ORIGINAL PAPER

Lıgia Lima Æ Ana Seabra Æ Paula Melo Æ Julie Cullimore

Helena Carvalho

Phosphorylation and subsequent interaction with 14-3-3 proteinsregulate plastid glutamine synthetase in Medicago truncatula

Received: 16 June 2005 / Accepted: 13 July 2005 / Published online: 1 September 2005� Springer-Verlag 2005

Abstract In this report we demonstrate that plastidglutamine synthetase of Medicago truncatula (MtGS2) isregulated by phosphorylation and 14-3-3 interaction. Toinvestigate regulatory aspects of GS2 phosphorylation,we have produced non-phosphorylated GS2 proteins byexpressing the plant cDNA in E. coli and performedin vitro phosphorylation assays. The recombinant iso-enzyme was phosphorylated by calcium dependent ki-nase(s) present in leaves, roots and nodules. Using an(His)6-tagged 14-3-3 protein column affinity purificationmethod, we demonstrate that phosphorylated GS2interacts with 14-3-3 proteins and that this interactionleads to selective proteolysis of the plastid located iso-form, resulting in inactivation of the isoenzyme. By sitedirected mutagenesis we were able to identify a GS2phosphorylation site (Ser97) crucial for the interactionwith 14-3-3s. Phosphorylation of this target residue canbe functionally mimicked by replacing Ser97 by Asp,indicating that the introduction of a negative chargecontributes to the interaction with 14-3-3 proteins andsubsequent specific proteolysis. Furthermore, we docu-ment that plant extracts contain protease activity thatcleaves the GS2 protein only when it is bound to 14-3-3proteins following either phosphorylation or mimickingof phosphorylation by Ser97Asp.

Keywords 14-3-3 proteins Æ Glutamine Synthetase ÆMedicago Æ Phosphorylation Æ Proteolysis

Abbreviations GS: glutamine synthetase Æ GS2: plastidGS Æ NR: nitrate reductase Æ Ni-NTA:nickel-nitrilotriacetic acid Æ 6x his-tag: six histidine tag

Introduction

Glutamine synthetase (E.C.6.3.1.2) is a key enzyme in thenitrogen assimilatory process, as it catalyses the first stepin the conversion of inorganic nitrogen (ammonium) intoan organic form (glutamine) (Lea et al. 1990). The com-plete understanding of the mechanisms controlling GSactivity in plants is of crucial importance due to the keyposition of this enzyme in the nitrogen assimilatorypathways. Plant GS is an octameric enzyme that occurs asdistinct isoenzymes located in the cytosol (GS1) and in theplastids (GS2). The isoenzymes are encoded by a smallmultigene family, whose members (3 to 6) show distinctpatterns of expression (Forde et al. 1989; Sakakibaraet al. 1992; Li et al. 1993; Dubois et al. 1996). Medicagotruncatula contains the smallest GS gene family identifiedto date in a higher plant, comprising only three expressedGS genes, MtGS1a and MtGS1b encoding cytosolicpolypeptides and MtGS2 encoding a plastid locatedpolypeptide (Carvalho and Cullimore 2003). It has beengenerally considered that transcriptional regulation of theGS gene family is the main regulatory point controllingGS activity in plant cells (Forde and Cullimore 1989;Walker and Coruzzi 1989; Cock et al. 1991; 1992; Rocheet al. 1993; Sukanya et al. 1994; Temple et al. 1995;Oliveira et al. 1997). However, evidence is accumulatingindicating the existence and importance of post-transla-tional mechanisms regulating GS activity (Hoelzle et al.1992; Temple et al. 1996; 1998; Ortega et al. 1999; 2001;2004; Palatnik et al. 1999). Recently, the possible post-translational regulation of the enzyme’s activity per se byreversible protein phosphorylation has emerged. Phos-phorylation of plant GS and interaction with 14-3-3proteins were first observed by Moorhead et al. (1999),and more recently this mechanism has been shown to be

L. Lima Æ A. Seabra Æ P. Melo Æ H. Carvalho (&)Instituto de Biologia Molecular e Celular Rua do Campo Alegre,823, 4150-180 Porto, PortugalE-mail: mhcarval@ibmc.up.ptTel.: +351-22-6074900Fax: +351-22-6099157

J. CullimoreLaboratoire des Interactions Plantes-MicroorganismesINRA-CNRS, BP 27, 31326 Castanet-Tolosan Cedex, France

Planta (2006) 223: 558–567DOI 10.1007/s00425-005-0097-8

involved in the regulation of cytosolic GS in senescingleaves of Brassica napus (Finnemann and Schjoerring2000) and in the green alga Chlamydomonas reihardtii(Pozuelo et al. 2001) and of plastidial GS from Nicotianatabacum (Riedel et al. 2001) and Hordeum vulgare (Manand Kaiser 2001).

