Plant Physiol, June 2000, Vol. 123, pp. 757-764
Deletion of the Nitrate Reductase N-Terminal Domain Still Allows
Binding of 14-3-3 Proteins but Affects Their Inhibitory
Properties1
Fiona
Provan,
Liv-Margareth
Aksland,
Christian
Meyer, and
Cathrine
Lillo*
School of Technology and Science, Stavanger College, Box 2557 Ullandhaug, N-4091 Stavanger, Norway (F.P., L.-M.A., C.L.); and
Unité de Nutrition Azotée des Plantes, Institut National de
la Recherche Agronomique, F-78026 Versailles cedex, France (C.M.)
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 (
NR) 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 NR. Electron transport in the heme-binding domain of NR 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 NR compared with NR. The NR 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.
1
This work was financially supported by the
Norwegian Research Council.
*
Corresponding author; e-mail cathrine.lillo{at}tn.his.no; fax 33-
1-3083-3099.
© 2000 American Society of Plant Physiologists
