Novel Protein Phosphorylation Site Identification in Spinach Stroma

Dec 22, 2005 - In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We iden...
1 downloads 0 Views 222KB Size
Download PDF
Novel Protein Phosphorylation Site Identification in Spinach Stroma Membranes by Titanium Dioxide Microcolumns and Tandem Mass Spectrometry Sara Rinalducci,† Martin R. Larsen,‡ Shabaz Mohammed,‡ and Lello Zolla*,† Department of Environmental Sciences, Tuscia University, Viterbo, Italy, and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Received December 22, 2005

In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We identified seven previously unknown phosphorylation sites by taking advantage of TiO2 phosphopeptides enrichment coupled to mass spectrometric analysis. Upon illumination at 100 µmol m-2 s-1, two novel phosphopeptides belonging to the N-terminal region of Lhcb1 light-harvesting protein were detected: NVSSGSpPWYGPDR and TpVQSSSPWYGPDR. Moreover, three new threonine residues in CP43 (Thr-6, Thr-8, and Thr-346) and, for the first time, two amino acid residues of the N-terminus of Rieske Fe-S protein of the cytochrome b6f complex (Thr-2 and Ser-3) were revealed to be phosphorylated. Since Lhcb1 and CP43 have been reported as mobile proteins, it may be suggested that illumination derived phosphorylation, and consequently the addition of negatively charged groups to the protein, is a necessary condition to induce a significant protein structural change. Keywords: titanium dioxide • stroma membranes • chlorophyll-binding proteins • tandem mass spectrometry • phosphopeptides

Introduction Modification of proteins by phosphorylation plays an important role in biochemical processes in almost all classes of organisms. This reversible post-translational modification is involved in the regulation of enzyme activities and metabolic pathways, in the processes of signal transduction, the control of gene transcription and translation, and in the differentiation and proliferation of cells. In the case of the plant kingdom, a light- and redox-controlled protein phosphorylation system has evolved in plant thylakoid membranes for regulation of the photosynthetic process.1-5 A number of photosynthetic subunits are reversibly phosphorylated by thylakoid-bound kinases for which at least four different regulatory patterns have been described.6 These intrinsic protein kinases are activated by light or reducing conditions and controlled by the reduction of plastoquinone and its binding to the reduced cytochrome b6f complex.3,4,7,8 Additional modulation of protein phosphorylation in thylakoid membranes involves the thiol redox state9,10 as well as light-modulated conformational changes of substrate proteins.11-13 The protein dephosphorylation reactions are catalyzed by both integral thylakoid membrane and soluble chloroplast phosphatases.5,14 Thylakoid phosphoproteins include the main light harvesting complex of photosystem II * To whom correspondence should be addressed. Tuscia University, Largo dell′ Universita` snc, 01100 Viterbo, Italy. Tel: 0039 0761 357 100. Fax: 0039 0761 357 630. E-mail: [email protected]. † Tuscia University. ‡ University of Southern Denmark. 10.1021/pr050476n CCC: $33.50

 2006 American Chemical Society

(LHCII),4,15,16 the peripheral antenna protein CP29,17-20 and D1, D2, CP43, and PsbH core subunits.1,4,16,21-23 Recently, two additional phosphoproteins have been identified: the 9 kDa thylakoid-soluble phosphoprotein (TSP9)24 with yet unknown function and the 14 kDa thylakoid membrane phosphoprotein (TMP14)18 which recently has been demonstrated to be a novel subunit of plant photosystem I.25 Moreover, the same authors found evidence for protein phosphorylation in the PsaD subunit of photosystem I.18 In general, the role of phosphorylation for these proteins is not completely understood. Reversible phosphorylation of LHCII participates in the balancing of the absorbed light energy distribution between the two photosystems:3 as a consequence of phosphorylation, up to 80% of this antenna complex may be transferred to the stroma lamellae.26,27 Phosphorylation at threonine 83 in the minor chlorophyll a/b protein CP29 has been associated with the resistance of maize plants to cold stress,17,28 whereas the CP29 phosphorylation site mapped to threonine 6 in Arabidopsis thaliana has been found under normal plant growth conditions.18 Moreover, it has been suggested that in Chlamydomonas reinhardtii a hyper-phosphorylated form of the CP29 protein may aid the functional coupling of phosphorylated LHCII to PSI.20 In the case of core proteins, the functional meaning of phosphorylation is less clear. As an example, phosphorylation of D1 has been reported either to prevent29 or to promote30 its light-induced turnover, whereas phosphorylation of other core subunits might have a role in the PSII repair cycle and in monomer/dimer interconversion.31 Journal of Proteome Research 2006, 5, 973-982

973

Published on Web 03/23/2006

research articles In almost all of the identified thylakoid phosphoproteins the phosphorylation sites were found exposed to the outer surface of thylakoids,4 therefore such polypeptides could be “shaved” from the surface of thylakoids by a specific proteolytic enzyme, e.g., trypsin.16,18,32 Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics studies. Several analytical techniques exist for the analysis of protein phosphorylation, such as (i) 32Plabeling, (ii) Edman sequencing, and (iii) mass spectrometric methods. The latter can be used not only for sequencing of peptides but also as a selective detection method for phosphorylated peptides by using methodologies such as precursor ion and neutral loss scanning.33-35 The significant improvements in sensitivity and duty cycle over recent years in mass spectrometry has made it the de facto standard for interrogating the ‘phospho-proteome’. However, phosphopeptides are often present in small amounts and need selective isolation or enrichment before identification by mass spectrometry, especially from complex peptide mixtures. The most common strategies to enrich for phosphopeptides revolve around immobilized metal affinity chromatography (IMAC).36,37 Recently, a promising new strategy was introduced by Pinkse et al.,38 and subsequently modified by Larsen et al.,39 where titanium dioxide (TiO2) was used for enrichment of phosphorylated peptides prior to ESI liquid chromatography and/or MALDI tandem MS. In this paper we report identification of seven previously unknown phosphorylation sites in stromal proteins from Spinacia oleracea. The selectivity and practicality of using TiO2 micro-columns for selective purification of phosphopetides allowed the successful analysis of stroma membranes purified by digitonin from illuminated spinach leaves. Novel phosphorylation sites were found in the light-harvesting apparatus of photosystem II (Lhcb1 proteins), in the PSII reaction center protein CP43, and in the cytochrome b6f Rieske FeS protein. Interestingly, more than one phosphorylation site was revealed on the same protein, and in some cases at different domains. Precise identification of new phosphorylation sites could bring significant biological insights about the cellular mechanisms of signaling activation and inhibition, in particular the existence of different kinases and/or different pathways.

