title: ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles.
abstract: Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
authors.affiliation: Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy.
authors.forename: Alexis
authors.initials: A
authors.lastname: De Angeli
authors.forename: Oscar
authors.initials: O
authors.lastname: Moran
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wege
authors.forename: Sophie
authors.initials: S
authors.lastname: Filleur
authors.forename: Geneviève
authors.initials: G
authors.lastname: Ephritikhine
authors.forename: Sébastien
authors.initials: S
authors.lastname: Thomine
authors.forename: Hélène
authors.initials: H
authors.lastname: Barbier-Brygoo
authors.forename: Franco
authors.initials: F
authors.lastname: Gambale
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000465:Algorithms; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
chemical_list: C407524:ATCLC-A protein, Arabidopsis; D029681:Arabidopsis Proteins; D018118:Chloride Channels; D009566:Nitrates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate