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• W2109556543 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. 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. The PII signal transduction protein of Arabidopsis thaliana forms an arginine-regulated complex with plastid N-acetyl glutamate kinase. VOLUME 281 (2006) PAGES 5726-5733Journal of Biological ChemistryVol. 281Issue 33PreviewPAGE 5726: Full-Text PDF Open Access 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 (1Forchhammer K. FEMS Microbiol. Rev. 2004; 28: 319-333Crossref PubMed Scopus (205) Google Scholar, 2Ninfa A.J. Jiang P. Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (211) Google Scholar, 3Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (352) Google Scholar, 4Ninfa A.J. Atkinson M.R. Trends Microbiol. 2000; 8: 172-179Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). 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) 3The abbreviations used are: 2KG, 2-ketoglutarate; NAG, N-acetyl glutamate; NAGK, NAG kinase; ITC, isothermal titration calorimetry; MALDI-TOF, matrix assisted laser desorption ionization time-of-flight; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; Ni-NTA, nickel-nitrilotriacetic acid. 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 (3Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (352) Google Scholar, 5Magasanik B. Trends Microbiol. 2000; 8: 447-448Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In terms of metabolic sensing, cyanobacterial PII plays a similar role, but in this case, covalent modification is by phosphorylation (6Forchhammer K. Tandeau de Marsac N. J. Bacteriol. 1994; 176: 84-91Crossref PubMed Scopus (141) Google Scholar). To date, the processes known to be regulated by PII in cyanobacteria are: ammonium-dependent nitrate/nitrite uptake (7Lee H.M. Flores E. Forchhammer K. Herrero A. Tandeau De Marsac N. Eur. J. Biochem. 2000; 267: 591-600Crossref PubMed Scopus (63) Google Scholar), high affinity bicarbonate transport (8Hisbergues M. Jeanjean R. Joset F. Tandeau de Marsac N. Bedu S. FEBS Lett. 1999; 463: 216-220Crossref PubMed Scopus (69) Google Scholar), regulation of the global transcriptional activation by NtcA (9Paz-Yepes J. Flores E. Herrero A. FEBS Lett. 2003; 543: 42-46Crossref PubMed Scopus (50) Google Scholar, 10Fadi Aldehni M. Sauer J. Spielhaupter C. Schmid R. Forchhammer K. J. Bacteriol. 2003; 185: 2582-2591Crossref PubMed Scopus (62) Google Scholar), and arginine biosynthesis (11Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (110) Google Scholar). In eukaryotes, PII has only been identified in plants and red algae (12Moorhead G.B. Smith C.S. Plant Physiol. 2003; 133: 492-498Crossref PubMed Scopus (41) Google Scholar), 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 (13Smith C.S. Morrice N.A. Moorhead G.B. Biochim. Biophys. Acta. 2004; 1699: 145-154Crossref PubMed Scopus (36) Google Scholar). Like the bacterial protein, plant PII binds 2-oxoglutarate, but only after binding ATP first, and thus likely functions to sense plastid energy and 2KG status (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar). PII transcripts appear to be present in all plant organs, and PII protein levels do not change during day/night cycles or N-nutrition (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar, 15Ferrario-Mery S. Bouvet M. Leleu O. Savino G. Hodges M. Meyer C. Planta. 2005; : 1-12Google Scholar). Although PII T-DNA knock-out lines show increased sensitivity to nitrite and slight alternations in carbon metabolite and amino acid levels during altered N-nutrition (15Ferrario-Mery S. Bouvet M. Leleu O. Savino G. Hodges M. Meyer C. Planta. 