Experimental Section Chemicals and Materials. Magnesium chloride, sodium chloride, sucrose, sodium fluoride, Na-EDTA, hepes, digitonin, phosphoric acid, trifluoroacetic acid, as well as analytical-grade acetonitrile were obtained from Sigma-Aldrich (Milan, Italy). Modified trypsin was purchased from Promega (Madison, WI), chymotrypsin from La Roche (Basel, Switzerland). GELoader tips were from Eppendorff (Eppendorf, Hamburg, Germany). 2,5-Dihydroxybenzoic acid (DHB) was from Fluka (St. Louis, MO). The 3M Empore C8 disk was from 3M Bioanalytical Technologies (St. Paul, MN). Syringe for HPLC loading (P/N 038030, N25/500-7C PKT 2) were from SGE (Victoria, Australia). The water was from a Milli-Q system (Millipore, Bedford, MA). Titanium dioxide beads were obtained from a disassembled TiO2 cartridge (4.0 mm ID - 5020-08520-5u-TiO2) purchased from GL sciences Inc, Japan. All other chemicals and reagents were of the highest grade commercially available. Light Treatments of the Leaf Disks. Spinach (Spinacia oleracea) leaf disks (diameter 2.7 cm), punched from darkadapted, fully expanded leaves and floating on distilled water in a Petri dish, were illuminated in a growth chamber under a 974

Journal of Proteome Research • Vol. 5, No. 4, 2006

Rinalducci et al.

photon flux densities (PFDs) of 100 µmol m-2 s-1 for 1 h at 23 °C. A metal-halide lamp HQI-T 250W daylight served as a light source. For analysis of thylakoid phosphoproteins, the leaf disks were rapidly frozen in liquid nitrogen and stored at -80 °C until isolation of thylakoid membranes. Isolation of Thylakoids and Preparation of Stroma Lamellae Membranes. Thylakoid membranes were isolated according to the method of Aro et al.32 Frozen leaf disks were mixed with ice-cold isolation buffer containing 50 mM HEPES-NaOH, pH 7.5, 300 mM sucrose, 5 mM MgCl2, 1 mM Na-EDTA, 10 mM NaF, and 1% (w/v) bovine serum albumin and thereafter rapidly homogenized. The homogenates were filtered through Miracloth and centrifuged at 1500 × g for 4 min. The pellets were washed with 10 mM HEPES-NaOH, pH 7.5, 5 mM sucrose, 5 mM MgCl2, and 10 mM NaF and pelleted at 3000 × g for 3 min. Thylakoid pellets were resuspended to a concentration of 200 µg of chlorophyll/ml in 10 mM HEPES-NaOH, pH 7.5, 100 mM sucrose, 5 mM NaCl, 10 MgCl2, and 10 mM NaF. Subfractionation of thylakoid membranes into grana and stroma lamellae with digitonin method were performed as described earlier.27 Recrystallized digitonin (1% in distilled water) was added to the stirred membranes to give a final concentration of 0.4%. The 2-min detergent treatment was terminated by a 10-fold dilution of the sample with resuspension buffer at 4 °C. Differential centrifugation according to Anderson and Boardman40 yielded pellets following 1000 × g for 10 min, 10 000 × g for 30 min, 40 000 × g for 30 min, and 140 000 × g for 60 min. The preparations were protected from light and kept icecold during the isolation procedure. Chlorophyll Determinations. The chlorophyll content of the isolated membranes was determined according to the method of Porra et al.41 In-Solution Protein Digestion. Preparation of the surfaceexposed peptides from stroma lamellae was performed according to Vener et al.16 with minor modifications as follows: isolated stroma membranes were washed twice with 25 mM NH4HCO3 (pH 8.0) by centrifugation and resuspended in the same buffer to a concentration of 1.8-2.0 mg of chlorophyll/ mL. The suspension was incubated with sequencing-grade modified trypsin (Promega, Madison, WI) or chymotrypsin (La Roche, Basel, Switzerland) (8 µg of enzyme/mg of chlorophyll) at 22-23 °C for 2 h. Note that the proteolytic treatment was not performed at 37 °C because a number of PSII phosphoproteins are rapidly dephosphorylated by the heat-shockactivated membrane protein phosphatase.32 The digestion products were acidified with 5% formic acid (FA), frozen, thawed, and clarified at 14 000 × g for 10 min. The supernatant containing released peptides was collected for further analysis. Purification of Phosphorylated Peptides Using TiO2 Microcolumns. TiO2 microcolumns with a length of approximately 3 mm were packed in GELoader Tips according to Larsen et al.39 A small plug of C8 material was stamped out of a 3M Empore C8 extraction disk using a HPLC syringe needle and placed at the constricted end of the GELoader tip. The C8 disk serves only as a frit to retain the titanium dioxide beads within the GELoader tip. The TiO2 beads were suspended in 80% acetonitrile/0.1% TFA and an aliquot of this suspension (depending on the size of the column) was loaded onto the GELoader tip. Gentle air pressure created by a plastic syringe was used to pack the column as previously described.42,43 The peptide solution (5-10 µL) was loaded onto TiO2 columns in DHB solutions (200 mg/mL in 80% acetonitrile/0.1%TFA). The

