title: Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization.
abstract: Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Qinli
authors.initials: Q
authors.lastname: Wang
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Wangxi
authors.initials: W
authors.lastname: Luo
authors.forename: Ruili
authors.initials: R
authors.lastname: Li
authors.forename: Qihua
authors.initials: Q
authors.lastname: He
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Fang
authors.forename: Roberto De
authors.initials: RD
authors.lastname: Michele
authors.forename: Cindy
authors.initials: C
authors.lastname: Ast
authors.forename: Nicolaus
authors.initials: N
authors.lastname: von Wirén
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video / Q000379:methods; D009154:Mutation; D030821:Plants, Genetically Modified; D055503:Protein Multimerization; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D027682:Cation Transport Proteins; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D033922:Clathrin Heavy Chains; D049452:Green Fluorescent Proteins
keywords: FCS; VA-TIRFM