2005; : 1-12Google Scholar), no molecular targets have been firmly established for plant PII. Two preliminary yeast two-hybrid studies have indicated that N-acetyl glutamate kinase (NAGK) is a PII interactor in plants (16Burillo S. Luque I. Fuentes I. Contreras A. J. Bacteriol. 2004; 186: 3346-3354Crossref PubMed Scopus (103) Google Scholar, 17Sugiyama K. Hayakawa T. Kudo T. Ito T. Yamaya T. Plant Cell Physiol. 2004; 45: 1768-1778Crossref PubMed Scopus (72) Google Scholar). We searched for PII-interacting proteins by performing affinity chromatography with plant PII and have identified the major PII receptor of A. thaliana as the plastid enzyme N-acetyl glutamate kinase. Biochemical studies showed that PII alters the kinetic properties of NAGK and that the downstream end product of this metabolic pathway (arginine) promotes the interaction of PII and NAGK. Interestingly, arginine levels are barely detectable in plants during the light period and are high during the dark (18Coruzzi G.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2003https://doi.org/10.1199/tab.0010Crossref Google Scholar), suggesting arginine inhibition of NAGK during the light and relief by PII in the dark. Affinity Chromatography—A. thaliana PII minus the chloroplast transit peptide was expressed and purified as described (19Smith C.S. Zaplachinski S.T. Muench D.G. Moorhead G.B. Protein Expression Purif. 2002; 25: 342-347Crossref PubMed Scopus (17) Google Scholar). After dialysis into PBS, 2 mg of PII and 2 mg of Fraction V BSA (Sigma) were separately coupled to 1 ml of CH-Sepharose (Amersham Biosciences) by following the manufacturer’s instructions (coupling was >95%). A. thaliana suspension cells were grown in culture, harvested, and lysed as described in Smith et al. (13Smith C.S. Morrice N.A. Moorhead G.B. Biochim. Biophys. Acta. 2004; 1699: 145-154Crossref PubMed Scopus (36) Google Scholar). Typically, ∼300 g of cells were lysed in a French press with 1 volume of 25 mm Tris/Cl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 1 mm benzamidine, 0.1 mm PMSF, 0.1% (v/v) 2-mercaptoethanol, and 1% (w/v) polyvinylpyrrolidone and clarified by centrifugation at 35,000 rpm for 30 min in a Ti-45 rotor. Following filtration through Miracloth (Calbiochem), the crude extract (∼1900 mg of protein) was then split in half and mixed end-over-end at 4 °C with 1 ml of PII or BSA affinity matrix that was previously equilibrated in buffer A (25 mm Tris/Cl, pH 7.5, 1 mm benzamidine and 0.1 mm PMSF). After 4 h, the mixture was poured into a column and washed with 300 ml of buffer A plus 150 mm NaCl, and protein was eluted with 8 ml of buffer A plus 1.5 m NaCl. Eluted protein was concentrated in a Centriprep 10 (Amicon) and then in a Centricon 10 to 25 μl and boiled in SDS-mixture. In all cases, PII- and BSA-Sepharose chromatographies were performed identically to allow direct comparison. Mass Spectrometry and Edman Sequencing—PII- and BSA-binding proteins were run on SDS-PAGE and Coomassie Blue-stained, and bands were excised, trypsin was digested, and proteins were identified by MALDI-TOF mass spectrometry (20Tran H.T. Ulke A. Morrice N. Johannes C.J. Moorhead G.B. Mol. Cell Proteomics. 2004; 3: 257-265Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The only proteins indicated (see Fig. 1A) are those for which a high level of confidence for identification was obtained (see supplementary Table 1). In all cases, the identified protein was the best match to the peptide mass data, the identified protein predicted mass was very near the observed mass on the gel, and the percentage of peptide coverage was high, ranging from 11 to 62% of the total number of residues using a cut-off of less than 35 ppm. For N-terminal sequencing, the PII-Sepharose-eluted proteins were run on SDS-PAGE, blotted to PVM, stained