New Phosphorylation Sites in Spinach Stroma Lamellae

columns were washed with 10 µL of the DHB solution and 20 µL of 80% acetonitrile/0.1% TFA. The bound peptides were eluted using 3 µL NH4OH, pH 10.5 and the eluate was analyzed directly by LC- or MALDI-MS/MS after acidification. Nano-Flow Liquid Chromatography Electrospray Tandem Mass Spectrometry Analysis (LC-ESI-MS/MS). Automated nanoflow liquid chromatography/tandem mass spectrometric analysis was performed using a QTOF Ultima mass spectrometer (Micromass UK Ltd., Manchester, UK) employing automated data dependent acquisition (DDA). A nanoflow-HPLC system (Ultimate; Switchos2; Famos; LC Packings, Amsterdam, The Netherlands) was used to deliver a flow rate of 2 µL/min (loading) and 100 nL/min (elution). Loading was accomplished by using a flow rate of 2 µL/min onto a homemade 2 cm fused silica precolumn (75 µm i.d.; 375 µm o.d.; Resprosil C18-AQ, 3 µm (Ammerbuch-Entringen, DE) using autosampler. Sequential elution of peptides was accomplished using a linear gradient from Solution A (0.6% acetic acid) to 40% of Solution B (80% acetonitrile; 0.5% acetic acid) in 40 min over the precolumn in-line with a homemade 10-15 cm resolving column (50 µm i.d.; 375 µm o.d.; Resprosil C18-AQ, 3 µm (AmmerbuchEntringen, Germany). The resolving column was connected using a fused silica transfer line (20 µm I. D.) to a distally coated fused silica emitter (New Objective, Cambridge, MA) (360 µm OD/20 µm ID/10 µm tip ID) biased to 1.8 kV. The mass spectrometer was operated in the positive ion mode and data dependent analysis was employed (three most abundant ions in each cycle): 1 s MS (m/z 350-1500) and 3 × 2 s MSMS (m/z 50-2000, continuum mode), 30 s dynamic exclusion. External mass calibration using NaI resulted in mass errors of less than 50 ppm, typically 5-15 ppm in the m/z range 50-2000. Raw data were processed using Protein Lynx Global Server Protein Lynx (smooth 3/2 Savitzky Golay and center 4 channels/80% centroid) and the resulting MS/MS data set exported in the Micromass pkl format. Automated peptide identification from raw data was performed using an in-house MASCOT server (v. 2.0) (Matrix Sciences, London, UK) using the NCBI nonredundant protein database using the following constraints: only tryptic peptides up to two missed cleavage sites were allowed; 20 ppm tolerance for MS and (0.2 Da for MS/MS fragment ions; carbamidomethyl cysteine (C) was specified as a fixed modification, deamidation (NQ) phosphorylation (S and T and Y) and methionine oxidation (M) were specified as variable modifications. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). MALDI-MS was performed using a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK). All spectra were obtained in positive reflector mode. Mass spectrometric data analysis was performed using the software MassLynx 3.5. Sequence analysis and peptide assignment were accomplished using the GPMAW software (http:// welcome.to/gpmaw). For analysis of phosphorylated peptides, DHB (20 mg/mL) in 50% acetonitrile, 1% phosphoric acid was used as the matrix. Note that inclusion of 1% phosphoric acid in the MALDI matrix solution increases the relative abundance of multiphosphorylated peptides.44

Results Within each chloroplast, the thylakoids form a continuous network which contains two distinct types of membrane domains, the stacked grana thylakoids and the unstacked stroma thylakoids. Photosystem I (PSI) is predominantly local-

research articles ized in stroma lamellae and peripheral membranes of the grana, while photosystem II (PSII) is mainly located in grana appressions. It is well documented that under illumination, the light harvesting proteins of PSII undergo phosphorylation and migrate to PSI.3,45 Reversible phosphorylation also controls the photoinhibition-repair cycle of the PSII reaction center subunits, in particular migration of the damaged PSII to stromaexposed thylakoids can be regarded as the first step in the PSII repair cycle.46 With the aim to study the role of photosynthetic protein phosphorylation either regarding the mechanisms of light energy distribution balancing between the two photosystems or the turnover processes, thylakoids from illuminated spinach leaves were fractionated by digitonin and stroma membranes were investigated to identify phosphorylation sites by mass spectrometry. Illumination of spinach leaves was performed at 100 µmol m-2 s-1 for 1 h, since it was previously demonstrated that under these experimental conditions maximum phosphorylation occurs.47 Knowing that protein phosphorylation in thylakoids is restricted to the outer surfaceexposed regions of the membrane proteins allowed the “exposed” peptides to be released by the stroma samples being introduced to trypsin.16,18,32 The characterization of the thylakoid phosphopeptides by mass spectrometry has previously been performed by using IMAC enrichment with immobilized Fe(III) and Ga(III) cations.16,18 Recently, a new and selective enrichment strategy was developed based on titanium dioxide (TiO2)38,39 and further improved with respect to reduced nonspecific binding by peptide loading in 2,5-dihydroxybenzoic acid (DHB).39 However, despite the improved enrichment procedure, few nonphosphorylated peptides can still be present in the fractions, such as the highly acidic peptide ion corresponding to the sequence GGGSDKKDDDVNAFTPDT (NCBI accession no. gi|129073) belonging to the 6.7 kDa outer envelope membrane protein (data not shown). In the present work, we enriched for phosphorylated peptides from stroma membranes by using this method and analyzed by both MALDI and nano LC-ESI Q-TOF tandem MS. For phosphopeptide detection we utilized the preferred loss of the phosphate group upon collision-induced dissociation (CID). In positive ion tandem MS, an intense neutral loss of 98 Da corresponding to H3PO4 is observed for serine and threonine phosphorylated peptides.34 Preliminary experiments of the phosphorylation status of these photosynthetic proteins showed the presence of CP43 (1+, 1385.65 m/z), D1 (1+, 980.47 m/z), D2 (1+, 710.32 m/z), and Lhcb1.3 (1+, 1501.59 m/z) phosphorylated forms in darkadapted samples (data not shown). Interestingly, a higher number of phosphopetides were detected after leaf illumination. Table 1 lists the light-activated phosphopeptides identified in the stroma samples after TiO2 enrichment and MS/MS analyses. However, besides the previously known phosphopeptides belonging to photosynthetic proteins,15,16,18,21,22,24 a number of new phosphopeptides were detected, which will be discussed separately below. New Phosphorylation Sites in Light Harvesting Protein Lhcb1. MALDI Q-TOF sequencing of the tryptic mono-charged peptide at 1501.59 m/z identified it as NVSSGSpPWYGPDR, where Sp designates the phosphorylated amino acid (Figure 1A). Mascot sequence similarity searching found that this sequence belongs to the light harvesting chlorophyll a/b binding protein Lhcb1 from Prunus persica (NCBI accession number gi|556367). To verify this result, we analyzed the mixture of peptides with nano LC-ESI-Q-TOF MS/MS. The electrospray tandem MS Journal of Proteome Research • Vol. 5, No. 4, 2006 975

research articles

Rinalducci et al.

Table 1. Phosphorylation Sites Identified by Tandem MS after Titanium Dioxide Enrichmenta identified phosphopeptides

parent protein

sequence

m/z

charge state

Ac-TpAILER Ac-TpAILERR Ac-TpIAVGK Ac-TpLFNGTLTLAGR ATQTpVESSSR ATpQTpVESSSR Ac-RRTpVK Ac-RKTpAGKPKTVQSSSPWc KGTpVSIPSK NVSSGSpPWYGPDR

824.37 980.47 710.32 1385.65 1145.44 1225.41 781.40 1879.85 995.47 1501.59 751.29 780.30 788.81 887.38 880.93 791.81 580.24

1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 2+ 1+ 2+ 2+ 2+ 1+ 2+ 2+ 3+

TpVQSSSPWYGPDR SPTpGEVIFGGETMoxR Ac-TpLFNGtpLc NGtpLTpLAGRDQETTGFc ATpspIPADNVPDMoxQK ATpspIPADNVPDMoxQKR

