A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes

Abstract

The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.

Protein phosphorylation plays a major regulatory role in all cellular functions, from gene expression to signaling and metabolic control. A unique light- and redox-controlled protein phosphorylation system has evolved in plant thylakoid membranes for regulation of the photosynthetic process (1, 2). Intrinsic protein kinases in chloroplast thylakoid membranes (3–5) are activated by light or reducing conditions and controlled by the reduction of plastoquinone and its binding to the reduced cytochrome bf complex (6, 7). Additional modulation of protein phosphorylation in thylakoid membranes involves the thiol redox state (8, 9) as well as light-modulated conformational changes of substrate proteins (10). Activated thylakoid kinases phosphorylate the membrane proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) as well as a number of still unidentified protein substrates (2, 11–13). The protein dephosphorylation reactions are catalyzed by both integral thylakoid membrane and soluble chloroplast phosphatases (2, 14). The reversible phosphorylation of LHCII polypeptides helps balance the distribution of absorbed light energy between the two photosystems (1, 15–17). Phosphorylation of the core subunits of PSII controls their maintenance and turnover, with dephosphorylation of the D1 and D2 proteins being a signal for their proteolytic degradation (18, 19). Two other subunits of PSII, the chlorophyll a-binding protein CP43 and the 9-kDa PsbH gene product, are also phosphorylated in thylakoid membranes (20, 21). All of the thylakoid phosphoproteins identified so far are hydrophobic integral membrane proteins phosphorylated at threonine residues at or near their N termini (12, 21, 22).

One of the phosphopeptides in the thylakoid membrane, the so-called 12-kDa phosphoprotein (23, 24), has, however, resisted identification with respect to its sequence, corresponding gene, and functional significance. An earlier N-terminal sequencing of this protein from spinach revealed a unique stretch of amino acids that did not match any protein in the databases (24). The protein is phosphorylated by the intrinsic redox-controlled protein kinase of the thylakoid membrane both in vivo and in vitro (23, 24). The general characteristics for redox-controlled phosphorylation of the 12-kDa phosphoprotein as well as for its dephosphorylation and sensitivity to inhibitors closely follows those observed for the LHCII polypeptides, but not those of the PSII phosphoproteins (8, 24–26). A phosphoamino acid analysis showed that this protein, like other thylakoid proteins, was phosphorylated at threonine residue(s) (23). However, in marked contrast to the other thylakoid phosphoproteins that are integral to the membrane, the 12-kDa phosphoprotein is a peripheral thylakoid protein. The 12-kDa protein from barley thylakoids was found partially in the soluble stroma fraction, and thus it was speculated that the nonphosphorylated protein is associated with the membrane but released on phosphorylation (23). The 12-kDa phosphoprotein from spinach thylakoid membranes was also released by washings at high-salt and/or low-detergent concentration (24).

In this paper, we report an extensive mass spectrometric sequencing of the 12-kDa phosphoprotein from spinach as well as the isolation and sequencing of the corresponding cDNA. We determine that the real molecular mass of the protein is equal to 8,640 Da and demonstrate the partial release of the protein from the membrane on light-induced phosphorylation. Due to these characteristics, we designate the protein as a thylakoid soluble phosphoprotein of 9 kDa (TSP9). We reveal that TSP9 is a plant-specific protein, identify the exact positions of multiple phosphorylation sites in the spinach TSP9, and discuss its possible involvement in plant cell signaling.

Materials and Methods

Preparation of Thylakoids.

Spinach (Spinacea oleracea) was grown hydroponically at 20–25°C and a light intensity of 400- to 500-μmol photons m⁻²⋅s⁻¹ with a 10/14-h light/dark regime. Thylakoid membranes were prepared from 7-wk-old dark-adapted spinach leaves, as described (27). The membranes were resuspended in 0.1 M sorbitol/25 mM tricine, pH 7.9/5 mM MgCl₂/10 mM KCl/10 mM NaF (buffer A).