with Amido Black, and washed with water to visualize the 33-kDa protein. This band was excised and submitted to Edman chemistry as described (21Moorhead G. MacKintosh C. Morrice N. Cohen P. FEBS Lett. 1995; 362: 101-105Crossref PubMed Scopus (82) Google Scholar). Cloning, Expression, and Purification of A. thaliana N-Acetyl Glutamate Kinase and Non-tagged PII—The N-acetyl glutamate kinase gene was PCR-amplified (to start from the predicted chloroplast cleavage site Ala50) from an A. thaliana silique cDNA library template obtained from the A. thaliana Biological Resource Centre, Ohio State University, using the following primers: 5′-TATAAGATCTCAGCCACCGTATCAACACCAC-3′ and 3′-TATAGAATTCTTATCCAGTAATCATAGTTCCAGC-5′. After digestion with EcoRI and BglII, the PCR product was cloned into EcoRI-BglII-restricted pRSET A plasmid (Invitrogen) to produce a fusion construct that coded for NAGK and a 6-histidine tag on the N terminus with a predicted mass of ∼36 kDa. This construct was transformed into E. coli DH5α cells, and positive transformants were sequenced at the University of Calgary DNA sequencing facility and confirmed to have identical amino acid sequence as A. thaliana NAGK. The resulting plasmid was used for the overexpression of His6-NAGK fusion protein in E. coli Rosetta gami pLysRARE cells (Novagen). The transformed bacterial cells were grown at 37 °C for 26 h. The cells from 1 liter of culture were harvested by centrifugation and resuspended in 50 ml of bacterial lysis buffer (25 mm Tris/Cl, pH 7.5, 150 mm NaCl, 10 mm imidazole, pH 7.5, 0.5 mm PMSF, 0.5 mm benzamidine, 5 μg/ml leupeptin). Cells were lysed by two passes through a French pressure cell at 1000 p.s.i., and cell debris was removed by centrifugation at 35,000 rpm for 45 min at 4 °C in a Ti-45 rotor. The soluble recombinant NAGK protein was bound to a 10-ml Ni-NTA agarose (Qiagen) column pre-equilibrated with bacterial lysis buffer. The matrix was washed with 25 mm Tris/Cl, pH 7.5, 1 m NaCl, 30 mm imidazole, pH 7.5, 0.1% (v/v) Tween 20, 0.5 mm PMSF, and 0.5 mm benzamidine. The His6-NAGK protein was eluted with 50 mm Tris/Cl, pH 7.5, 150 mm NaCl, 300 mm imidazole, pH 7.5, 0.5 mm PMSF, and 0.5 mm benzamidine. Peak fractions were pooled and dialyzed against PBS plus 50% (v/v) glycerol. The purified expressed protein was confirmed to be NAGK by MALDI-TOF mass spectrometry as described above. One liter of culture produced ∼45 mg of pure NAGK. A non-tagged PII protein was made by PCR amplification from the plasmid described in Ref. 19Smith C.S. Zaplachinski S.T. Muench D.G. Moorhead G.B. Protein Expression Purif. 2002; 25: 342-347Crossref PubMed Scopus (17) Google Scholar, cloned into the pET3a vector, and after sequence verification, used to transform E. coli Rosetta gami pLys cells. Protein was expressed by growing cells in Terrific broth at 25 °C for 16 h in 0.1 mm isopropyl-1-thio-β-d-galactopyranoside. After harvesting cells and French press lysis as above, the protein was purified by sequential chromatography on Macro-Prep High S matrix (Bio-Rad) and phenyl-Sepharose (Amersham Biosciences). One liter of cells yielded 10 mg of pure PII. Reverse Affinity Chromatography—The A. thaliana PII cloned without a tag or transit peptide (32 μg) was mixed with either His6-NAGK (65 μg) or His6-PR65α(65 μg) for 10 min in a total volume of 100 μl with 25 mm Tris/Cl, pH 7.5, 150 mm NaCl, 5% (v/v) glycerol and then mixed with 25 μl of Ni-NTA-agarose end-over-end for 1 h at 4°C. After pelleting, beads were washed two times with 500 μl of PBS, two times with 25 mm Tris/Cl, pH 7.5, 0.05% (v/v) Nonidet P-40, and 20 mm imidazole, and two times again with PBS, and then proteins were released from the matrix by the addition of 0.3 m imidazole, pH 7.5. The