D1 D1 D2 CP43 PsbH PsbHb Lhcb2 Lhcb1.2 TSP9 Lhcb1.3 Lhcb1.2 CP43 CP43 CP43 Rieske Fe-S Rieske Fe-S

a Ac designates acetylated N-termini; Tp indicates the main phosphorylated Thr; tpindicates the secondary phosphorylated Thr; Sp indicates the phosphorylated Ser; spindicates the secondary phosphorylated Ser; Mox indicates the oxided Met. b Doubly phosphorylated N-terminal peptide of PsbH protein. c Chymotryptic peptide.

spectrum for the same peptide belonging to Lhcb1 (2+, 751.29 m/z) is shown in Figure 1B. Much more pronounced b and y ion series were obtained, e.g. the presence of the ion at m/z 301.22 evidently rules out the serine 4 as potential phosphorylation site, as well as the signal corresponding of the doubly charged y8 fragment ion (y82+ ) 480.22 m/z) confirming the assignment of the phosphorylation to serine 6. Automatic sequencing by LC-ESI-MS/MS also identified the sequence of the phosphopeptide TpVQSSSPWYGPDR (doubly charged molecular ion with m/z 780.30), whose CID spectrum is displayed in Figure 2. The peptide tandem mass spectra contained a highly abundant neutral loss peak (corresponding to the signal at m/z 731.35) indicative of phosphorylation. The presence of N-terminal b2-b6 ions with neutral loss of 98 Da (in particular b2 ) 183.10 m/z; b3 ) 311.17 m/z) localizes the phosphorylation site to the first threonine. It is known that the Lhcb1 protein exists in spinach in three distinct isoforms that differ in their amino terminus48 called Lhcb1.1 (Ac-RK SAGKPK NVSSGSPWYGPDR), Lhcb1.2 (AcRK TAGKPK TVQSSSPWYGPDR) and Lhcb1.3 (Ac-RK TAGKPK NVSSGSPWYGPDR). In earlier experiments, the light-induced phosphorylation of light-harvesting proteins was localized to the threonine or serine 3.15 Interestingly, the newly identified phosphorylated peptide TpVQSSSPWYGPDR belongs to Lhcb1.2 isomer, whereas the sequence NVSSGSpPWYGPDR can be ascribed either to Lhcb1.1 or to Lhcb1.3 isoforms. However, previous investigations performed by both intact mass measurements and immunoblotting rule out a phosphorylated form of the Lhcb1.1 protein.27 New Phosphorylations Sites in CP43 Protein. The most characterized thylakoid phosphoproteins have been shown to be phosphorylated at or close to the N-terminus49 generally exposed to the stromal side. Interestingly, our experimental data revealed for the first time a novel phosphorylation site in a lumen-exposed loop of CP43 protein close to its C-terminal. Figure 3 shows the ESI tandem mass spectrum of the doubly charged ion at m/z 788.81 matching to the sequence SPTpGEVIFGGETMoxR in the CP43 protein. This peptide is located 976

Journal of Proteome Research • Vol. 5, No. 4, 2006

in position 344-357 of the CP43 amino acid sequence (NCBI accession no. gi|7443192, Spinacia oleracea), and we found the presence of both a phosphorylation and an oxidation. In particular, neutral loss of H3PO4 from the doubly charged parent ion produced the signal at m/z of 739.84 indicating phosphorylation. The peptide contains two potential phosphorylation sites (serine 1 or threonine 3), but the presence of the b2 ion with m/z 185.08 clearly rules out serine 1 whereas the b3 (m/z 268.13) fragment ion perfectly corresponds to a phosphorylated threonine residue after the neutral loss of 98 Da. Concerning the well-known N-terminal phosphorylation of CP43 protein, we isolated and sequenced the tryptic peptide at 1385.65 m/z (Ac-TpLFNGTLTLAGR). A careful analysis of the MS/MS spectrum obtained suggested there was a mixture of isomeric peptides. In addition to the presence of the well documented phosphorylation on threonine 1 there was the possibility of phosphorylations on threonines 6 and 8 indicated by some low abundant b and y ions (data not shown). To confirm this, we performed an in-solution chymotryptic digestion and two different peptides were detected: Ac-TpLFNGtpL (887.38 m/z); NGtpLTpLAGRDQETTGF (880.932+ m/z) where Tp indicates the main phosphorylation site, whereas the lowercase residue corresponds to a secondary phosphorylation site. The registered m/z values of 887.38 and 880.93 fit with the addition of only one phospho group, but in the ion MS/MS spectra we found the presence of b and y ion fragments at significant levels produced from CID of several isomeric phosphorylated peptides. Figure 4A shows the fragment ion spectrum of the chymotryptic mono-charged peptide at 887.38 m/z obtained on a MALDI Q-TOF. The elimination of phosphoric acid (98 Da) from the precursor ion gives rise to the intense signal at 789.43 m/z. The N-terminal acetylation was easily assigned based on the detection of a complete b series with addition of 42 Da. Concerning the phosphorylation, we can surmise that threonine 1 is the main phosphorylation site (clear signals produced after the 98 Da neutral loss at b2(239.15), b3(386.22), b4(500.26), b5(557.27), and b6(658.34) m/z) but several peaks suggest a second minor phosphorylation site on threonine 6 that could represent up to 20% of the total CP43 phosphorylated pool (based on ion signals). First, the 215.14 m/z ion corresponds to the y2 fragment after the neutral loss from the threonine 6. Second, the prominent signal at 641.32 m/z could be assigned to an internal β-elimination reaction from the same residue. A confirmation of this behavior was obtained by analyzing the fragmentation pattern of the second phosphorylated peptide with the calculated molecular mass of 1759.86 Da, the 16-amino acid-long sequence shown in Figure 4B. This amino acid stretch was identified as the chymotryptic peptide aa 8-23 of the CP43 protein and its sequence partially overlaps the one of the peptides examined above. The fragment ion spectrum shown in Figure 4B corresponds to the doubly protonated molecular ion of this peptide at 880.93 m/z, and it is also present in the beta-eliminated form at 831.95 m/z. In this case, the phosphorylation site is localizated to threonine 5, however the pronounced peaks at 255.13 and 368.22 m/z designate the threonine 3 as a secondary phosphorylated amino acid residue. New Phosphorylations Sites in Rieske FeS Protein. Our MS/ MS analyses identified the sequence of the previously unknown phosphopeptide ATpspIPADNVPDMoxQK belonging to Rieske FeS protein from the cytochrome b6/f-complex of spinach thylakoids. Both doubly (791.81 m/z) and triply (plus a C-

New Phosphorylation Sites in Spinach Stroma Lamellae

research articles

Figure 1. Identification of the NVSSGSpPWYGPDR phosphopeptide in the Lhcb1 protein. Panel A, MALDI-Q-TOF tandem MS spectrum of the phosphopeptide with parent molecular ion at 1501.60 m/z. The indicated differences of 80 and 98 Da correspond to the neutral losses of HPO3 and H3PO4, respectively. Panel B, The product ion spectrum obtained after nanoelectrospray ionization and CID of the same peptide. The selected doubly charged molecular ion with m/z 751.29 is indicated along with the ion that underwent the neutral loss of phosphoric acid (m/z 702.30). The detected b (N-terminal) and y (C-terminal) fragment ions are labeled in the spectra. Fragment ions corresponding to the loss of ammonia (17 Da) and water (18 Da) are marked with asterisks and superscript 0, respectively.

