The regulatory phosphorylated serine in full-length nitrate reductase is necessary for optimal binding to a 14-3-3 protein

Surface plasmon resonance studies and activity assays were used to examine the interaction between tobacco nitrate reductase (NR) from transgenic Nicotiana plumbaginifolia lines and the yeast 14-3-3 protein BMH1. Binding of BMH1 to NR was phosphorylation dependent. The 14-3-3 binding of a mutant tobacco NR protein from a transgenic N. plumbaginifolia line (S521D) lacking the conserved phosphorylation site was reduced by 50%, relative to the wild-type. Assays of NR activity confirmed that 14-3-3 inhibition of the enzyme activity was dependent on 14-3-3 binding.