following metabolites were included in the incubation mixture to test for their ability to disrupt or enhance the PII-NAGK interaction: Glu, Gln, Asp, ATP, ADP, carbamoyl phosphate, citrulline, and Lys (all at 5 mm), 2KG, AMP, NaNO2 (all at 1 mm), NH4Cl (20 mm), 2KG + ATP (1 and 5 mm, respectively), MgCl2 (10 mm), MgCl2 + ATP (10 and 5 mm, respectively), MgCl2 + ATP (10 and 5 mm, respectively), N-acetyl glutamate (NAG) (20 mm), and Arg (1, 5, 10, and 20 mm). In one series of experiments in which 5 mm arginine was included in the incubation buffer, the same concentration of arginine was included in all wash steps. Antibody Production and Localization of NAGK to Chloroplasts—Recombinant NAGK purified by Ni-NTA-agarose (see Fig. 1B) was used to raise antibodies in a rabbit using standard procedures (19Smith C.S. Zaplachinski S.T. Muench D.G. Moorhead G.B. Protein Expression Purif. 2002; 25: 342-347Crossref PubMed Scopus (17) Google Scholar). The antibodies were used in Western blots and immunofluorescence experiments as 5000- and 200-fold diluted crude serum, respectively. For immunological staining of fixed cells, A. thaliana suspension cells were grown as described previously (13Smith C.S. Morrice N.A. Moorhead G.B. Biochim. Biophys. Acta. 2004; 1699: 145-154Crossref PubMed Scopus (36) Google Scholar), harvested, and allowed to settle by gravity. Once settled, the supernatant was removed, and the cells were fixed by adding a freshly prepared 4% (w/v) formaldehyde solution made in PBS. Cells were fixed for 15 min before being washed with PBS (3× 1 ml). The cell wall was partially digested with 500 μl of a 0.1% (w/v) pectolyase Y-23 (Seishen Pharmaceutical Co.) solution made in PBS for 15 min at 30 °C. The cell membrane was permeabilized by incubation with 1% (v/v) Triton X-100 for 5 min at room temperature. The cells were then washed with PBS (3× 1 ml), blocked in 2% (w/v) BSA in PBS for 10 min, and then incubated overnight at 4 °C in 200-fold diluted crude serum specific for NAGK or PII (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar). The following day, the cells were washed with PBS (3× 1 ml) and incubated with anti-mouse secondary antibody conjugated to the fluorophore Alexa Fluor 488 (Molecular Probes) and in blocking buffer for 1 h at room temperature. The samples were washed again in PBS (3× 1 ml) before being analyzed with a fluorescence microscope (Leica DMR) using the fluorescein isothiocyanate filter set. For the observation of chloroplasts, chlorophyll fluorescence was achieved using the UV filter set. Images were captured using a cooled CCD camera (Retiga 1350 EX, Qimaging), and image enhancement and deconvolution confocal algorithm manipulations were performed using the Openlab software package (Version 3.0, Improvision). Pseudocoloration and image manipulation was performed in PhotoShop. Gel Filtration Chromatography—Protein samples were diluted into Superdex 200 column buffer (25 mm Tris/Cl, pH 7.5, 150 mm NaCl, 5% (v/v) glycerol) to a final volume of 45 μl, centrifuged at 14,000 rpm for 10 min, passed through a Costar 0.22-μm Spin-X filter, and then chromatographed on a Superdex 200 PC 3.2/30 column (Amersham Biosciences) equilibrated at room temperature in column buffer. The fast protein liquid chromatography flow rate was 0.40 μl/min, and 0.10-ml fractions were collected. For experiments that included Mg-ADP, the sample was made 2 mm MgCl2 and 1 mm ADP and chromatographed in column buffer that included 0.4 mm MgCl2 and 0.2 mm ADP. The Superdex 200 PC 3.2/30 column was calibrated from a standard plot of Kav versus molecular mass for ribonuclease A (13.7 kDa), ovalbumin (43 kDa), bovine serum albumin (66 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (669 