Figure 2. Identification of the TpVQSSSPWYGPDR phosphopeptide in the Lhcb1 protein. ESI-Q-TOF tandem MS spectrum of the doubly charged parent molecular ion with 780.30 m/z. The detected b (N-terminal) and y (C-terminal) fragment ions are labeled in the spectrum. Fragment ions corresponding to the loss of water (18 Da) are marked with the superscript 0. The indicated difference of 49 correspond with the neutral loss of phosphoric acid from the doubly charged parent ion at m/z 780.30.

terminal arginine, 580.24 m/z) charged forms of this peptide were subjected to CID and the results are displayed in Figure 5A and B, respectively. The series of N-terminal fragments with

the neutral loss pattern localized the phosphorylation site to threonine 2 or serine 3. The presence of 242.11 and 224.10 m/z signals strongly suggested the serine as phosphorylation site Journal of Proteome Research • Vol. 5, No. 4, 2006 977

research articles

Rinalducci et al.

Figure 3. Identification of SPTpGEVIFGGETMoxR phosphopeptide in the CP43 protein. ESI-Q-TOF tandem MS spectrum of the doubly charged parent molecular ion with m/z ) 788.81 (shown by an arrow). The indicated differences of 49 and 32 Da correspond to the neutral losses of H3PO4 and CH3SOH, respectively. The detected b (N-terminal) and y (C-terminal) fragment ions are labeled in the spectrum. Fragment ions corresponding to the loss of ammonia (17 Da) and water (18 Da) are marked with asterisks and superscript 0, respectively. The superscript p designates the b ions with the phosphate group.

representing the b3 loss of 98 and 18 Da, respectively. However, closer examination of the spectrum in the region m/z 200450 revealed the ion at 253.06 m/z corresponding to b2 + 80 Da and the ion at 225.10 m/z corresponding to a2 + 80 Da (the 225.10 m/z signal height should be significantly smaller if it was the isotopic peak for 224.10 m/z ion, see inset of Figure 5A). These ions, jointly with the signal at 155.03 m/z, suggested that the threonine is phosphorylated. On the other hand, it can be noted that also the peak at 340.10 m/z (b3 + 80 Da) was present, providing further evidence to suggest phosphorylation at the serine. From these analyses, it can be suggested that there are two mono-phosphorylated versions of this peptide.

Discussion The choice to investigate stroma instead of the entire thylakoid is due to the fact that upon illumination at 100 µmol m-2 s-1 some proteins migrate from the grana toward the stroma in phosphorylated form.3,45 Moreover, stroma can easily and rapidly be divided from grana upon treatment by digitonin, in as little as 3 min the separation is complete. Such speed reduces the probabilities that phosphorylated proteins have time to retreat to the grana. Once the stromal proteins were separated, the surface-exposed portions of each protein were cleaved by trypsin and/or chymotrypsin as described previously16,18,32 and the resultant peptide mixtures were directly analyzed for the presence of phosphorylation. Through the phosphopeptide enrichment strategy described above, we identified seven previously unknown phosphorylation sites in spinach stroma membranes; five of them were on threonines reinforcing the consolidated evidence of a high specificity of protein kinases in thylakoid membranes toward these amino acid residues in the substrate proteins.15,16,18,20-22,24,28 Interestingly, this study did not reveal new phosphorylated proteins, with the exception of Rieske subunit, but the new phosphorylations regard the well-known Lhcb1 and CP43 proteins, on which we revealed the presence of multiphosphorylatable sites. These new phosphopetides are directly related to the light stimulus. Thus, light induces a high increase in the phosphorylation status, as these phosphopetides are absent in dark-adapted sample or their relative abundance below the level of detection. These interesting evidence might lead to 978

Journal of Proteome Research • Vol. 5, No. 4, 2006

further progress in unraveling the complex mechanisms of the thylakoid kinase activation that are still elusive. Light-Harvesting Complex. We isolated and sequenced two different phosphopeptides belonging to type I of the PSII lightharvesting complex (Lhcb1 protein): NVSSGSpPWYGPDR and TpVQSSSPWYGPDR, located at the N-terminal region of the protein. This domain of LHCII plays a substantial role in the subunit-subunit interactions responsible for supramolecular organization and regulation of photosynthetic protein complexes. In fact, trimerization is a specific process that depends on particular protein sequences present in both N-terminal and C-terminal domains.50,51 The structural basis for trimeric/ monomeric switch is unclear, but an altered arrangement of the N-terminal hydrophilic domain in LHCII, triggered by its phosphorylation, has been proposed.52 The addition of negatively charged phosphate groups to the LHCII complex causes it to dissociate from PSII and favors its migration out of the grana.53 Previous studies directed toward understanding phosphorylation of the LHCII have concentrated on a single phosphorylation site located at position 3 of the Lhcb1 amino acid sequence, although the position of the modified residue(s) appears to vary according to the species. Interestingly, a study with a series of recombinant pea Lhcb1 proteins, each missing an N-terminal segment including the known phosphorylation site (Thr-5), demonstrated that the LHCII proteins can be phosphorylated at one or more different sites, although the amino acid residue(s) undergoing the phosphorylation remain to be identified.54 This study agrees with our results defining for the first time the additional phospho-amino acid residues in the N-terminal of spinach Lhcb1 protein (Ser-14 in Lhcb 1.3; Thr-9 in Lhcb1.2). On the other hand, the LHCII, beside light capture, is involved in a number of regulatory processes that may require a more flexible behavior of the apoprotein. Recently, the conformational distribution of the N-terminal region of the major LHCII has been characterized by EPR measurements giving rise to the conclusion that this domain exists in at least two conformational states having very likely a functional significance since they may correspond to trimeric and monomeric state of the antenna complex.13 The model proposed by Jeschke et al. suggested that the phosphorylation of Thr-3, which is known to control the switch trimer/

