Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Lack of evidence for phosphorylation of Arabidopsis thaliana PII: implications for plastid carbon and nitrogen signaling
Introduction
The phosphorylation of proteins is a common regulatory event found in organisms across the three domains of life and is likely the most prevalent protein modification that takes place in eukaryotic cells [1]. The addition of phosphate to a protein is known to potentially alter biological activity, increase or decrease the rate of proteolytic turnover, promote shuttling between subcellular compartments, and facilitate protein:protein interactions [1]. The phosphorylation of proteins in prokaryotes is thought to be less common than compared to eukaryotes. Yet, recent genomic surveys for protein kinases and phosphatases indicate that protein phosphorylation may be more common in prokaryotes than originally thought [2].
Before committing to metabolic and cellular events linked to carbon and nitrogen metabolism, most organisms assess their carbon and nitrogen status through key metabolite levels [3], [4], [5], [6]. In enteric bacteria this function is performed primarily by a trimeric protein with ~12-kDa subunits that is referred to as PII. The best-characterized example occurs in E. coli, where the carbon status molecule, 2-oxoglutarate, is allosterically perceived by PII. PII also interprets the cellular nitrogen status, but in this case the nitrogen signal molecule, glutamine, is allosterically interpreted by another protein (uridylyltransferase/uridylyl-removing enzyme) which then (de)uridylylates PII appropriately. Thus, the nitrogen status is conferred by the covalent modification of PII. The combined result of these two signals, carbon status and nitrogen status, then affects the ability of PII to interact with and control two other proteins: adenylyltransferase and NRII [7], [8], [9]. The bifunctional adenylyltransferase regulates the activity of glutamine synthetase by addition or removal of an AMP moiety while the protein kinase/phosphatase NRII controls the transcription of the nitrogen sensitive regulon, which includes glutamine synthetase. In cyanobacteria, as exemplified by Synechococcus PCC 7942, the PII protein is not uridylylated, but phosphorylated on a T-loop serine located two residues away from the tyrosine modified in E. coli PII. When Synechococcus PCC 7942 is grown in the presence of ammonium, PII is not phosphorylated. Cell culture in the presence of nitrate or no nitrogen source leads to increasing degrees of PII phosphorylation reflecting the nitrogen regime [10], [11]. The protein kinase and phosphatase that modify cyanobacterial PII display reciprocal activities in the presence and absence of ATP and 2-oxoglutatrate. These two small molecules allosterically bind PII controlling the ability of the kinase or phosphatase to act upon it [12], [13], [14]. The protein targets of cyanobacterial PII are not known.
Plant PII is localized to the chloroplast and the protein shares 50% and 54% sequence identity to E. coli and Synechococcus/Synechocystis PII, respectively [15], [16]. Early work by Heish et al. [15] revealed that Arabidopsis PII mRNA was induced by light and further regulated by carbon and nitrogen metabolites. More recently, we have demonstrated that plant PII migrates as a 17-kDa protein during denaturing gel electrophoresis [15], [17], chromatographs as a ∼50-kDa protein on gel filtration and models well to the E. coli PII structure [18]. These data suggest that, like bacterial PII, plant PII exists as a trimer. Arabidopsis PII selectively binds the adenylates ADP and ATP with high affinity and the binding constant determined for 2-oxoglutarate indicates that it likely functions as a carbon sensor in the chloroplast [18].
To date, it is not known if plant PII is regulated by covalent modification. The tyrosine that is uridylylated in E. coli PII is not conserved in plants, but the T-loop serine that is modified in certain cyanobacteria (Fig. 2A) is maintained in all plant species examined to date. Furthermore, a preliminary phylogenetic analysis demonstrates that plant PII proteins always group closest to cyanobacterial PII (data not shown). Because of the important role played by covalent modification in prokaryotic PII, we speculated that plant PII may be regulated by phosphorylation as well [16], [18]. We have employed a number of techniques to examine the phospho-status of Arabidopsis thaliana PII under various carbon and nitrogen regimes. All of our data suggest that plant PII is not regulated by phosphorylation and we discuss the implications of this observation.
Section snippets
Cloning and overexpression of A. thaliana PII
The high-level expression vector encoding the A. thaliana PII gene product (GLB1) minus the transit peptide region was constructed and recombinant A. thaliana PII minus the transit peptide (PII-tp) was purified as described by Smith et al. [17].
A. thaliana cell culture conditions
Our photoheterotrophic wild-type A. thaliana Columbia ecotype suspension cells were cultured in liquid Murashige and Skoog (MS) media in the presence of 3% sucrose, harvested and stored as described [17]. In order to grow the suspension cells in various
In vivo labeling
We initiated our work by performing time course experiments to identify the optimal 32Pi labeling time for our photoheterotrophic plant suspension cell culture. Maximal protein labeling appeared between 4 and 6 h (data not shown) and therefore all subsequent labeling experiments were 4 h in length. PII was immunoprecipitated from 32Pi-labeled cells cultured in complete media that contains both ammonium and nitrate as a nitrogen source. The enriched 17-kDa band was confirmed to be PII by Western
Conclusions
It is well established that in enteric bacteria the interpretation of cellular nitrogen status occurs primarily through the covalent modification of PII via the uridylyltransferase/uridylyl-removing enzyme [4], [5], [6]. Under conditions of poor nitrogen status, i.e. low glutamine, E. coli PII is uridylylated and this governs its ability to control the signaling cascade to turn on glutamine synthetase activity and transcription of the ‘nitrogen sensitive regulon’ [4], [5], [6]. More recent work
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of Canada. We thank Hue Tran for assistance in preparing figures.
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