Phosphorylation of Thylakoid Membranes and Isolation of TSP9-Enriched Fractions.

For the preparation of TSP9, 200 ml of thylakoid membranes at a chlorophyll concentration of 0.5 mg/ml was phosphorylated by illumination at 120-μmol photons m⁻²⋅s⁻¹ for 30 min in the presence of 0.5 mM ATP in buffer A. The membranes were centrifuged at 5,000 × g for 5 min, washed once in buffer A, and then resuspended in 150 ml of 2 M NaBr in the same buffer and incubated on ice for 45 min with occasional stirring. After this treatment, the membranes were centrifuged at 30,000 × g for 50 min. The supernatant was collected and concentrated to a final volume of 1 ml by filtration through a membrane (Amicon) with a 3-kDa cutoff. The concentrate was dialyzed against 0.1 M Tris⋅HCl, pH 7.5/5 mM MgCl₂/10 mM NaF to remove NaBr. For partial fractionation of the released proteins, 0.5 ml of the concentrated sample was applied to a 5-ml Sephadex G-75 (Pharmacia) column equilibrated with 25 mM NH₄HCO₃, and 0.5-ml fractions were collected. ³²P-labeling of thylakoid proteins was performed by illumination in the presence of 0.25 mM ATP containing [γ-³²P]ATP (0.02 mCi/mg chlorophyll). The reaction was stopped by the addition of 25 mM EDTA. For identification and sequencing, the proteins were separated on 12–22% SDS/PAGE gradient gels.

N-Terminal Sequencing.

N-terminal microsequencing was essentially carried out according to Matsudaira (28) by using a Procise sequencer from Applied Biosystems.

Protein Digestion with Trypsin.

For in-gel digestion, the protein bands were excised from the gel and treated with trypsin (Sequencing Grade Modified, Promega) essentially according to the procedure described by Shevchenko et al. (29). For digestion of soluble proteins, the fractions after chromatography on Sephadex G-75 were dried in a vacuum centrifuge, and then proteins were dissolved in 25 mM NH₄HCO₃ up to 1-mg/ml concentration. The cysteine residues were reduced by 1 mM DTT and alkylated by 3 mM iodoacetamide. The trypsin (Sequencing Grade Modified, Promega) was added to 0.02-mg/ml concentration, and digestion was performed for 24 h at 37°C.

Matrix-Assisted Laser Desorption Ionization (MALDI)–Time of Flight (TOF) Mass Spectrometry.

The analyses were carried out on a Voyager-DE Pro (Applied Biosystems). For peptide fingerprinting analyses, equal volumes of sample and α-cyano-4-hydroxycinnamic acid in 70% (vol/vol) acetonitrile with 0.3% (vol/vol) trifluoroacetic acid were spotted on the target plate. Reflector mass spectra were recorded by using the instrument settings recommended by Applied Biosystems and were calibrated externally. For mass measurements of intact proteins, equal volumes of sample and sinapinic acid in 70% (vol/vol) acetonitrile with 0.3% (vol/vol) trifluoroacetic acid were spotted on the target plate, and a linear detector was used for recording of the spectra. The accelerating voltage was set at 25 kV, grid voltage at 95%, guide wire voltage at 0.05%, and an extraction delay time of 800 ns was used.

Electrospray Ionization Tandem Mass Spectrometry.

The spectra were acquired on a hybrid mass spectrometer API Q-STAR Pulsar i (Applied Biosystems) equipped with a nanoelectrospray ion source (MDS Protana, Odense, Denmark). The nanoelectrospray capillaries were loaded with 2 μl of peptide solutions in water with 1% formic acid for positive ion mode analyses or in water with 25 mM NH₄HCO₃ for negative ion mode analyses. The collision-induced decomposition of selected precursor ions was performed by using the instrument settings recommended by Applied Biosystems.

PCR-Mediated Isolation of cDNAs.