New Phosphorylation Sites in Spinach Stroma Lamellae

research articles

Figure 4. Identification of N-terminal phosphopeptides in the CP43 protein. Panel A, MALDI-Q-TOF tandem MS spectrum of the chymotryptic phosphopeptide Ac-TpLFNGtpL where the lowercase t designates the secondary phosphorylation site and Ac- indicates acetylation. The indicated differences of 98 and 18 Da correspond to the neutral losses of H3PO4 and water, respectively. Panel B, Tandem MS spectrum obtained after nanoelectrospray ionization and CID of the peptide NGtpLTpLAGRDQETTGF. The indicated difference of 49 Da correspond to the neutral loss of H3PO4 from the doubly charged parent molecular ion at 880.93 m/z. The detected b (Nterminal) and y (C-terminal) fragment ions are labeled in the spectra. Gray b and y ions correspond to the fragments due to the secondary phosphorylation site. Fragment ions corresponding to the loss of ammonia (17 Da) and water (18 Da) are marked with asterisks and superscript 0, respectively.

monomer,55 causes a conformational change of the protein moving the N-terminal domain from a position above the protein core to a position sideways from it.13 In agreement, a combined NMR and Fourier transformed infrared spectroscopy study of a peptide consisting of the 15 N-terminal residues of LHCII and its counterpart phosphorylated at Thr-5 indicated a similar conformational switching.52 CP43 Protein. CP43 protein is an intrinsic chlorophyllbinding PSII core subunit classified as a photosynthetic phosphoprotein alongside D1, D2, and PsbH. Our mass spectrometric analysis confirmed that CP43 is phosphorylated at the N-terminal Thr-1 residue located on the stromal side of the thylakoid membrane.16,22 We detected three additional new phosphorylation sites in this internal antenna protein, two of which are present at the N-terminus (Thr-6 and Thr-8) exposed to the stroma16,22 and the other one at the hydrophilic loop E (Thr-346) exposed to the lumen.56 One of the functions of CP43 is to transfer excitation energy from the Chl a/b binding proteins to the PSII reaction center, in the case of higher plants and green algae, and from phycobilisomes in the case of cyanobacteria and red algae.57 So far no specific role has been ascribed to phosphorylation of CP43, although this protein is

located inside the reaction center. However, the presence of three new phosphorylation sites in this protein will require refinement of its probable role. A number of published experimental data suggests that phosphorylation of the PSII core proteins regulates the PSII photoinhibition-repair cycle.31,58 It is assumed that under severe photoinhibition, that is when the “repair sites” in stroma exposed membranes are fully occupied, the integrity of photodamaged PSII complexes in the grana regions is ensured by phosphorylation of the PSII core proteins; D1, D2, and CP43.31,58 The phosphorylation extent of PSII core phosphoproteins increases in light-exposed chloroplasts or intact leaves. Under unstressed conditions, their complete phosphorylation is generally attained at irradiances corresponding to those experienced by the plant during growth or at irradiances exceeding those.47 In particular, light-induced exposure of the phosphorylation sites of the CP43 antenna has been demonstrated and it increases with the light intensity in the range of 20-100 µmol m-2 s-1.59 On the basis of the accepted model of the phosphorylation-regulated LHCII-PSII interaction, a similar situation may occur within the PSII core complex. Thus, energy transfer between CP43 and the PSII reaction center could be down-regulated when both proteins Journal of Proteome Research • Vol. 5, No. 4, 2006 979

research articles

Rinalducci et al.

Figure 5. Identification of N-terminal phosphopeptides in the Rieske Fe-S protein. Panel A, Tandem MS spectrum obtained after nanoelectrospray ionization and CID of the peptide ATpspIPADNVPDMoxQK. The indicated difference of 49 Da correspond to the neutral loss of H3PO4 from the doubly charged parent molecular ion at 791.81 m/z. Inset displays a zoom of the spectrum in the region of m/z 200-400 for indication of some fragment ions suggesting two mono-phosphorylated versions of this peptide. Panel B points out the tandem MS spectrum obtained after nanoelectrospray ionization and CID of the peptide ATpspIPADNVPDMoxQKR. The shown difference of 33 Da corresponds to the neutral loss of H3PO4 from the triply charged parent molecular ion at 580.24 m/z. The detected b (Nterminal) and y (C-terminal) fragment ions are labeled in the spectra. Fragment ions corresponding to the loss of water (18 Da) are marked with superscript 0.

are phosphorylated and the electrostatic and/or conformational changes of the phosphoproteins may alter their interaction, reduce energy transfer and/or increase fluorescence quenching.59 Cytochrome b6f Rieske Fe-S Protein. For the first time a phosphorylated form of the cyt b6f Rieske Fe-S subunit has been detected. MS/MS experiments revealed that both Thr-2 and Ser-3 were phosphorylated. Besides its role in photosynthetic electron transport, the cyt b6f complex is also recruited for redox sensing and signal transduction in chloroplasts.60 The cyt b6f complex plays a key role in the supramolecular reorganization of the photosynthetic apparatus upon state transitions, i.e., charges in the redox state of plastoquinones (PQs) that bind the cyt b6f complexes allow a photosynthetic cell to adapt to changes in light quality as well as to changes in intracellular ATP levels.61 At the molecular level, plastoquinol binding at the Q0 site of cyt b6f complexes on the lumen side of the thylakoid membranes activates a kinase that phospho980

Journal of Proteome Research • Vol. 5, No. 4, 2006

rylates the light-harvesting antenna on the stromal side of membranes.5,7,8 It is assumed that the mechanism for which the plastoquinol binding is transduced across the membrane for kinase activation involves the Rieske Fe-S protein undergoing a transient conformational change through a distalproximal movement of the extramembrane segment.5,8 This proposal was further clarified recently by Finazzi et al. indicating that “locking” of the Rieske protein in either the distal or proximal positions prevents kinase activation.62 Interestingly, a new cyt b6f component in Chlamydomonas, the subunit V, was identified as a phosphoprotein63 although the site of phosphorylation remains unknown. Thus, the phosphorylation of subunit V, concomitant with the conformational changes induced by the movement of the Rieske protein, may be involved in the release of the activated kinase.62 Similarly, phosphorylation in the N-terminal of Rieske protein discovered in our work could be involved in the complex conformational changes that are essential for kinase activation in plants. This