kDa), and blue dextran (void). The mass of NAGK (n = 5), His6-PII (n = 7) and the NAGK-His6-PII complex (n = 3) was determined and is presented as the mean ± S.E. Isothermal Titration Calorimetry—ITC experiments were performed as described in Smith et al. (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar) using His6-NAGK and His6-PII without transit peptides, in the following: 25 mm Tris/Cl, pH 7.5, 5% (v/v) glycerol, and 150 mm NaCl. Each run was corrected for heat of dilution. Enzyme Kinetics—For enzyme kinetics, all assays were done in duplicate in three or more separate experiments, except in the assays where Km and Vmax values were examined for arginine-inhibited NAGK, plus and minus PII. Here, each assay was done in triplicate. NAGK activity assays with purified proteins were determined as described by Heinrich et al. (11Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (110) Google Scholar). Unless stated otherwise, the reaction mixture consisted of 300 mm NH2OH · HCl, pH 7.5, 20 mm Tris/Cl, pH 7.5, 20 mm MgCl2,40mm NAG, and 10 mm ATP and NAGK diluted into 25 mm Tris/Cl, pH 7.5, and 150 mm NaCl such that each assay contained 0.81 μg of enzyme. Reactions were initiated by the addition of reaction mixture, and the incubation was carried out at 37 °C in a volume of 0.10 ml and terminated after 20 min by the addition of 0.1 ml of stop mixture (1:1:1 of 5% (w/v) FeCl3 · 6H2Oin0.2 m HCl, 8% (w/v) trichloroacetic acid, and 0.3 m HCl), and after standing for 5 min at room temperature, the tubes were centrifuged for 1 min at 14,000 rpm. Under these reaction conditions, the assay was linear up to at least 30 min. Blank reactions were performed by omitting N-acetyl glutamate from the assay. One unit of enzyme activity is defined as one mmol of product produced in 1 min calculated with a molar absorption coefficient of 456 m-1 cm-1 at 540 nm for the N-acetylglutamylhydroxamate-Fe3+ complex. The non-linear regression program GraphPadPrism 4.0 (GraphPad Software, San Diego, CA) was used for estimating the Km and Vmax values for all data except the data obtained from arginine inhibition. The catalytic efficiency and kcat of NAGK were calculated using Kcat/Km and Vmax/[NAGK]total, respectively. The Vmax and Km values for NAGK were determined by holding the concentration of the second substrate at a saturating level (NAG at 40 mm or ATP 10 mm (MgCl2 at 20 mm)). The varied substrate was omitted when carrying out blank assays. To study the effect of PII on the activity of NAGK, various amounts of A. thaliana PII (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar) were added into the diluted NAGK fraction, and the mixture was placed at 4 °C for 10 min to allow complex formation. The reaction was initiated by the addition of the reaction mixture into the NAGK-PII complex mixture. For determining the effect of PII on the Km and Vmax for NAG and ATP, PII was added into the diluted NAGK such that the molar ratio of PII-polypeptide to NAGK-polypeptide was 2:1 (based on a Bradford assay with BSA as standard) and allowed to interact at 4 °C for 10 min. For studying the NAGK activity in the presence of PII and its effect or 2KG, PII was incubated with 5 mm ATP and 1 mm 2KG for 3 min at 4 °C followed by the addition of NAGK. For studying the Km and Vmax of NAGK for the substrate ATP, PII was preincubated with 0.05 mm ATP and 1 mm 2KG. The effect of arginine on NAGK was studied under saturating substrate conditions (10 mm ATP and 40 mm NAG). The effect of arginine was also studied at Km conditions in which the concentration of NAG was set to 40 mm and the concentration of ATP was set to the Km value (1.74 mm). For either condition, various amounts of arginine were added into the NAGK fraction, and