Although the regulation of GS by phosphorylation inplants has only recently been shown, considerable datahas accumulated showing that the activities of other keyenzymes involved in carbon and nitrogen metabolism aremodulated by reversible phosphorylation, suggestingthat this mechanism may contribute to the coordinationof the carbon and nitrogen assimilation pathways inplants. The control of metabolic flux involves the regu-lation of both the activity and the levels of rate-limitingenzymes in the pathway. One means by which plantscontrol protein levels is through regulated protein deg-radation and certain post-translational events, such asphosphorylation and 14-3-3 association, influence thesusceptibility of important metabolic enzymes to prote-olysis. Phosphorylation of sucrose synthase and pyruvatekinase is associated with degradation of the enzymes(Hardin et al. 2003; Hardin and Huber 2004; Tang et al.2003). Furthermore, sucrose-phosphate synthase (SPS)and nitrate reductase (NR) are well studied enzymeswhich are know to be regulated by a rapid and reversiblepost-translational mechanism involving a phosphoryla-tion/dephosphorylation cycle and 14-3-3 interaction(Toroser et al. 1998; Huber et al. 1996). Modulation byphosphorylation/14-3-3 binding and enzyme degrada-tion seem to be closely related. However, in the case ofNR, it is still not clear whether 14-3-3 binding protectsphosphorylated NR from degradation or constitutes asignal for proteolysis (Cotelle et al. 2000; Mackintoshand Meek 2001; Kaiser et al. 2002).

Material and methods

Plant material

Plants of Medicago truncatula Gaertn. (cv. Jemalong J5)obtained from J. Cullimore (see author list) were grownin aeroponic conditions at 21�C, with a relative humidityof 75% and a 14 h light period at 200 lmol.m�2 s�1, inthe growth medium described by Lullien et al. (1987).For nodule induction the growth medium was replacedwith fresh medium lacking a nitrogen source, two daysbefore inoculation with Sinorhizobium meliloti strainRCR 2011. Nodules were harvested 14 days after inoc-ulation. Leaves and roots were collected from non-inoculated plants. All plant material was immediatelyfrozen in liquid nitrogen and stored at �80�C.

Production of plant GS2 and 14-3-3 proteins in E. coli

The M. truncatula isoenzyme GS2 (without the plastidtargeting signal) was expressed in E. coli using the

expression vector pTrc99A (Amersham Biosciences) aspreviously described (Melo et al. 2003). The cloneMtBB06G11 containing a full length cDNA for a 14-3-3isoform, was selected from the Medicago truncatula ESTlibrary database (Journet et al. 2002; http//medica-go.toulouse.inra.fr). The coding region was excised fromthe plasmid as an NcoI- XhoI fragment and introducedinto the NcoI– SalI sites of the expression vectorpTrc99A, allowing in frame ligation. Protein expressionfrom the trc promoter of each construct was induced byadding 1 mM IPTG to the growth media.

To produce (His)6-GS2 and (His)6-14-3-3 proteins,the cDNAs were subcloned into the expression vectorpET 28a (Novagen). The cDNA inserts of pTrc-GS2and pTrc-14-3-3 were removed from the above describedconstructs as NcoI- PstI fragments and introduced asblunt fragments into the NdeI (blunt) site of pET28a,allowing the production of GS2 and 14-3-3 proteinscontaining an N-terminal extension of 6 histidines. Theconstructs were confirmed by restriction analysis andDNA sequencing. The plasmids pET-GS2 and pET-14-3-3 were independently transformed into the bacterialstrain BL21 (DE3) (Novagen) and protein expressionwas induced by adding 1 mM IPTG to the growthmedia.

Site-directed mutagenesis

The QuickChange Site-Directed Mutagenesis Kit(Stratagene) was used to mutate Ser97 to Ala and Asp inM. truncatula GS2 protein. Briefly, mutations weregenerated by PCR amplification using either the plasmidpET-GS2 or pTrc-GS2 as template and the appropriateprimers containing the desired mutation. The sense se-quences of the primers used were: S97A – 5¢-GTG CGCAGC AAA TCT AGA ACC ATA GCA AAG CCTGTT-3¢ and S97D – 5¢- G CGC AGC AAA TCT AGA

ACC ATA GAT AAG CC �3¢, where the underlinebases represent the nucleotide changes to mutate Ser97(codon TCA) to Ala (codon GCA) or Asp (codon GAT)and to create a XbaI restriction site (bold) to facilitatethe initial screening for mutations. The PCR amplifiedproducts were transformed into E. coli XL1-Blue su-percompetent cells. Plasmid DNA was extracted andsequenced to verify production of the desired mutations.