research articles

New Phosphorylation Sites in Spinach Stroma Lamellae

consolidate the finding that a fraction of the cytochrome b6f complex migrates to stroma lamellae during state I to state II transition.64 Despite efforts of many research groups toward the elucidation of thylakoid protein phosphorylation, the progress in the identification and characterization of the kinases as well as the elucidation of their precise specificity toward each of the different membrane intrinsic substrates, has been limited. The evidence of a multiphosphorylation may allow further insights and perhaps the elucidation of the role played by the light acclimation processes in the context of the transition states between photosystem II and photosystem I. In fact, both Lhcb13 and CP4358 are mobile proteins which leave the PSII to migrate toward PSI upon illumination. Probably, this event requires a significant alteration in the surface charge of protein and the addition of more negatively charged phosphate groups to the protein leads to significant structural changes, resulting in the movement of phosphorylated proteins away from the grana stacks.65 We determined that five of the newly identified phosphopeptides were phosphorylated at threonine residues, whereas only two at serines. These data seem to reinforce the evidence for a higher specificity of thylakoid kinases toward the Thr residues in the substrate proteins.15,16,18,20-22,24,28 Moreover, other special behavior of thylakoid kinases is their light activation and redox regulation.49 Among all putative thylakoid kinases, so far five protein kinases associated with the photosynthetic membranes have been identified in plants: the family of three thylakoid-associated kinases (TAKs)66,67 and two kinases STN768 and STN869 that have significant sequence identity with chloroplast protein kinase Stt7 from the green alga Chlamydomonas reinhardtii.70 The Stt7, STN7, and TAKs kinases were found essential for phosphorylation of the LHCII amino terminus and the photosynthetic state transitions,66-68,70 although in the latter case the authors do not exclude that other substrates could occur.67 Curiously, TAKs themselves have been shown to be prone to phosphorylation66,67 and they may be part of a kinase cascade that could initiate with the signal from cyt b6f to phosphorylate a thylakoid substrate. It is also possible that the different TAKs are responsive to different light conditions.67 The STN8 was discovered to be specific for phosphorylation of PSII core proteins69 with particular selectivity toward the N-terminal residues of D1, D2, CP43, and the Thr-4 of PsbH. Interestingly, the light-activated STN8 kinase is able to phosphorylate the Thr-4 in PsbH only if the Thr-2 is prior phosphorylated, indicating the existence of another kinase phosphorylating the Thr-2. In this complicated scenery, it is not surprising that our newly detected phosphorylation sites could be the product of activity of different kinases and/or different pathways. On the other hand, dephosphorylation under preferential excitation of PSI is more pronounced for the LHCII than for the PSII core proteins, indicating a more rapid inactivation of the LHCII kinases as compared with that of PSII core protein kinases.71 Although it is possible that different protein kinases responding to redox conditions may be involved in the phosphorylation of PSII core proteins, it is also possible that illumination may affect the exposure of various membrane-integrated phosphoproteins to the protein kinase/phosphatases in different ways.49 This could result in the presence of multiphosphorylation sites in a protein which may play different physiological roles. However, to better understand and discriminate the role of each phosporylation it is of great importance to use investigative methods which allow researchers to recognize all the

chemical modifications present in a protein. Thus, improving the analysis of phosphorylated peptides either by improving the purification strategy or the mass spectrometric analysis will help revealing the role of phosphorylation in cellular pathways, including the light-harvesting complex. Abbreviations. CID, collision-induced dissociation; CP, chlorophyll protein; Cyt, cytochrome; DHB, 2,5-dihydroxybenzoic acid; ESI, electrospray ionization; HPLC, high performance liquid chromatography; IMAC, immobilized metal affinity chromatography; LC, liquid chromatography; LHC, lightharvesting complex; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; PQ, plastoquinone; PS, photosystem; Q-TOF, quadrupole-time-of-flight; TFA, trifluoracetic acid; TiO2, titanium dioxide.

Acknowledgment. We thank Anna Maria Timperio and Corrado Ciambella from Tuscia University for their precious help as well as Elisabetta Boeri Erba from the Protein Research Group at University of Southern Denmark. Special thanks to Kate Rafn and Lene Skou for technical assistance in mass spectrometric measurements. We would also like to thank Prof. Peter Roepstorff for providing interesting proposals to this experimental work. This work is supported by CIB (Consorzio Interuniversitario per le Biotecnologie), the Italian Ministry of University Research (MIUR-PRIN 2004) and the Danish Research Agency for funds to the Danish Biotechnology Instrument Center. M.R.L. was sponsored by the Danish Natural Science Research Council. References (1) Bennett, J. Nature 1977, 269, 344-346. (2) Allen, J. F.; Bennett, J.; Steinback, K. E.; Arntzen, C. J. Nature 1981, 291, 25-29. (3) Allen, J. F. Biochim. Biophys. Acta 1992, 1098, 275-335. (4) Bennett, J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991, 42, 281-331. (5) Vener, A. V.; Ohad, I.; Andersson, B. Curr. Opin. Plant Biol. 1998, 1, 217-223. (6) Pursiheimo, S.; Martinsuo, P.; Rintamaki, E.; Aro, E.-M. Plant Cell Environ. 2003, 26, 1995-2003. (7) Vener, A. V.; van Kan, P. J.; Rich, P. R.; Ohad, I.; Andersson, B. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1585-1590. (8) Zito, F.; Finazzi, G., Delosme, R.; Nitschke, W.; Picot, D.; Wollman, F. A. EMBO J. 1999, 18, 2961-2969. (9) Carlberg, I.; Rintama¨ki, E.; Aro, E.-M.; Andersson, B. Biochemistry 1999, 38, 3197-3204. (10) Rintama¨ki, E.; Martinsuo, P., Pursiheimo, S., Aro, E.-M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11644-11649. (11) Zer, H.; Vink, M.; Keren, N.; Dilly-Hartwig, H. G.; Paulsen, H.; Herrmann, R. G.; Andersson, B.; Ohad, I. Proc. Natl. Acad. Sci. USA 1999, 96, 8277-8822. (12) Zer, H.; Vink, M.; Shochat, S.; Herrmann, R. G.; Andersson, B.; Ohad, I. Biochemistry 2003, 42, 728-738. (13) Jeschke, G.; Bender, A.; Schweikardt, T.; Panek, G.; Decker, H.; Paulsen, H. J. Biol. Chem. 2005, 280, 18623-18630. (14) Hammer, M. F.; Markwell, J.; Sarath, G. Plant Physiol. 1997, 113, 227-233. (15) Michel, H.; Griffin, P. R.; Shabanowitz, J.; Hunt, D. F.; Bennett, J. J. Biol. Chem. 1991, 266, 17584-17591. (16) Vener, A. V.; Harms, A.; Sussman, M. R.; Vierstra, R. D. J. Biol. Chem. 2001, 276, 6959-6966. (17) Bergantino, E.; Dainese, P.; Cerovic, Z.; Sechi, S.; Bassi, R. J. Biol. Chem. 1995, 270, 8474-8481. (18) Hansson, M.; Vener, A. V. Mol. Cell. Proteomics 2003, 2, 550559. (19) Turkina, M. V.; Villarejo, A.; Vener, A. V. FEBS Lett. 2004, 564, 104-108. (20) Kargul, J.; Turkina, M. V.; Nield, J.; Benson, S.; Vener, A. V.; Barber, J. FEBS J. 2005, 272, 4797-4806. (21) Michel, H. P.; Bennett, J. FEBS lett. 1987, 212, 103-108. (22) Michel, H.; Hunt, D. F.; Shabanowitz, J.; Bennett, J. J. Biol. Chem. 1988, 263, 1123-1130.