Total RNA was prepared from spinach leaves according to Qiagen (Chatsworth, CA) procedure by using RNeasy Plant Mini Kit and treated with DNase I (DNA-free, Ambion). The corresponding single-stranded cDNAs were obtained by using oligo(dT)₁₅ primer and Reverse Transcription System (Promega). The following primers for PCR reactions were constructed: for ferredoxin-thioredoxin reductase (FTR) subunit A: forward, 5′-CTCCGATGACAACAGGTGTG-3′ and reverse, 5′-GACAACAAGGAAGAAGAAAGACAA-3′; and for TSP9: forward, 5′-ACTCAATCAGCCCAAGGAGA-3′ and reverse, 5′-AAGACACTGCAGGAAACTGAGA-3′. The PCR reactions were performed by using TaKaRa Ex TaqDNA polymerase (Takara Shuzo, Kyoto), and PCR products were purified by using QIAquick PCR Purification Kit (Qiagen). Sequencing of the PCR products was done with Thermo Sequenase Cy5 Dye Terminator Kit on ReproGel High resolution using Alf Express II sequencer (Amersham Pharmacia Biotech).

Results

Isolation and Sequencing of the 12-kDa Phosphoprotein (TSP9).

The characteristics that define the 12-kDa thylakoid phosphoprotein are: (i) phosphorylation by a redox-dependent kinase in the thylakoid membrane; (ii) a relative electrophoretic mobility during SDS/PAGE corresponding to a molecular mass of about 12 kDa; and (iii) release from the membrane by salt-wash treatment (23, 24). To characterize this protein further, we first phosphorylated isolated spinach thylakoids by illumination in the presence of radioactive ATP. The autoradiogram in Fig. 1A shows the major phosphorylated proteins including TSP9, as well as several unassigned phosphoproteins labeled to a lower extent. TSP9 migrates during SDS/PAGE above the phosphorylated PsbH protein (7.6 kDa), and its relative molecular mass corresponds to 12 kDa, in agreement with previous observations (23, 24). Notably, we observe a partial release of the phosphorylated TSP9 from the membrane during light-induced phosphorylation of the thylakoids (Fig. 1B). The release under the present experimental conditions corresponds to ≈15% of the labeled TSP9 (data not shown). As can be seen in Fig. 1B, this release is specific for TSP9. No other thylakoid protein phosphorylated by redox-dependent protein kinases releases from the membrane into the soluble fraction. Thus, TSP9 is unique as a soluble thylakoid protein discharging from the membranes in phosphorylated form.

Figure 1.

Phosphorylation and release of TSP9 from the thylakoid membrane. (A) Autoradiogram of ³²P-labeled thylakoid proteins separated by SDS/PAGE after light-induced phosphorylation of the membranes in the presence of [γ-³²P]ATP. The positions of major known thylakoid phosphoproteins are indicated. (B) The autoradiogram of ³²P-labeled proteins released from the membranes during illumination of thylakoids for the times indicated above the lanes. (C) Coomassie-stained gel of the proteins released from phosphorylated thylakoid membranes by washing with 2 M NaBr. The position of the TSP9 double band is indicated.