the mixture was allowed to stand for 5 min before initiating the reaction by adding substrates. The Km and Vmax values for NAGK in which NAG was the varied substrate were studied under three different concentrations of arginine, 0.16, 0.32 and 0.64 mm. Hill plots, with the equation log [Vo/(Vn-Vo)] = N log [NAG]0-log K′, were performed for each set of data. The Vmax values were estimated from Eadie-Hofstee plots, and the Km values were estimated from plots of velocity versus substrate concentration. To test the effect of arginine on the PII-NAGK complex, PII was first added into the diluted NAGK solution, and the two proteins were placed at 4 °C for 10 min to allow complex formation followed by the addition of arginine. The enzyme mixture was allowed to stand for 5 min before the initiation of assays. Identification of A. thaliana PII-binding Proteins—To identify PII-binding proteins, an extract from A. thaliana was incubated with PII coupled to Sepharose beads. After extensive washing, bound proteins were eluted with 1.5 m NaCl and run on SDS-PAGE (Fig. 1A). No proteins were eluted from the control matrix (BSA-Sepharose), whereas several proteins were consistently enriched on the PII matrix. The eluted proteins were identified by MALDI-TOF mass spectrometry and are indicated on the gel (Fig. 1A, and see also Supplemental Table 1). The major PII-binding protein of 33 kDa was identified as a putative plastid N-acetyl glutamate kinase (gi 15230338). Other minor bound proteins were the cytosolic α, β, β′, and γ subunits of the coatomer protein complex I vesicle coat protein complex and several of the smaller cytosolic 60 S ribosomal proteins. ChloroP analysis (ChloroP 1.1 Server) of the NAGK sequence predicts that this gene product is chloroplast-localized, and cleavage of the chloroplast transit peptide is between Lys49 and Ala50 likely, yielding a protein of 31.1 kDa. Edman sequencing of the PII-purified NAGK yielded the sequence 51TVSTPPSI58. This confirmed that mature NAGK has its putative chloroplast transit peptide cleaved, but between Ala50 and Thr-51, and thus likely localizes to the chloroplast. This is consistent with the expected localization of a PII-interacting protein. Being the most abundant PII-binding protein and likely residing in the same compartment, we further characterized the interaction of PII and NAGK. A. thaliana NAGK was cloned minus the chloroplast transit peptide, expressed in E. coli as a 6-histidine fusion protein, and purified (Fig. 1B). NAGK and PII Are Chloroplast-localized Proteins—The chloroplast localization of NAGK and PII was confirmed by immunolocalization experiments using antibodies raised against each protein. The NAGK antibody was first tested by probing Western blots of pure enzyme and an A. thaliana crude fraction. The diluted crude serum could easily detect less than 1 ng of recombinant NAGK and one major band of 33 kDa in a crude extract (data not shown). Both PII and NAGK stained in small structures that resemble chloroplasts and were subsequently colocalized with chlorophyll fluorescence (Fig. 2). The immunolocalization of PII and NAGK to the chloroplast is consistent with previous biochemical studies of A. thaliana PII (22Hsieh M.H. Lam H.M. van de Loo F.J. Coruzzi G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13965-13970Crossref PubMed Scopus (198) Google Scholar) and the predicted chloroplast transit peptides of both PII and NAGK. Reverse Affinity and Gel Filtration Chromatography Confirms an Interaction between PII and NAGK—Knowing that PII and NAGK localize to the same compartment supports the idea that they are true interacting partners. To further confirm an interaction between PII and NAGK, we performed the reverse experiment in