Evidence that NR inactivation requires NRphosphorylation and 1433binding

When extracts were rapidly prepared from illuminated or darkened leaves and NR activity was measured in the presence of excess divalent cations, activity was usually twice as high in extracts from illuminated leaves than from darkened leaves. These changes in NR activity were not affected by desalting of extracts; thus they were not due to some direct allosteric effects of metabolites on NR (Kaiser and Brendle-Behnisch 1991). Importantly, these activity changes were observed only in buffers containing divalent cations in excess, but were absent in the presence of excess EDTA. Apparently divalent cations were required to keep NR in its "inactivated state". NR activity from "light extracts" could be rapidly decreased in vitro by incubation with MgATP. Based on such observations, we suggested already in 1991 that NR was probably inactivated by protein phosphorylation (Kaiser and Spill 1991). Subsequently, Huber et al. (1992) and MacKintosh (1992) presented direct evidence that NR was indeed more phosphorylated in dark than in light, consistent with the activity measurements. Some years later, a Ser-543 residue in the hinge 1 region was identified as the site of regulatory phosphorylation (Douglas et al. 1995, Bachmann et al. 1996a, Su et al. 1996).

On a first attempt to fractionate a leaf extract into a protein kinase free NR fraction and a kinase fraction, we realised that just mixing a kinase fraction with the NR fraction was sufficient to phosphorylate NR upon addition of y-32P-ATP, but without inactivating it (Glaab and Kaiser 1995). Inactivation of phosphorylated NR (P-NR) occurred only after addition of yet another protein fraction which lacked protein kinase activity. Similar observations were independently reported by MacKintosh's group, and the unknown protein was subsequently termed "IP" or "NIP", which stand for "inhibitor protein". Characterisation of that "IP" showed that it belonged to a family of binding proteins known as 14-3-3, for which 14 isoforms have been found in Arabidopsis (Bachmann et al. 1996b, Moorhead et al. 1996, Bachmann et al. 1998, MacKintosh 1998) (Figure 2).

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