To obtain a TSP9-enriched fraction for further analysis, we washed the phosphorylated membranes with 2 M NaBr. This treatment has previously been shown to completely release TSP9 from the membrane (24). The released proteins were concentrated, dialyzed, and separated on SDS/PAGE. Fig. 1C shows a doublet protein band in the stained gel corresponding to TSP9 in the autoradiogram. Each of the two protein bands was excised from the gel and digested with trypsin. The resultant peptides were analyzed by MALDI-TOF mass spectrometry, which gave an identical pattern of peptides for both bands (data not shown). The peptide-mass fingerprint analysis (30) performed with these peptides did not match any known protein in the databases. Thus, we performed an extensive sequencing of TSP9. The N-terminal sequence (peptide 1 in Table 1) was obtained after transfer of the protein to a polyvinylidene difluoride membrane and sequencing by Edman degradation (28). The TSP9 peptides after in-gel digestion by trypsin were subjected to collision-induced fragmentation by using nanospray-quadrupole-TOF hybrid mass spectrometry. The high-quality fragmentation spectra allowed de novo sequencing of most of the peptides originating from TSP9, as summarized in Table 1. One of the tryptic peptides is overlapping with the N-terminal sequence obtained by Edman degradation (Table 1, peptide 2). Two pairs of peptide sequences are also partially overlapping with each other due to alternative tryptic cleavages (Table 1, peptides 3 and 4 and peptides 5 and 6). The peptides from TSP9 did not correspond to any known protein in the databases. Thus, we concluded that TSP9 is a previously uncharacterized protein and that it has at least two isoforms that can be separated during electrophoresis.

Table 1.

Sequences of the peptides from TSP9

No.	Sequence	Theoretical mass	Experimental mass
1	AAKGTAETKQEKSFVDWLLG
2	SFVDWLLGK	1,063.58	1,063.53
3	ITKEDQFYETDPILR	1,866.94	1,866.86
4	EDQFYETDPILR	1,524.71	1,524.66
5	KDGDGGVFGGLFAK	1,366.70	1,366.65
6	DGDGGVFGGLFAK	1,238.60	1,238.55
7	GGDVK	474.25	474.25
8	GGtTSGK	686.27	686.31
9	KGtVSIPSK	995.51	995.51
10	SSGStSGK	789.30	789.33

The N-terminal sequence (peptide 1) was obtained by Edman degradation. Peptides 2–10 from TSP9 digested with trypsin have been sequenced by using collision-induced fragmentation mass spectrometry. A single-letter amino acid code is used; the lowercase t designates phosphothreonine residues. The shown peptide masses are monoisotopic. 

Identification of TSP9 as a Plant-Specific Protein.

The initial use of the TSP9 peptide sequence information (Table 1) in a translated nucleotide blast search (tblastn) (31) gave a surprising result. All of the peptides showed a high degree of identity to a long “untranslated” 3′-trailer region of a cDNA clone (X77161) for FTR subunit A from spinach (32). However, the TSP9 peptides were found in different reading frames of the cDNA, suggesting some sequencing mistakes. Also, the apparent “polycistronic” cDNA clone suggested the possible formation of artifacts during the preparation of the spinach cDNA library (32). Thus, we prepared total RNA from spinach leaves, obtained the corresponding single-stranded cDNAs, and performed PCR-mediated isolation of cDNAs by using primers for FTR and TSP9 based on the sequence of the published FTR cDNA clone (32). The use of mixed primers for FTR (forward) and TSP9 (reverse) yielded no PCR product. A 600-bp PCR product with a sequence corresponding to the subunit A of FTR (32) was obtained with primers for FTR (data not shown). The use of the primers for TSP9 resulted in a 400-bp PCR product that encoded a full length 103-aa-long precursor protein for TSP9 (Fig. 2). The sequence of the protein that corresponds to the N-terminal peptide of TSP9 begins at the 21st amino acid of this translation product, and the sequenced peptides cover 95% of its length (Fig. 2). The presence of two aspartates (D) in the peptides from the C terminus of the protein (Fig. 2; peptides 5 and 6 in Table 1) instead of two asparagines (N) encoded in the cDNA (Fig. 2) could result from a common deamidation reaction, which has also been observed in the case of other thylakoid phosphoproteins (12). The molecular mass of the predicted mature protein is 8,640 Da, a value confirmed by MALDI-TOF mass spectrometry of the isolated TSP9 (Fig. 4A, see below). Thus, we designate this protein thylakoid soluble phosphoprotein of 9 kDa (TSP9). Due to the large content of positively charged amino acids, TSP9 is a basic protein with a calculated isoelectric point as high as 9.8.

Figure 2.

The sequence of TSP9 deduced from the cDNA encoding a full-length precursor protein aligned with the peptide sequences from the isolated protein (Table 1). The nucleotide sequence is in the upper line (lowercase, the start and stop codons are underlined), the precursor protein (uppercase) and the peptide sequences (uppercase, bold) are in the lower lines, respectively. The numbers to the left correspond to the amino acid positions in the mature TSP9. The threonine residues phosphorylated in TSP9 are designated by the lowercase bold t.

Figure 4.

Identification of three phosphorylated forms of TSP9 and the corresponding phosphorylation sites. (A) The MALDI-TOF spectrum of the intact TSP9. The masses of non-, mono-, di- and triphosphorylated forms of TSP9 are indicated. (B and C) The product ion spectra obtained by positive mode electrospray ionization and collision-induced dissociation of phosphorylated peptides 8 (B) and 9 (C) (Table 1). The peptide sequences are shown in respective spectrum with the low case t designating phosphorylated threonine residues. The parent molecular ions (M+H)⁺ with m/z 687.3 (B) and (M+2H)²⁺ with m/z 498.7 (C) are indicated along with the b- (N-terminal) and y- (C-terminal) fragment ions. The fragment ions produced by neutral loss of H₃PO₄ (mass 98) are marked with asterisks. Only fragment ions that help to localize the phosphorylation sites are labeled in the spectra. (D) The product ion spectrum of the doubly charged negative ion [(M−2H)²⁻, m/z 393.7] of peptide 10 (Table 1). The characteristic fragments −79 m/z (PO), −97 m/z (H₂PO), and –708.4 m/z [(M−2H)²⁻- PO]^- are indicated. The ion with m/z –180.0 corresponds to a fragment of phospho-threonine, designated by lowercase t in the shown peptide sequence.

A database protein blast search (31) with the TSP9 sequence revealed two homologous proteins annotated as “unknown:” a 100-aa-long protein encoded by a single-copy gene At3g47070 in chromosome 3 of Arabidopsis and a 119-aa-long protein (GenBank accession no. AAK82437) encoded by a single-copy gene in chromosome 3 of rice. To get further insight into the presence of TSP9-like proteins in other organisms, we also used its amino acid sequence to search the EST databases, resulting in the identification of mRNAs with a high degree of similarity to that of TSP9 in 20 different plant species. Notably, no homologous ESTs were found in species other than plants. Fig. 3 shows the alignment of TSP9 with the deduced amino acid sequences for the proteins from a few representatives of different plants. The alignment of the TSP9 sequence with the putative homologues from 20 plants is shown in Fig. 5, which is published as supporting information on the PNAS web site, www.pnas.org. Most of the ESTs originate from higher plants, among which M. crystallinum (iceplant) shows the highest similarity with 64% of the amino acids identical to the spinach protein. One gymnosperm, Cryptomerica japonica, and one lower plant, Marchanthia polymorpha (liverwort), are also represented among these ESTs, showing a lower but still significant similarity to spinach TSP9. The estimated reading frames of the ESTs lie between 78 and 122 amino acids, suggesting putative polypeptides with a mass of 8–10 kDa. The most conserved region is found in the central part of the protein, corresponding to positions 30–59 in spinach TSP9 (Fig. 3). The N-terminal regions of the translated proteins lack negative charges and are abundant in serine and threonine residues (Fig. 3), characteristic for chloroplast targeting signal peptides (33, 34), and in good agreement with the experimental localization of TSP9 (“the 12-kDa phosphoprotein”) to the thylakoid membrane of chloroplasts in a number of plant species (6, 23, 24, 35, 36).

Figure 3.

