The proline 160 in the selectivity filter of the Arabidopsis NO(3)(-)/H(+) exchanger AtCLCa is essential for nitrate accumulation in planta.
Nitrate, the major nitrogen source for plants, can be accumulated in the vacuole. Its transport across the vacuolar membrane is mediated by AtCLCa, an antiporter of the chloride channel (CLC) protein family. In contrast to other CLC family members, AtCLCa transports nitrate coupled to protons. Recently, the different behaviour towards nitrate of CLC proteins has been linked to the presence of a serine or proline in the selectivity filter motif GXGIP. By monitoring AtCLCa activity in its native environment, we show that if proline 160 in AtCLCa is changed to a serine (AtCLCa(P160S) ), the transporter loses its nitrate selectivity, but the anion proton exchange mechanism is unaffected. We also performed in vivo analyses in yeast and Arabidopsis. In contrast to native AtCLCa, expression of AtCLCa(P160S) does not complement either the ΔScCLC yeast mutant grown on nitrate or the nitrate under-accumulation phenotype of clca knockout plants. Our results confirm the significance of this amino acid in the conserved selectivity filter of CLC proteins and highlight the importance of the proline in AtCLCa for nitrate metabolism in Arabidopsis.
Wege
Stefanie
S
Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 1 Avenue de la Terrasse, Gif-Sur-Yvette Cedex, France.
Jossier
Mathieu
M
Filleur
Sophie
S
Thomine
Sébastien
S
Barbier-Brygoo
Hélène
H
Gambale
Franco
F
De Angeli
Alexis
A
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D017136:Ion Transport; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009154:Mutation; D009566:Nitrates / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
C407524:ATCLC-A protein, Arabidopsis; D029681:Arabidopsis Proteins; D018118:Chloride Channels; C089713:GEF1 protein, S cerevisiae; D009566:Nitrates; D029701:Saccharomyces cerevisiae Proteins; D011392:Proline