Deletion of the Nitrate Reductase N-Terminal Domain Still Allows Binding of 14-3-3 Proteins but Affects Their Inhibitory Properties

Abstract

Abstract Nitrate reductase (NR) is post-translationally regulated by phosphorylation and binding of 14-3-3 proteins. Deletion of 56 amino acids in the amino-terminal domain of NR was previously shown to impair this type of regulation in tobacco (Nicotiana plumbaginifolia) (L. Nussaume, M. Vincentez, C. Meyer, J.-P. Boutin, M. Caboche [1995] Plant Cell 7: 611–621), although both full-length NR and deleted NR (ΔNR) appeared to be phosphorylated in darkness (C. Lillo, S. Kazazaic, P. Ruoff, C. Meyer [1997] Plant Physiol 114: 1377–1383). We show here that in the presence of Mg2+ and phosphatase inhibitors, NR and endogenous 14-3-3 proteins copurify through affinity chromatography. Assay of NR activity and western blots showed that endogenous 14-3-3 proteins copurified with both NR and ΔNR. Electron transport in the heme-binding domain of ΔNR was inhibited by Mg2+/14-3-3, whereas this was not the case for NR. This may indicate a different way of binding for 14-3-3 in the ΔNR compared with NR. The ΔNR was more labile than NR, in vitro. Lability was ascribed to the molybdopterin binding domain, and apparently an important function of the 56 amino acids is stabilization of this domain.