Induction and growth of recombinant proteins

To produce his-tag GS2 and 14-3-3 proteins, E. coliBL21 (DE3) harbouring plasmids pET28a-GS2 orpET28a-14-3-3 were grown at 37�C in LB mediumsupplemented with kanamycin (100 lg/nm) until an ODof 0.5 at 600 nm. The incubation was prolonged for 3 to5 h in the same media supplemented with 1 mM IPTGto induce the expression of the recombinant proteins. Toproduce non-tag GS2 and 14-3-3 proteins, E. coliXL1-blue were transformed with plasmids pTrc-GS2 or

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pTrc-14-3-3 and grown as described above on LBmedium supplemented with ampicillin (100 lg/nm).

Preparation of soluble protein extracts from E. coliand plant tissues

Plant material was homogenized at 0 to 4�C in a mortaland pestle with two volumes of an extraction buffercontaining 10 mM Tris-HCl pH 7.5, 5 mM sodiumglutamate, 10 mM MgSO4, 1 mM DTT, 10% (v/v)glycerol, 0.05% (v/v) Triton X-100 and protease inhib-itor cocktail specific for plant extracts which contains: 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF),bestatin, pepstatinA, leupeptin, E-64, and 1,10-phenan-throline (Sigma Aldrich). E. coli cells were collected bycentrifugation (13 000 g, for 15 min), pellets were fro-zen in liquid nitrogen and ground with alumina type V(Sigma Aldrich) in a mortal and pestle with two volumesof the same extraction buffer. The homogenates werecentrifuged at 13 000 g for 20 min, at 4�C and the su-pernatants desalted on P10 Sephadex columns (Amer-sham Biosciences). Soluble protein concentration wasmeasured using the Bio-Rad dye (BioRad) reagent andBSA as a standard. Plant extracts used as kinase sourcewere homogenized using the same extraction buffer,centrifuged for 20 min at 13 000 g and further clarifiedby ultracentrifugation at 100 000 g for 1 h at 4�C. Thedesalted samples containing 20% glycerol were stored at�80�C until use.

Determination of GS activity

GS activity was determined using the transferase assay(Cullimore and Sims 1980). One unit of transferaseactivity is equivalent to 1 lmol min�1 c-glutamylhydroxamate produced at 30�C.

Gel electrophoresis

Proteins were analysed in 12% SDS- polyacryamidegel electrophoresis (SDS-PAGE). For two-dimensionalgel electrophoresis, protein samples (200 lg) wereapplied overnight to 13 cm IPG strips pH 4-7 (Amer-sham Biosciences) by in-gel rehydration. The rehy-drated gels were subjected to isoelectric focusing in aMultiphor II unit (Amersham Biosciences) accordingto the manufacturer’s instructions. After the firstdimension, the strips were incubated for 15 min in anequilibration buffer consisting of 50 mM Tris-HClpH 7.5, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDSand DTT (3.5 mg/ml) and for 15 min in the samebuffer containing iodoacetamide (45 mg/ml) instead ofDTT supplemented with bromophenol blue. Seconddimensional SDS-PAGE was performed in 12%acrylamide gels.

Immunoblotting

Proteins separated by SDS-PAGE were electroblottedonto nitrocellulose membrane (Schleicher & Schuell)using a semi-dry transfer system (Bio-Rad). The mem-branes were incubated with primary antibodies: poly-clonal anti-GS antibody (Cullimore and Miflin 1984) oranti-14-3-3 antibody (Moorhead et al. 1999). The poly-peptides were detected with secondary peroxidase-con-jugated IgGs (Vector Laboratories).

Column affinity purification and in vitrophosphorylation of his-tag GS2

His-tag GS2 proteins were extracted from E. coli, boundto nickel-nitrilotriacetic acid (Ni-NTA) his-bind resin(Novagen) according to the manufacturer’s protocols.In vitro phosphorylation assays were performed byincorporation of [c�32P] ATP (11.1x1013 Bq/mmol) in areaction mixture consisting of 10 mM Tris/HCl pH 7.5,5 mM MgCl2, 0.1 mM CaCl2, 0.5 lM microcystin-LR,18.5 Bq of [c�32] ATP, 20 lM ATP and 40 lg totalsoluble protein of extracts of leaves, nodules or rootsused as kinase source. The reaction was incubated for30 min at 30�C with gentle shaking. After brief centri-fugation, the bound proteins were sequentially washedwith buffer A (500 mM NaCl, 20 mM imidazole,20 mM Tris-HCl, pH 7.9) and buffer B (500 mM NaCl,60 mM imidazole, 20 mM Tris-HCl, pH 7.9), and elutedwith buffer C composed of 500 mM NaCl, 1 M imid-azole and 20 mM Tris-HCl pH 7.9. The phosphoryla-tion products were resolved by SDS-PAGE and the gelstained with coomassie brilliant blue R-250 thoroughlydestained and dried. The dried gels were analysed byphosphorimaging.

