Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties

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 (DeltaNR) 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 Mg(2+) 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 DeltaNR. Electron transport in the heme-binding domain of DeltaNR was inhibited by Mg(2+)/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 DeltaNR compared with NR. The DeltaNR 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.

Figure 1

Protein-gel blots showing NR, ΔNR, and 14-3-3 proteins after Blue Sepharose chromatography of extracts from C1, del7 (d7), and del8 (d8) plants. Affinity-purified NR and ΔNR were subjected to SDS-PAGE. NR purified in the presence (lane 1) and absence of Mg²⁺ (lane 2). ΔNR (d7) purified in the presence (lane 3) and absence of Mg²⁺ (lane 4). Lanes 5 and 6 show ΔNR from del7 and del8 purified in the presence of Mg²⁺, and a different assay for detecting 14-3-3 protein was used (see “Materials and Methods”). Samples tested in lanes 1 through 4 were eluted with KNO₃ from the Blue Sepharose column and concentrated on Centricon 30. Samples tested in lanes 5 and 6 were eluted with NADH from the Blue Sepharose column and concentrated on Centricon 100. Amounts of protein added to wells 1 through 4 were approximately 6 μg and for wells 5 and 6, approximately 2 μg. Following transfer of the proteins to a nitrocellulose membrane, the membrane was cut in two halves. Top, Upper half of the membrane was probed with antiserum raised against squash NR. Bottom, Lower half of the membrane was probed with antiserum raised against spinach 14-3-3 proteins.

Figure 2

Stability of NR and ΔNR in fractions of freshly prepared affinity-purified enzyme eluted with 0.3 m KNO₃. ΔNR not desalted (●), NR not desalted (○), ΔNR desalted (▵), and 0.3 m KNO₃ was added to desalted ΔNR (□). Storage temperature was 25°C. The experiment was repeated three times with different enzyme preparations. Data presented represent (▵) NR prepared in phosphate buffer and in the absence of Mg²⁺, however, the same results were obtained for preparations made in HEPES buffer in the presence or absence of Mg²⁺ and phosphatase inhibitors. se is indicated when exceeding the size of the symbol.