title: Identification of structural elements involved in fine-tuning of the transport activity of the rice ammonium transporter OsAMT1;3.
abstract: Ammonium transporters (AMTs) are major routes for plant uptake of the NH4+-form nitrogen. Plant AMTs mediate predominantly the uptake of NH4+and to a lesser extent, its organic analog methylammonium (MeA+). Mutagenesis studies on potential phosphorylation residues have achieved solid recognition that alteration of the phosphorylation status can result in allosteric regulation and impair the functionality of plant AMTs. However, molecular insights to the fine-tuning of a functional ammonium transporter remain less clear. In this report, we demonstrate that the rice root expressed OsAMT1;3 (Oryza sativa ammonium transporter 1;3) functions as a typical high-affinity NH4+transporter and is weakly permeable to MeA+using growth assays in NH4+uptake defective yeast cells and electrophysiological measurements in Xenopus oocytes. Upon screening of six point mutations generated with the transporter, we identified two amino acid residues involved in the functional modulation of OsAMT1;3. The H199E mutation caused loss of transport activity whereas other five mutations retained the functionality of OsAMT1;3. Furthermore, the L56F mutation enabled respectively 5- and 3.5 -fold increased capability for NH4+and MeA+uptake with several-fold decreased affinity (Km) and accelerated Vmaxvalues. Surprisingly, yeast cells expressing the L56F mutation shown increased Na+toxicity leading to a speculation that enhanced Na+permeation occurred with this mutation. The phenomenon was further supported by the observation of significant Na+uptake current in oocytes. Our results seemingly support a speculation that the L56F mutation of OsAMT1;3 widens the substrate passage tunnel and allows enhanced permeability to NH4+and larger ions MeA+and Na+.
authors.affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Dongli
authors.initials: D
authors.lastname: Hao
authors.affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China.
authors.forename: Shunying
authors.initials: S
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China. Electronic address: yhsu@issas.ac.cn.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Su
mesh_terms: D000641:Ammonia / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004594:Electrophysiology / Q000379:methods; D005260:Female; D008744:Methylamines / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D012275:Oryza / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D017354:Point Mutation; D012964:Sodium / Q000378:metabolism / Q000493:pharmacokinetics; D014981:Xenopus; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D027682:Cation Transport Proteins; D008744:Methylamines; D010940:Plant Proteins; C088952:ammonium transporters, plant; D000641:Ammonia; D012964:Sodium; C027451:methylamine
keywords: Functional modulation; OsAMT1;3; Point mutations; Substrate permeability