Journal of Proteome Research • Vol. 5, No. 4, 2006 981

research articles (23) Andreucci, F.; Barbato, R.; Picollo, C.; Segalla, A. J. Exp. Bot. 2005, 56, 1239-1244. (24) Carlberg, I.; Hansson, M.; Kieselbach, T.; Schroder, W. P.; Andersson, B.; Vener, A. V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 757762. (25) Khrouchtchova, A.; Hansson, M.; Paakkarinen, V.; Vainonen, J. P.; Zhang, S.; Jensen, P. E.; Scheller, H. V.; Vener, A. V.; Aro, E.M.; Haldrup, A. FEBS Lett. 2005, 579, 4808-4812. (26) Delosme, R.; Olive, J.; Wollman, F. A. Biochim. Biophys. Acta 1996, 1273, 150-158. (27) Timperio, A. M.; Zolla, L. J. Biol. Chem. 2005, 280, 28858-28866. (28) Testi, M. G.; Croce, R.; Polverino-De Laureto, P.; Bassi, R. FEBS lett. 1996, 399, 245-250. (29) Koivuniemi, A.; Aro, E.-M.; Andersson, B. Biochemistry 1995, 34, 16022-16029. (30) Callahan, F. E.; Ghirardi, M. L.; Sopory, S. K.; Mehta, A. M.; Edelman, M.; Mattoo, A. K. J. Biol. Chem. 1990, 265, 15357-15360. (31) Baena-Gonzalez, E.; Barbato, R.; Aro, E.-M. Planta 1999, 208, 196-204. (32) Aro, E.-M.; Rokka, A.; Vener, A. V. Methods Mol. Biol. 2004, 274, 271-285. (33) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1993, 4, 710-717. (34) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413-3421. (35) Zappacosta, F.; Huddleston, M. J.; Karcher, R. L.; Gelfand, V. I.; Carr, S. A.; Annan, R. S. Anal. Chem. 2002, 74, 3221-3231. (36) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250-254. (37) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (38) Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal. Chem. 2004, 76, 3935-3943. (39) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Mol. Cell. Proteomics 2005, 4, 873-886. (40) Anderson, J. M.; Boardman, N. K. Biochim. Biophys. Acta 1966, 112, 403-410. (41) Porra, R. J.; Thompson, W. A.; Kriedemann, P. E. Biochim. Biophys. Acta 1989, 975, 384-394. (42) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105-116. (43) Larsen, M. R.; Cordwell, S. J.; Roepstorff, P. Proteomics 2002, 2, 1277-1287. (44) Kjellstrom, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109-5117. (45) Haldrup, A.; Jensen, P. E.; Lunde, C.; Scheller, H. V. Trends Plant Sci. 2001, 6, 301-305. (46) Melis, A. Trends Plant Sci. 1999, 4, 130-135. (47) Rintama¨ki, E.; Salonen, M.; Suoranta, U. M.; Carlberg, I.; Andersson, B.; Aro, E.-M. J. Biol. Chem. 1997, 272, 30476-30482.

982

Journal of Proteome Research • Vol. 5, No. 4, 2006

Rinalducci et al. (48) Walcher, W.; Timperio, A. M.; Zolla, L.; Huber, C. G. Anal. Chem. 2003, 75, 6775-6780. (49) Aro, E.-M.; Ohad, I. Antioxid. Redox Signal. 2003, 5, 55-67. (50) Hobe, S.; Forster, R.; Klingler, J.; Paulsen, H. Biochemistry 1995, 34, 10224-10228. (51) Kuttkat, A.; Hartmann, A.; Hobe, S.; Paulsen, H. Eur. J. Biochem. 1996, 242, 288-292. (52) Nilsson, A.; Stys, D.; Drakenberg, T.; Spangfort, M. D.; Forsen, S.; Allen, J. F. J. Biol. Chem. 1997, 272, 18350-18357. (53) Andersson, B.; Aakerlund, H. E.; Jergil, B.; Larsson, C. FEBS Lett. 1982, 149, 181-185. (54) Dilly-Hartwig, H.; Allen, J. F.; Paulsen, H.; Race, H. L. FEBS Lett. 1998, 435, 101-104. (55) Allen, J. F.; Forsberg, L. Trends Plant Sci. 2001, 6, 317-326. (56) Sayre, R. T.; Wrobel-Boerner, E. A. Photosynth. Res. 1994, 40, 1119. (57) Barber, J. Biochim. Biophys. Acta 1998, 1365, 269-277. (58) Aro, E.-M.; Suorsa, M.; Rokka, A.; Allahverdiyeva, Y.; Paakkarinen, V.; Saleem, A.; Battchikova; N.; Rintama¨ki; E. J. Exp. Bot. 2005, 56, 347-356. (59) Vink, M.; Zer, H.; Herrmann, R. G.; Andersson, B.; Ohad, I. Photosynth. Res. 2000, 64, 209-219. (60) Allen, J. F. Trends Plant Sci. 2004, 9, 130-137. (61) Bulte, L.; Gans, P.; Rebeille, F.; Wollman, F. A. Biochim. Biophys. Acta 1990, 1020, 72-80. (62) Finazzi, G.; Zito, F.; Barbagallo, R. P.; Wollman, F. A. J. Biol. Chem. 2001, 276, 9770-9774. (63) Hamel, P.; Olive, J.; Pierre, Y.; Wollman, F. A.; de Vitry C. J. Biol. Chem. 2000, 275, 17072-17079. (64) Vallon, O.; Bulte, L.; Danese, P.; Olive, J.; Bassi, R.; Wollman, F. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8262-8266. (65) Bennett, J. Biochem. J. 1983, 212, 1-13. (66) Snyders, S.; Kohorn, B. D. J. Biol. Chem. 1999, 274, 9137-9140. (67) Snyders, S.; Kohorn, B. D. J. Biol. Chem. 2001, 2764, 32169-32176. (68) Bellafiore, S.; Barneche, F.; Peltier, G.; Rochaix, J. D. Nature 2005, 433, 892-895. (69) Vainonen, J. P.; Hansson, M.; Vener, A. V. J. Biol. Chem. 2005, 280, 33679-33686. (70) Depege, N.; Bellafiore, S.; Rochaix, J. D. Science 2003, 299, 15721575. (71) Carlberg, I.; Andersson, B. Photosynth. Res. 1996, 47, 145-156.

PR050476N