Evaluation of GS2-14-3-3 protein interaction

As a first approach to evaluate an interaction between14-3-3 proteins and plant GS, (His)6-14-3-3 proteinsfrom E. coli were bound to Ni-NTA his-bind resincolumns and incubated with leaf extracts for one h at30�C, with gentle shaking. The columns were sequen-tially washed with 4 ml buffer A and with 2.4 ml ofbuffer B, and the bound proteins eluted with 2.4 ml ofelute buffer C, as previously described. Control col-umns without his-tag 14-3-3 isoform were also incu-bated with leaf extracts to ensure the specificity of theinteraction. In a second experiment, His-tag GS2 wasbound to Ni-NTA resin and in vitro phosphorylated byincubation with a leaf extract used as kinase source for30 min at 30�C. After washing with buffer A, the col-umns were incubated with the E. coli produced 14-3-3proteins (without the his-tag) for 30 min at 4�C.Finally, the columns were washed and the proteinseluted as described.

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Results

2-DE analysis of GS polypeptides in leavesof M. truncatula

Initial evidence that GS is subjected to post-translationalmodifications was obtained by GS western blot analysisof two-dimensional gels (Fig. 1). There are only threeexpressed GS genes in Medicago truncatula, encodingone plastid located GS polypeptide of 42 kDa (GS2) andtwo cytosolic peptides (GS1a and GS1b) which have thesame molecular mass of 39 kDa but different pI: 5.54and 5.36 respectively (Carvalho and Cullimore 2003).However, following 2-DE a GS-specific antibody de-tected four isoelectric variants of 42 kDa and four of39 kDa indicating that both types of GS isoenzymesmay be subjected to post-translational modifications inleaves of M. truncatula, leading to polypeptides differingin charge.

In vitro phosphorylation of GS2

In order to evaluate the phosphorylation susceptibilityof GS2 by kinases present in different organs of theplant, we produced non-phosphorylated enzyme byexpressing the plant MtGS2 cDNA in E. coli, and per-formed in vitro phosphorylation assays using plantextracts as a source of kinase. MtGS2 cDNA was ex-pressed in E. coli with an N-terminal 6x his-tag, allowingthe purification by nickel-nitrilotriacetic acid (Ni-NTA)affinity. The recombinant His-tag GS2 enzyme wasfound to be catalytically active.

Initial studies were performed to evaluate the optimalATP concentration and incubation time required formaximal phosphorylation. In vitro phosphorylationassays were performed in the presence of CaCl2, MgCl2,

phosphatase inhibitors, leaf extracts as kinase sourceand increasing concentrations of ATP (cold ATP con-taining equal proportions of [c�32P] ATP). Maximalincorporation of 32P into GS2 protein was obtainedusing 20 lM ATP over 30 min of incubation with theleaf extract as source of kinase. Increasing ATP con-centration did not result in increased labelling (data notshown). Using these optimised conditions, in vitrophosphorylation assays were then performed using dif-ferent plant organs (leaves, nodules and roots) as asource of GS-kinase(s) (Fig. 2). Control assays wereperformed in the presence of [c�32P] ATP and absenceof plant extracts, which showed that the E. coli producedGS2 protein did not become phosphorylated in the ab-sence of plant kinases (Fig. 2, lane 1). A clearly 32P-labelled GS band was detected in all the assays per-formed with the different plant extracts demonstratingthatM. truncatulaGS2 is susceptible to phosphorylationby plant kinases present in leaves, nodules and roots.

Evaluation of the calcium dependence of GS kinase(s)

To evaluate whether the kinase(s) responsible for GS2phosphorylation is dependent on calcium, E. coli pro-duced GS2 isoenzyme was phosphorylated in vitro byincubation with desalted plant extracts in the presence of[c�32P] ATP and either CaCl2 or EGTA (Fig. 3). GS2appears to be phosphorylated by a calcium dependentprotein kinase(s) (CDPK) in leaf, nodule and root ex-tracts as phosphorylation was completely inhibited inthe presence of 0.5 mM EGTA.