which His-tagged NAGK was incubated with non-tagged PII and the complex was pulled out with Ni-NTA-beads. Fig. 3A shows that NAGK binds PII and that PII in the presence of an unrelated His-tagged protein (PR65) is not retained on the beads, nor is non-tagged PII retained on the beads alone (data not shown). The mass of the A. thaliana purified NAGK was determined by gel filtration chromatography after elution from the PII matrix. Peak fractions demonstrated a mass of ∼215 kDa (Fig. 3B). This 33-kDa band was confirmed to be NAGK by Western blot analysis (data not shown) and suggests that the native protein exists as a multimer of six or seven 33-kDa subunits. Fig. 3, C-F, show the elution profiles for recombinant PII alone, NAGK alone, and a mixture of PII plus excess NAGK. Tagged PII displayed a mass of ∼45 ± 0.5 kDa (14Smith C.S. Weljie A.M. Moorhead G.B. Plant J. 2003; 33: 353-360Crossref PubMed Scopus (70) Google Scholar), which is consistent with a mass of a trimer of 16-kDa subunits, whereas recombinant NAGK eluted at ∼276 ± 2 kDa, which is consistent with a multimer of six or seven 33-kDa subunits (the recombinant protein contains a tag of ∼5 kDa in addition to the native protein). If the recombinant protein exists as a hexamer, subtracting six tags gives a mass similar to the native protein, giving us confidence that the bacterial produced protein is behaving like the purified plastid enzyme. When the two proteins are mixed (Fig. 3, E and F), the PII elution profile shifts dramatically, suggesting that PII and NAGK form a complex that displays a mass of ∼300 ± 1.5 kDa. PII and NAGK chromatographed in the presence of 0.4 mm MgCl2 and 0.2 mm ADP displayed the same mass (data not shown). Isothermal Titration Calorimetry—ITC allowed us to further address the stoichiometry of binding for PII and NAGK. Titration of PII into a solution of NAGK (Fig. 4) gives an n value of 0.43, suggesting that each PII trimer binds ∼7 NAGK subunits. In the reverse experiment, in which NAGK was titrated into a solution of PII, n = 1.77, suggesting that about five NAGK molecules interact with each PII trimer. In combination with the gel filtration data, this supports the idea that one PII trimer interacts with one NAGK hexamer and that native NAGK is indeed a hexamer. Altered Kinetic Behavior of NAGK in the Presence of PII and Its Effector Molecule 2KG—NAGK catalyzes the second step in the arginine biosynthetic pathway (see Supplemental Fig. 1). The reaction catalyzed by NAGK involves two substrates, ATP and NAG, in which NAGK transfers the γ-phosphate group from ATP to NAG to form N-acetyl-γ-glutamyl phosphate. For free NAGK (not complexed with PII), the apparent Vmax and Km obtained when NAG was the variable substrate was 10.6 (±0.3 S.E.) units/mg and 7.08 (±0.49 S.E.) mm, respectively, yielding a catalytic efficiency (kcat/Km) of 872 s-1m-1. For ATP, the apparent Km of 1.74 (±0.21 S.E.) mm and Vmax of 9.99 (±0.29 S.E.) units/mg yields a catalytic efficiency of 3350 s-1m-1. The plot of velocity versus substrate concentration for NAG (Fig. 5A) displayed a classic Michaelis-Menten kinetic profile (a Michaelis-Menten profile was obtained for ATP as well). As shown in Fig. 5B, the V" @default.
• W2109556543 created "2016-06-24" @default.
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• W2109556543 date "2006-03-01" @default.
• W2109556543 modified "2023-10-13" @default.
• W2109556543 title "The PII Signal Transduction Protein of Arabidopsis thaliana Forms an Arginine-regulated Complex with Plastid N-Acetyl Glutamate Kinase" @default.
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• W2109556543 doi "https://doi.org/10.1074/jbc.m510945200" @default.
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