu S
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Mol Cell Biol. 2001 Sep;21(17):5733-41. doi: 10.1128/MCB.21.17.5733-5741.2001.
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 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 Deltavph1 mutant of S. cerevisiae and a Deltavma1 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).
酿酒酵母中的甲铵和铵（MEP）通透酶属于一类普遍存在的细胞质膜蛋白家族，该家族仅转运铵（NH₄⁺ + NH₃）。铵类似物[¹⁴C]甲铵（一种弱碱）的转运和积累促使人们提出该家族成员能够进行能量依赖的铵离子NH₄⁺浓缩。然而，在细菌中，谷氨酰胺合成酶将甲铵ATP依赖性转化为γ-N-甲基谷氨酰胺，这使得它无法用于评估跨细胞质膜的浓缩转运。我们已证实甲铵在酿酒酵母中不会被代谢，并表明它在丝状真菌粗糙脉孢菌中也很少被代谢。然而，它的积累依赖于液泡的能量依赖酸化。缺乏液泡H⁺-ATP酶活性的酿酒酵母Deltavph1突变体和Deltavma1突变体在甲铵积累方面存在很大（五倍或更大）缺陷，而初始转运速率几乎没有伴随缺陷。粗糙脉孢菌的vma-1突变体将甲铵大量代谢为甲基谷氨酰胺。因此，与细菌一样，在真菌中，随后甲铵的能量依赖利用使其无法用于评估跨细胞质膜的主动转运。在酸性液泡中隔离带电物质CH₃NH₃⁺需要质子梯度，这证明MEP蛋白的底物是不带电物质CH₃NH₂。由此推断，它们的天然底物是气体NH₃。我们推测MEP蛋白促进NH₃跨细胞质膜的扩散，并推测与MEP蛋白位于同一结构域家族的人类恒河猴蛋白促进CO₂的扩散。