Evaluation of GS2 interaction with 14-3-3 proteins

As 14-3-3 proteins are known to interact with severalphosphorylated targets, including cytosolic and plastid

Fig. 1 Two-dimensional PAGE profile of GS polypeptides inleaves. Soluble leaf proteins (200 lg) were separated by 2-D gelelectrophoresis in a pH gradient of 4 to 7 followed by SDS-PAGEon a 12% acrylamide gel. The GS polypeptides were detected withspecific anti-GS antibody by western analysis. The position of themolecular weight markers (kDa) are indicated on the left

Fig. 2 a, b In vitro phosphorylation of GS2. Phosphorylationassays were performed by incorporation of [c�32P]ATP into E. coliexpressed (His)6-GS2 in the absence (-K) or in the presence of totalsoluble extracts (40 lg) from leaves (L), nodules (N) and roots (R).The polypeptides were separated by SDS-PAGE and visualized bycoomassie staining (a). Phosphorimage of the same gel (b). Theposition of the molecular weight markers (kDa) are indicated onthe left

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GSs (Finneman and Schojoerring 2000; Pozuelo et al.2001; Riedel et al. 2001; Man and Kaiser 2001) a (His)6-tagged-14-3-3 protein affinity binding strategy was usedto examine a possible interaction between Medicagotruncatula GS2 and 14-3-3 proteins. Leaf extracts (whereGS2 is the most abundant GS isoform) were loaded on

affinity columns containing a Medicago truncatula(His)6-14-3-3 protein (Fig. 4). After incubation with theplant extracts, non-bound proteins were removed, thecolumns washed and elution of (His)6-14-3-3 interactingproteins performed. The initial leaf extracts (Fig. 4, lane1), the non-bound plant proteins (Fig. 4, lane 2) and theeluted fractions (Fig. 4, lane 3) were analysed by westernblot using anti-14-3-3 (Fig. 4a) and anti-GS (Fig. 4b)antibodies. A single GS polypeptide of around 40 kDa,was specifically eluted with the 14-3-3 proteins. No GSactivity was detected in this eluted fraction. As this GSpolypeptide is bigger than GS1, this protein is morelikely to correspond to a selective product of GS2 deg-radation induced by 14-3-3 binding. No GS polypeptideswere detected in the eluted fraction from control col-umns (resin without 14-3-3), incubated with the plantextracts, ensuring that the GS polypeptide is a result of14-3-3 interaction and not unspecifically bound to theresin (data not shown).

To confirm that GS2 is actually interacting with 14-3-3, and to evaluate if this interaction is dependent on GSphosphorylation, Ni-NTA columns were loaded withnon-phosphorylated (Fig. 5, lane 2) or in vitro phos-phorylated (Fig. 5, lane 3) (His)6-GS2 and incubatedwith the M. truncatula 14-3-3 isoform (expressed with-out the (His)6-tag). A control assay was performed withphosphorylated (His)6-GS2 suppressing the final incu-bation with 14-3-3 proteins to evaluate whether thecleaved GS peptide is dependent on 14-3-3 interaction(Fig. 5, lane 1). The eluted fractions (Fig. 5, lanes 1, 2

Fig. 4 a, b Analysis of GS polypeptides purified by 14-3-3 proteinaffinity chromatography. AM. truncatula (His)6-14-3-3 protein wasproduced in E. coli, bound to Ni-NTA columns and incubated withleaf extracts. The initial plant extracts (1), the non-bound plantproteins (2) and the eluted fractions (3) were analysed by westernblot using anti-14-3-3 (a) and anti-GS (b) antibodies

Fig. 5 a, b Evaluation of GS2-14-3-3 interaction by GS2 affinitychromatography. Ni-NTA resin columns were loaded with E. coliproduced (His)6-GS2, either non-phosphorylated (2) or in vitrophosphorylated, using a leaf extract (3). The bound proteins weresubsequently incubated with the M. truncatula 14-3-3 protein(expressed without the His-tag). A control assay was performedusing phosphorylated GS2 but suppressing the final incubationwith 14-3-3 proteins (1). The eluted fractions (1, 2, 3) were analysedby western blot with anti-14-3-3 (a) and anti-GS antibodies (b)

Fig. 3 Evaluation of the calcium dependence of GS2 kinase(s).Recombinant (His)6-GS2 was in vitro phosphorylated by incuba-tion with [c�32P]ATP and 40 lg of desalted extracts from leaves,nodules or roots in the presence of 0.1 mM CaCl2 or 0.5 mMEGTA. The polypeptides were separated by SDS-PAGE, visualizedby coomassie staining and phosphorimaging

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and 3) were analysed by western blot with anti-14-3-3(Fig. 5a) and anti-GS antibodies (Fig. 5b). 14-3-3 is onlydetected when incubated with GS in a phosphorylatedform (Fig. 5a, lane 3) and a low molecular weight GS2polypeptide is detected as a result of this interaction,being absent when 14-3-3 is not present (Fig. 5b, lane 1)or when GS2 is not phosphorylated (Fig. 5b, lane 2).These results indicate that M. truncatula GS2 is able tobind 14-3-3 only when it is phosphorylated and thisinteraction appears to induce a selective proteolysis ofthe plastidial GS enzyme.

