A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylationand 14-3-3 binding

It has previously been shown that the N-terminal domain of tobacco (Nicotia na tabacum) nitrate reductase (NR) is involved in the inactivation of the e nzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincent z, 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 long er regulated by light-dark transitions. In this study smaller deletions wer e performed in the N-terminal domain of tobacco NR that removed protein mot ifs conserved among higher plant NRs. The resulting truncated NR-coding seq uences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotian a plumbaginifolia. We found that the deletion of a conserved stretch of aci dic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivatio n by phosphorylation in the dark. Therefore, the removal of this acidic str etch 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 t runcated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.