A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.
It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.
Pigaglio
E
E
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Centre de Versailles, F-78026 Versailles cedex, France.
Durand
N
N
Meyer
C
C
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D003624:Darkness; D015870:Gene Expression / Q000528:radiation effects; D017343:Genes, Plant; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D017931:DNA Primers; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D009565:Nitrate Reductases; D050901:Nitrate Reductase