The eluted fractions of (His)6-GS2 columns wereanalysed for GS activity using the transferase assay. Asignificant loss of enzyme activity (90% reduction inrelation to the control) was observed upon incubation ofphosphorylated (His)6-GS2 with 14-3-3, further sug-gesting that the cleaved GS2 polypeptide, which is de-tected after 14-3-3 incubation, is totally inactive; theremaining GS activity is probably due to non-cleavedGS2 still present in the eluted fraction (Fig. 5b, lane 3).Similarly, in vitro phosphorylation of non-his-taggedGS2, following incubation with 14-3-3 proteins (Fig. 6)provoked a loss of GS2 activity that could be correlatedwith the appearance of the proteolytic product, con-firming that the reduction in GS2 activity was in fact dueto proteolysis rather than inactivation of the enzyme byphosphorylation and/or 14-3-3 binding. The loss in GSactivity as well as the appearance of the proteolyticproduct were more pronounced when the phosphataseswere inhibited by the presence of microcystin.

Identification of Ser97 as a regulatory phosphorylationsite in GS2

Amino acid sequence analysis of Medicago truncatulaGS2 reveals a potential 14-3-3 binding site that closelyresembles the 14-3-3-binding motif described by Muslinet al. 1996 (RSXS*XP, where S* corresponds to thephosphorylated serine). The phosphorylated serine inthe 14-3-3 binding sequence corresponds to Ser97 in M.truncatulaGS2 protein (Table 1). To investigate whetherthis residue is a phosphorylation site and to evaluate theimportance of this residue for GS2-14-3-3 interaction,we have mutated serine 97 to alanine (S97A) by directed

mutagenesis. The incorporation of 32P-from c�32P-ATPby leaf kinases was around 2–fold lower in the mutantGS2-S97A compared to non-mutated protein indicatingthat Ser97 is a phosphorylation site in GS2 (Fig. 7).Other phosphorylation site(s) are present as the mutatedGS2-S97A protein could still be phosphorylated byplant kinases present in the leaf extract used as kinasesource.

To evaluate the importance of Ser97 for the interac-tion between GS2 and 14-3-3s, (His)6-GS2-S97A wasbound to Ni-NTA resin, in vitro phosphorylated andincubated with the M. truncatula 14-3-3 isoform

Fig. 6 a, b Evaluation of the effects of phosphorylation and 14-3-3binding on GS activity. E. coli produced GS2 (without the his-tag)(GS2) was phosphorylated by incubation with a leaf extract (GS2P)and with 14-3-3 proteins in the absence (GS2P-14-3-3) or presenceof microcystin (GS2P-14-3-3 + Mc). At various incubation timesaliquots were taken to assay GS activity (a) and for GS westernblot analysis (b)

Table 1 Alignment of putative14-3-3-binding motif of severalplant chloroplastic glutaminesynthetase protein sequences.

Source Deduced Sequence Accession No.

Medicago truncatula RSKSRTIS97KPVEHPS AAO37651Medicago sativa RSKSRTIS97KPVEHPS Q9XQ94Oriza sativa RSKSRTIS97KPVEDPS P14655Phaseolus vulgaris RSKSRTIS98KPVEHPS P15102Pisum sativum RSKSRTIS99KPVSHPS P08281Zea mays RSKSRTIS92KPVEDPS P25462Lotus japonicus RSKSRTIS99KPVSHPS AAL67439Nicotiana tabacum RSKSRTIS101KPVKHAS S22527Hordeum vulgare RSKSRTIS103KPVEDPS P13564

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(expressed without the (His)6-tag) (Fig. 8). The non-mutated (His)6-GS2 was analyzed in parallel. 14-3-3western blot analysis (Fig. 8b) revealed that 14-3-3proteins were unable to bind to the mutated protein(Fig. 8b, lane 3), being only detected after incubationwith non-mutated GS2 (Fig. 8b, lane 2) and becausethere was no interaction between 14-3-3 and the mutatedGS2, no GS2 cleavage product was detected (Fig. 8a,lane 4). These results clearly show the crucial role ofSer97 for 14-3-3 binding and subsequent regulation byselective proteolysis.

Leaf extracts contain protease(s) activitytoward GS2-14-3-3 complex

The results presented above raised a question regardingthe origin of the relevant protease, whether it is a plantor bacterial protease. To address this question, the GS2regulatory residue Ser97 was substituted by Asp (GS2-S97D) by directed mutagenesis, expecting that theintroduction of a negative charge at position 97, ratherthan the presence of phosphate per se, would mimicphosphorylation of the enzyme. Using this strategy wewere able to evaluate the interaction between GS2 and14-3-3 avoiding the incubation with a plant extract,previously used as a kinase(s) and potentially a prote-ase(s) source.