Alignment of spinach TSP9 with the amino acid sequences of putative homologous proteins deduced from ESTs of eight different plants. The arrow indicates the start of mature spinach TSP9. The alignment is done by using the CLUSTALW program (48). The threonine residues phosphorylated in TSP9 are indicated by the lowercase t above the sequence. The GenBank accession nos. are: Mesembryanthemum cristallinum, BE130175; Citrus sinensis, BQ623922; Glycine max, BG157421; Lycopersicon esculentum, BG643569; Vitus vinifera, BQ797569; Arabidopsis thaliana, AV533911; Allium cepa, AA451548; Marchantia polymorpha, AU081993.

The Phosphorylation Pattern of TSP9.

The analysis of the peptides obtained after in-gel digestion of TSP9 did not reveal any phosphorylated peptides from this protein. To circumvent a possible dephosphorylation or poor extraction of the phosphopeptides during the procedure, we decided to analyzed TSP9 without prior electrophoresis and in-gel digestion. To this end, the concentrated proteins released from the thylakoid membrane by NaBr-washing (Fig. 1C) were subjected to size-exclusion chromatography on a Sephadex G-75 column to get a partial protein fractionation. TSP9 was found in the fractions containing low molecular-weight proteins and was largely depleted of the main contaminants, the α and β subunits of the ATP synthase (data not shown). This result indicates that TSP9 does not form any high molecular-weight complexes with other proteins, when released from the thylakoid membrane by high salt. Direct MALDI-TOF analysis of the fractions containing TSP9 resulted in two valuable observations. Firstly, the molecular mass of TSP9 is determined to be 8,641 Da (Fig. 4A), which is almost equal to the molecular mass of 8,640 Da calculated from the amino acid sequence deduced from the cDNA for the mature protein (Fig. 2). Secondly, the mass spectrum reveals the existence of three protein species with the masses ≈80, 160, and 240 Da higher, respectively, than that of TSP9 (Fig. 4A). The mass increment of 80 Da is characteristic for the addition of one phosphate group. Thus, the present MALDI-TOF data provide the first evidence, to our knowledge, for the existence of TSP9 in non-, mono-, di-, and triphosphorylated forms. Notably, TSP9 is the first protein associated with plant photosynthetic membranes found to become triply phosphorylated.

To map the phosphorylation sites, the Sephadex G-75 fractions containing TSP9 were digested with trypsin and the resulting peptides were directly analyzed by nanospray-quadrupole-TOF mass spectrometry. For selective identification of phosphopeptides in a complex mixture containing peptides from TSP9 and other low molecular mass proteins, we first performed precursor-ion scanning in negative mode. The precursor-ion technique allows for selective determination of the mass-to-charge ratios just for phosphorylated peptides that produce a diagnostic phosphoryl ion (PO) −79 m/z (37, 38). This approach revealed the presence of three phosphopeptides with masses of 686.3, 995.5, and 789.3 atomic units (data not shown), which corresponded to monophosphorylated peptides 8, 9, and 10 from TSP9, respectively (Table 1). Peptides 8 and 9 contain three potential phosphorylation sites, and peptide 10 contains five. Thus, determination of the exact phosphorylation sites in these peptides is a challenging task. To achieve that, each of these peptides was subjected to the collision-induced fragmentation in positive ionization mode and sequenced. The fragmentation spectra for peptides 8 and 9 (Fig. 4 B and C) allowed determination of the phosphorylated residues. A phosphoester bond between a phosphoryl group and a peptide is less stable than a peptide bond, which leads to a prominent neutral loss of phosphoric acid (H₃PO₄, 98 Da) from the positively charged peptide ions (12, 37, 38). In Fig. 4B, this loss is evident from the appearance of the fragment ion 589.3, which is 98 m/z below the selected singly charged molecular ion of phosphopeptide 8 (M+H⁺ = 687.3). In Fig. 4C, the neutral loss at 49 m/z ({[M+2H⁺]-98}/2 = 449.7) below the selected doubly charged phosphopeptide ion ([M+2H⁺]/2 = 498.7) is observed. Each of these peptides contains three possible sites for phosphorylation of serine or threonine residues. However, the neutral loss of the phosphoric acid occurs only in the fragment ions containing phosphorylated residue. The series of y (C-terminal) and b (N-terminal) fragment ions without the neutral loss (originated from nonphosphorylated fragments of the peptide) together with the distinct ions that underwent the neutral loss (originated from phosphorylated peptide fragments; y and b are marked with asterisks) in each spectrum allowed unambiguous identification of the phosphorylation sites (Fig. 4 B and C). These results revealed phosphorylation of TSP9 at two distinct sites corresponding to threonine residues 53 and 60 in the sequence of the mature TSP9 (Fig. 2).

