A two-lane mechanism for selective biological ammonium transport

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process

Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation

Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily

Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH3/NH4+) by the distinct members of this family is still a matter of controversy. Using Saccharomyces cerevisiae cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH4+/NH3 conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in N-15 relative to N-14 when compared to the external ammonium source. We observed N-15 depletion over a wide range of external pH, indicating its independence of NH3 formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K+. We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor N-14 over N-15, owing to the dissociation of NH4+ into NH3+ H+ in the protein, leading to N-15 depletion in the cell and allowing NH3 passage or NH3/H+ cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH4+/K+ ratio, allowing organisms to specifically scavenge NH4+. We show that N-15 isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.I. A. was supported by a postdoctoral fellowship from the Government of Navarra, Spain (Anabasid outgoing Programme, 2011) and by a postdoctoral fellowship from the Portuguese Fundaçao para a Ciencia e a Tecnologia (SFRH/BPD/90436/2012). A.M.M. is a senior research associate of the Belgian Fonds de la Recherche Scientifique Fonds de la Recherche Scientifique-FNRS (grants CDR J017617F, PDR T011515F, and ARC) and a WELBIO investigator, and M.B. is a scientific research worker supported by WELBIO. This work was also developed in the context of the following projects: PTDC/BIA-BEC/099323/2008 and PTDC/AGR-PRO/115888/2009 to cE3c and FCUL, UID/DTP/04138/2013 to iMed. ULisboa, and AGL2015-64582-C3-1-R and AGL2012-37815-C05-05 to UPNa

α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Arrestins, known regulators of endocytosis, take on novel functions in nutrient-regulated endosomal recycling. Yeast α-arrestins, Aly1 and Aly2, redistribute the Gap1 permease from endosomes to the cell surface and interact with clathrin/AP-1. Aly2 is regulated by the Npr1 kinase and acts through mechanisms distinct from Aly1

The general amino acid permease FfGap1 of <em>Fusarium fujikuroi</em> Is sorted to the vacuole in a nitrogen-dependent, but Npr1 kinase-independent manner.

The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1&Delta; mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast

Correction : a two-lane mechanism for selective biological ammonium transport

Williamson G, Tamburrino G, Bizior A, Boeckstaens M, Mirandela GD, Bage MG, Pisliakov A, Ives CM, Terras E, Hoskisson PA, Marini AM, Zachariae U, Javelle A. 2020. A two-lane mechanism for selective biological ammonium transport. eLife 10:e57183. doi: 10.7554/eLife.57183 Published 14 July 2020 In Figure 4 panel B, we inadvertently used the same image to represent the lack of yeast growth for both D160A and D160E variants of AmtB. This has been corrected and D160E now has the appropriate image. As both the original and corrected panel show the same result, the text and figure legend remain unchanged. The article has been corrected accordingly