The GS2-S97D mutant enzyme was loaded into anaffinity column containing the Medicago truncatula(His)6-14-3-3 protein (Fig. 9). Western blot analysisusing anti-14-3-3 antibodies (Fig. 9 b) revealed that theAsp substitution does mimics Ser97 phosphorylation asGS2-S97D was able to bind 14-3-3 (Fig. 9b, lane 2)however, no GS2 cleavage product was detected(Fig. 9a, lane 2). In contrast, when GS2-S97D wasincubated with a leaf extract, the GS2 proteolytic frag-ment was detected (Fig. 9a, lane 3), clearly demon-strating that the protease responsible for GS2proteolysis is of plant origin and present in leaf extracts.The activity of this protease appears to be unaffected byconventional protease inhibitors, since all the experi-ments were performed in the presence of a proteaseinhibitor cocktail with broad specificity for the inhibi-tion of serine, cysteine and aspartic proteases, metallo-proteases and aminopeptidases.

Fig. 7 a, b In vitro phosphorylation of GS2-S97A mutant enzyme.E. coli expressed (His)6-GS2 and (His)6-GS2-S97A were bound toNi-NTA resin and in vitro phosphorylated. The eluted polypep-tides were separated by SDS-PAGE and visualized by coomassiestaining (a). Phosphorimage of the same gel (b)

Fig. 8 a, b Evaluation of GS2-S97A-14-3-3 interaction by affinitychromatography. (His)6-GS2 (a, lanes 1 and 3; b, lane 2) and(His)6- GS2-S97A (a, lanes 2 and 4; b, lane 3) were bound to Ni-NTA resin and in vitro phosphorylated, using a leaf extract. Thebound proteins were subsequently incubated with theM. truncatula14-3-3 protein (expressed without the His-tag). The initial GS and14-3-3 extracts (a, lane1 and 2; b, lane 1) and the eluted fractions (a,lane3 and 4; b, lane 2 and 3) were analysed by western blot withanti-GS (a) and anti-14-3-3 antibodies (b)

Fig. 9 a, b Cleavage of GS2-S97D by a protease(s) present in leafextracts. (His)6-GS2-S97D protein was directly loaded into a(His)6-14-3-3 Ni-NTA columns (a, lane 2; b, lane 2) or incubatedwith a leaf extract before loading into the (His)6-14-3-3 column (a,lane 3; b, lane 3). Lanes 1 – Initial GS and 14-3-3 extracts. Lanes 2and 3 – Eluted fractions

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Comparison of the GS activity in the two elutedfractions represented in figure 9 (lanes 2 and 3) also re-vealed that the plant protease activity is necessary for theinactivation of the enzyme. A 60% reduction is GS ac-tivity was observed when the complex GS2S97D-14-3-3was incubated with the protease contained in the leafextract (Fig. 9, lane 3), suggesting that the inactivation isdue to GS2 cleavage rather than the formation of theGS2S97D-14-3-3 complex per se.

Discussion

In this study we provide evidence that the M. truncatulaGS2 is regulated by phosphorylation of residue Ser97and subsequent binding to 14-3-3 proteins, which in-duces a selective proteolysis of the GS2 protein, resultingin an inactive cleavage product. These findings suggestthat GS2 activity is regulated post-transcriptionally by amechanism analogous to that reported for another keyenzyme in nitrogen metabolism: nitrate reductase. It iswell established that NR activity is highly regulated byphosphorylation and 14-3-3 interaction. 14-3-3 bindingto NR not only inhibits enzyme activity but also influ-ences it’s susceptibly to degradation (Mackintosh andMeek 2001; Weiner and Kaiser 1999; Huber et al. 1996).Like nitrate reductase, GS occupies a key, possibly ratelimiting, position in nitrogen metabolism and it is con-ceivable that the two enzymes are regulated by similarmechanisms.

In barley leaves, a similar behaviour between NR andthe plastid GS has been reported. When young leaves ofbarley were fed with AICAR, a component that mimicsthe effects of 5¢-AMP in vivo, an increase in GS2 activityand protein stability was observed and the authorssuggest that GS2 and NR are regulated by phosphory-lation and 14-3-3 interaction and that this interactioninitiates protein degradation (Man and Kaiser 2001).Reversible phosphorylation and 14-3-3 binding havealso been implied in the regulation of cytosolic GSduring light/dark transitions in leaves of Brassica napusand a tentative model has been proposed (Finnemannand Schjoerring 2000). According to this model, in thedark ATP/AMP levels are high and GS1 is phosphory-lated and binds 14-3-3 proteins, which confers protec-tion against degradation. Conversely, in the light GS1would be unphosphorylated and more susceptible todegradation (Finnemann and Schjoerring 2000).