The collision-induced fragmentation of peptide 10 (Table 1) in positive ionization mode did not allow unequivocal distinguishing of which of four serines or one threonine residue is phosphorylated (data not shown). To narrow down the site of phosphorylation in this peptide, we performed a collision-induced fragmentation of the negatively charged peptide. The fragmentation spectra of negative peptide ions are more complex than those for the positive ions but allow gathering of additional structural information (39). Particularly, the negative fragments of phosphoserine or phosphothreonine at −166.0 m/z or −180.0 m/z, respectively, could be observed (A.V.V., unpublished data). Accordingly, the fragmentation spectrum of peptide 10 revealed the internal fragment ion corresponding to phosphothreonine at −180.0 m/z but no signal at −166.0 m/z (Fig. 4D). Because of the single threonine residue in this peptide, we concluded that this residue corresponding to Thr-46 in the mature protein (Fig. 2) is phosphorylated. Thus, we have located all three phosphorylation sites to the central part of TSP9. This is an additional unique characteristic of TSP9 in contrast to the other known thylakoid phosphoproteins, which are phosphorylated at their N termini (12, 20, 22).

Discussion

We have determined the sequence of a protein that for a long time has been recognized as the “12-kDa phosphoprotein” and which has been shown to undergo redox-dependent phosphorylation in thylakoid membranes from barley (23), pea (36), as well as spinach (24). This polypeptide has no analogy to any other known protein in the databases. The protein consists of 83 amino acids, and its calculated and experimentally determined molecular mass is 8,640 Da. Accordingly, we have named the protein thylakoid soluble phosphoprotein of 9 kDa (TSP9). The name also reflects the finding that TSP9 is not an integral membrane protein and is partially released from the thylakoids into the chloroplast stroma as a soluble protein on light-induced phosphorylation.

TSP9 is a plant-specific protein. Nuclear single-copy genes encoding homologous proteins are found on the third chromosome in Arabidopsis and rice. Both genes were identified in the genome sequencing projects and were annotated as “unknown” proteins because of lack of homology to any classified protein domains. Additionally, the existence of genes encoding proteins homologous to TSP9 is evident in at least 20 different plant species, through the identification of cognate ESTs. All these ESTs encode putative precursor proteins with signal peptides. Although computer programs (33, 34) predict the chloroplast targeting for only one-half of these precursor proteins, TSP9 has been experimentally localized to thylakoid membranes of plant chloroplasts by a number of research groups (23, 24, 35, 36). No genes encoding proteins similar to TSP9 are found in any species other than plants. Surprisingly, no similar genes are present in the sequenced genomes of cyanobacteria and among the currently annotated genes from algae, implying that TSP9 is a lately evolved protein, which is probably specifically involved in regulation of the photosynthetic process in plants. The possible absence of TSP9 in algae could give this phosphoprotein a unique place in the protein phosphorylation network of oxygenic photosynthetic organisms. Phosphorylation of PSII proteins is absent in cyanobacteria but occurs in plants and green algae (19). TSP9 could represent a distinction between the regulatory phosphorylation in algae and plants.

