ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles.
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.
De Angeli
Alexis
A
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy.
Moran
Oscar
O
Wege
Stefanie
S
Filleur
Sophie
S
Ephritikhine
Geneviève
G
Thomine
Sébastien
S
Barbier-Brygoo
Hélène
H
Gambale
Franco
F
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
C407524:ATCLC-A protein, Arabidopsis; D029681:Arabidopsis Proteins; D018118:Chloride Channels; D009566:Nitrates; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate