The PII Signal Transduction Protein of Arabidopsis thaliana Forms an Arginine-regulated Complex with Plastid N-Acetyl Glutamate Kinase

Abstract

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through proteinprotein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.In prokaryotic organisms, the PII protein is recognized as the key mediator of energy, carbon, and nitrogen interactions and is referred to as the central processing unit of carbon:nitrogen metabolism (1-4). Escherichia coli PII is a 112-amino acid protein that as a homotrimer senses the cellular status of both ATP and the carbon skeleton 2-oxoglutarate (2KG) 3 via allosteric means. Nitrogen status is assessed through glutamine levels by covalent modification (uridylylation) of PII. This metabolic information is signaled to other proteins by proteinprotein interaction and produces an appropriate response that alters gene expression and the activity of glutamine synthetase (3, 5). In terms of metabolic sensing, cyanobacterial PII plays a similar role, but in this case, covalent modification is by phosphorylation (6). To date, the processes known to be regulated by PII in cyanobacteria are: ammoniumdependent nitrate/nitrite uptake (7), high affinity bicarbonate transport (8), regulation of the global transcriptional activation by NtcA (9, 10), and arginine biosynthesis (11).In eukaryotes, PII has only been identified in plants and red algae (12), and its sequence is highly conserved when compared with prokaryotic PIIs, with Arabidopsis thaliana PII being 50 and 55% identical to E. coli and Synechococcus elongatus PII, respectively. Plant PII proteins have a conserved N-terminal extension that functions as a chloroplast transit peptide, which is consistent with biochemical data indicating that PII resides in this compartment. We have previously shown that the plant PII protein is not regulated by phosphorylation (13). Like the bacterial...

The crystal structure of the complex of P II and acetylglutamate kinase reveals how P II controls the storage of nitrogen as arginine

“…The pull on D142 toward P II upon network completion may drag the lid toward its lowered position (16,17) found in the high-affinity form of the NAG site. This action explains why the lid is lowered in an empty NAG site of crystal II (data not shown), and the lack of effect of P II on the K m NAG in the absence of arginine in the NAGK of A. thaliana (7), which has D142 replaced by Asn.…”

“…The NAGK closely resembles other arginine-sensitive NAGKs (17) and appears representative of plant NAGKs, which are hexameric and similar in subunit mass (7,18) and sequence (Ϸ60% identity). Because plants and cyanobacteria have highly similar P II proteins (11,12) and conserve key interacting residues, the present structure should also represent the complex in plants.…”

“…The recently determined structure of the inhibitory complex of GlnK (a P II protein) with the ammonia channel AmtB of Escherichia coli (14,15) showed that the extended T-loop blocks the cytoplasmic opening of the ammonia channel, explaining channel function inhibition. This structure sheds no light on the P II -NAGK complex because P II activates NAGK (6,7) and because ADP was found in the GlnK-AmtB complex (15), whereas ADP prevents P II -NAGK complex formation (6).…”

“…The enzyme of arginine biosynthesis that is the target of arginine inhibition, N-acetyl-L-glutamate (NAG) kinase (NAGK) (1,(3)(4)(5), was found in cyanobacteria and plants (2,(4)(5)(6)(7)(8) to be a target of the carbon/ nitrogen P II signaling protein (9,10), forming with it a complex in which arginine inhibition is alleviated (6,7). P II signaling proteins are homotrimers of a 12-to 13-kDa subunit that interact with enzymes, transcription factors, and ammonia channels, regulating their activity (9,10) and carbon/ nitrogen homeostasis.…”

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

“…The T-loop domain, also the site of covalent modification, has been shown to be important in these interactions (17). The roles of PII proteins in regulation of nitrogen metabolism have also been studied in cyanobacteria (18) and Gram-positive bacteria (19)(20)(21), as well as in Archaea (22) and plastids (23). Although glutamine seems to be the primary nitrogen signal (reflecting excess) sensed by PII proteins in enteric bacteria (24), 2OG may be the main signal (reflecting nitrogen deficiency) in some cyanobacteria (25).…”