Among the three M. truncatula GS proteins only GS2contains a sequence -RTIS*KP, where S* correspondsto Ser97- very similar to a described 14-3-3-bindingmotif (RSXS*XP) (Muslin et al. 1996). This sequence isabsolutely conserved in the GS2 proteins of severaldicotyledonous and monocotyledonous plant species(Table 1). Mutation of the serine residue to alaninewithin the 14-3-3-binding motif in MtGS2 resulted in adecreased phospholabeling and abolished the interactionbetween GS2 and 14-3-3s. Substitution of serine 97 byaspartic acid was sufficient to mimic phosphorylation

and restore 14-3-3 binding, but the subsequent proteol-ysis of the protein could only be detected after incuba-tion with a plant extract, clearly indicating thatphosphorylation of Ser97 is a prerequisite for 14-3-3binding and that a plant protease is required for theselective proteolysis of the protein. In addition to Ser97,the protein contains other phosphorylated residue(s),which is consistent with the detection of more than twoGS2 isoelectric variants on 2D-gels. Also in tobaccoseveral GS2 polypeptides were detected on 2D-gels andsome of them cross-reacted with anti-phosphoserineantibodies suggesting multiple phosphorylation sites(Riedel et al. 2001). The authors report the presence of14-3-3 proteins inside the chloroplast and their interac-tion with GS2 (Riedel et al. 2001).

In M. truncatula 14-3-3s binding to the RTIS97 KP-binding motif in the N-terminal region of GS2 induces aselective proteolysis of the protein, resulting in an inac-tive cleavage product. We predict that the cleavage site islocated at the C-terminus of GS2 as the cleaved productremained bound to the his-binding column and the his-tag was introduced at the N-terminal end of the protein.It is noteworthy that cleavage of GS2 requires a proteasefrom plant origin, resulting in a limited proteolysis at aspecific site. This protease appears to be resistant to sixstandard protease inhibitors. Although this is an unu-sual feature for a protease, it is not without precedents.For example, the well studied plant protease CTPA isalso a carboxy-peptidase and it is known to be unaf-fected by a wide variety of protease inhibitors includingserine, cysteine and aspartic protease and metallopro-tease inhibitors (Yamamoto et al. 2001).

Proteolytic processes in the chloroplasts are wellrecognized, and are likely to play a role in determiningthe levels of key regulatory enzymes (Adam 1996). Be-sides the levels of proteolytic activities, the susceptibilityof the substrate proteins is also a contributing factor forprotein turnover. Covalent modifications such as oxi-dation of amino acid residues, phosphorylation orinteractions with other proteins have been found toinfluence the susceptibility of a protein to proteolysis(Thoenen and Feller 1998; Callis 1995). Plastidial iso-forms of GS appear to be far more susceptible to pro-teolysis than the cytosolic forms (Streit and Feller 1983).Some reports describe the appearance of a GS2 selectiveproteolytic product of around 40 kDa either associatedwith leaf senescence (Frohlich et al. 1994) or upon illu-mination of isolated chloroplasts (Roulin and Feller1997; Thoenen and Feller 1998). It is therefore con-ceivable that a selective proteolysis of GS2 followingphosphorylation and 14-3-3 interaction represents ageneral mechanism for the regulation of GS2 by proteinturnover in higher plants. Future research will bedeterminant to understand the implications of this newmechanism of GS2 regulation for nitrogen assimilationand place it into a physiological context.

In conclusion, our data support a two-step post-translational mechanism for regulation of plant GS2.Under certain conditions GS2 is phosphorylated at

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Ser97 by a calcium dependent plant kinase creating a 14-3-3 binding site. The phosphorylated GS2 bound to 14-3-3 proteins is recognized by an unknown plant proteasethat cleaves the enzyme near the C-terminal end. Wepostulate that phosphorylation induces a conforma-tional change of the protein that allows 14-3-3 bindingand subsequent proteolysis. This is supported by the factthat substitution of serine at position 97 to aspartic acidappears to mimic phosphorylation of the enzyme bypromoting 14-3-3 binding, indicating that the signifi-cance of phosphorylation is the introduction of a nega-tive charge rather than the presence of phosphate per se.

Acknowledgments We gratefully acknowledge Dr. Carol Mackin-tosh (MRC unit, University of Dundee, UK) for providing anti-14-3-3 antibodies. We are also extremely grateful to Michel Rossignoland Giselle Borderie (IFR40, Toulouse, France) for expert assis-tance in 2D electrophoresis. We are also grateful to Jorge Azevedoand Pedro Pereira (IBMC, Porto, Portugal) for helpful discussions.This work was supported by the Fundacao para a Ciencia eTecnologia (Project no. POC/PI/41433/2001)

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