Neither the spinach TSP9 nor the corresponding putative proteins from other plants appear to contain any conventional functional domains. All computer programs for protein secondary structure prediction at the ExPASy server (http://us.expasy.org/tools/) predict a random coil structure for most of the TSP9 sequence, especially for its central, most conserved part. The protein is hydrophilic and does not contain any transmembrane regions. A characteristic feature of TSP9 is the high isoelectic point, 9.8, which is rather unusual for thylakoid proteins and more comparable to that of the proteins interacting with nucleic acids. The positive charge of TSP9 under physiological conditions favors an electrostatic interaction with the surface of the thylakoid membrane, which has an overall negative charge (40). Any distinct partners interacting with TSP9 in the photosynthetic membrane have yet to be found. The neutralization of the total positive charge on light-induced phosphorylation of TSP9 could contribute to the release of the protein from the membrane. The dephosphorylation of the released TSP9 by soluble protein phosphatases in the chloroplast stroma compartment would lead to reverse association of the protein with the thylakoid membrane. We found three phosphorylated forms of TSP9 released from the thylakoids by the high-salt wash. In this context, the observed partial release of TSP9 during the light-induced phosphorylation is more consistent with the discharge of only the highly triply phosphorylated form of the protein from the membrane. Thus, along with the basic nature of TSP9, its multiple phosphorylation leading to a change in the localization resembles the characteristics of transcription/translation regulatory factors (41, 42).

Like other thylakoid phosphoproteins, TSP9 is phosphorylated at threonine residues by the redox-dependent protein kinase(s) after their activation either by light (23, 24, 36) or by reduction of the membrane plastoquinone pool in darkness (6). However, TSP9 differs significantly from all previously characterized thylakoid phosphoproteins and is, to our knowledge, the first characterized soluble thylakoid phosphoprotein, whereas all of the other phosphoproteins are integral membrane polypeptides. Also, TSP9 is the most heavily phosphorylated thylakoid polypeptide with three phosphorylation sites. Most thylakoid phosphoproteins are singly phosphorylated, and only the 9-kDa PsbH gene product has two phosphorylation sites (12, 43). The previously characterized thylakoid phosphoproteins constitute the major photosynthetic protein complexes (12, 13, 20–22), and the phosphorylation sites in analogous proteins from different plants are rather conserved (19). Comparison of the protein sequences deduced from ESTs of 20 different plants with spinach TSP9 does not show a high conservation for the phosphorylated threonine residues. This is yet an additional indication for the possible regulatory function of TSP9, because molecular details for regulation of gene expression are very diverse in different plant species and even cell types (41). The distinct properties of TSP9, including its phosphorylation and release from the membranes on illumination, make this protein a unique candidate for a role of a redox-mediating signaling factor.

The fate of TSP9 in the soluble compartment of chloroplasts remains to be revealed. However, the basic nature of the protein may determine binding to RNA or DNA molecules. We speculate that TSP9 could be a plant-specific transcription or translation regulatory factor that is released from the thylakoid membranes in response to reduction of the plastoquinone pool, which activates the membrane protein kinases. Importantly, the regulation of expression for both chloroplast- and nuclear-encoded photosynthetic genes by redox signals from thylakoid membranes has been well documented (41, 44–46). This regulation depends on the redox state of the plastoquinone pool in the photosynthetic membrane (45, 46) and on the activation of the membrane protein kinases (41, 47). As a result, plants adjust the stoichiometry of the two photosystems and the size of their light-harvesting antennae, thereby acclimating to changes in environmental light quality and intensity (41, 45). The possible existence of a protein factor that could be released from thylakoids after light-induced phosphorylation and that could connect the gene expression to the redox status of the plastoquinone pool has been proposed (44). The plant-specific protein TSP9 is the first apparent candidate, to our knowledge, for a role of such a signaling factor.

Abbreviations

PSII

photosystem II

CP43

chlorophyll a binding protein of PSII

D1 and D2

photosystem II reaction center proteins

FTR

ferredoxin-thioredoxin reductase

MALDI

matrix-assisted laser desorption/ionization

TOF

time-of-flight

TSP9

thylakoid soluble phosphoprotein of 9 kDa