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Originally published In Press as doi:10.1074/jbc.M505729200 on July 22, 2005

J. Biol. Chem., Vol. 280, Issue 39, 33679-33686, September 30, 2005
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STN8 Protein Kinase in Arabidopsis thaliana Is Specific in Phosphorylation of Photosystem II Core Proteins*

Julia P. Vainonen1, Maria Hansson, and Alexander V. Vener2

From the Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden

Received for publication, May 25, 2005 , and in revised form, July 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reversible protein phosphorylation is a key molecular mechanism regulating all aspects of physiology and development in eukaryotic cells. The genome sequencing of Arabidopsis thaliana has uncovered more than 1160 genes encoding for the protein kinases and phosphatases in this model plant (1), highlighting a new challenge in revealing of the substrates and functions for these catalysts of reversible protein phosphorylation. The task of identification of in vivo substrate specificities for individual protein kinases and phosphatases in Arabidopsis thaliana has recently became feasible because of two groundbreaking developments. First, the plant lines with knockouts of individual genes became public and commercially available (2). Second, new mass spectrometry-based analytical techniques permitted identification and mapping of in vivo phosphorylation sites in numerous proteins (as reviewed in Ref. 3). For the first time these technical advancements provide the possibility of unraveling the complex protein phosphorylation network in plant photosynthetic membranes, which regulates the adaptation of the photosynthetic apparatus and efficient energy utilization in response to light quality and intensity, ambient temperature, circadian rhythm, nutrient deficiency, and other stresses (4-6).

The major proteins undergoing reversible phosphorylation in the photosynthetic thylakoid membranes belong to photosystem II (PSII)3 and its light-harvesting antennae proteins LHCII (6-11). Phosphorylation status of these proteins is differentially regulated by light (12), redox state of the membrane electron carriers (13-15), the ferredoxin-thioredoxin system in chloroplasts (16, 17), temperature (18, 19), and endogenous circadian rhythm (20). Phosphorylation and dephosphorylation of PSII core proteins D1, D2, CP43, and PsbH ensures sustained functioning of this photosystem (4, 6). PSII is a subject for photoinhibition (21, 22), which is directly proportional to light intensity (23) and occurs because of inactivation of the D1 reaction center protein (24). This central functional subunit of PSII undergoes a very fast turnover (25, 26) that necessitates the repair cycle of PSII and depends on the phosphorylation status of D1 (27-29). Phosphorylation of PSII proteins at high irradiances controls stability of the photosystem, whereas stepwise dephosphorylation of CP43, D2, and D1 proteins is part of a repair cycle during lateral migration of PSII in the membrane and with its disassembly followed by degradation of photodamaged D1, which occurs only after complete dephosphorylation of this protein (4, 30). Reversible phosphorylation of LHCII is involved in the redistribution of light excitation energy between the two photosystems in the process called state transitions (5, 10, 31).

Five protein kinases associated with the photosynthetic membranes have been implicated in phosphorylation of thylakoid proteins in Arabidopsis thaliana: the family of three thylakoid-associated kinases (TAKs) (32, 33), and two kinases STN7 and STN8 (31) that have significant sequence identity with chloroplast protein kinase Stt7 from the green alga Chlamydomonas reinhardtii (10). Using genetic approaches in the green alga and T-DNA insertion lines in Arabidopsis, the Stt7 and STN7 kinases, respectively, were found essential for phosphorylation of LHCII and the photosynthetic state transitions (10, 31). Knock-out of STN7 in Arabidopsis abolished phosphorylation of LHCII, as judged by the assay with anti-phosphothreonine antibody and by radioactive labeling of thylakoid proteins (31). The growth of the stn7 mutant was not different from wild type at the normal growth conditions, but was slower when plants were exposed to changing light conditions. Neither phenotypic difference from the wild type nor change in LHCII phosphorylation was found for the plants with T-DNA insertions in the stn8 gene (31), leaving the function and substrate specificity of STN8 kinase unclear.

In this paper we present a comparative analysis of thylakoid protein phosphorylation in stn8 mutant lines and wild type Arabidopsis plants by mass spectrometry using the approach of relative quantification of phosphorylated peptides labeled with stable isotopes. We demonstrate that STN8 protein kinase is involved in phosphorylation of PSII core proteins but not in LHCII phosphorylation. We reveal that STN8 is a light-dependent protein kinase because it is essential for phosphorylation of PsbH protein at the Thr-4 position, which is strictly light-dependent. The in vivo substrate specificity of STN8 is found restricted toward the very N-terminal threonine residues of the membrane proteins and only the phosphorylation-primed N terminus of PsbH. Modeling of the structure for the STN8 catalytic domain shows extended peptide loops oriented toward the entrance in the protein kinase active site surrounded by basic amino acids, which can explain rather unique selectivity of STN8 to the protein substrates in the photosynthetic membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material—The seeds from A. thaliana wild type (ecotype Columbia) and two T-DNA insertion lines in Columbia background At5g01920 gene stn8 (SALK 060869 and SALK 064913, obtained from Salk Institute collection) were germinated and then cultivated hydro-ponically (34) for 12 weeks with a 8-h light/16-h dark photoperiod at photon flux density of 120 µmol m-2 s-1. Plants homozygous for the T-DNA insertion were identified based on PCR analysis with specific primers for stn8: 5'-TTGAGCTTCTCTGCAGCTTTG-3', forward; 5'-CTTGACCTGCGTTGGTTGATA-3', reverse; and T-DNA Left Border 5'-GCGTGGACCGCTTGCAACT-3'.

Extraction of RNA and RT-PCR Analysis—Total RNA of frozen leaf tissues was extracted with TRIzol (Invitrogen). After RNase-free DNase treatment, 1 µg of total RNA was used to synthesize cDNA using Super-Script III reverse transcriptase (Invitrogen) in a 40-µl reaction volume. 4 µl (1/10) of RT product was used for PCR amplification with STN8-specific and 18 S RNA control primers. The forward and reverse primers, respectively, for the 18 S RNA were 5'-CTGCCAGTAGTCATATGCTTGTC-3' and 5'-GTGTAGCGCGCGTGCGGCCC-3'. The forward and reverse primers, respectively, for the STN8 were 5'-AGATGGCCTCTCTTCTCTCTCC-3' and 5'-CCCGCCACCTATTAAGATCA-3'.

Preparation of Thylakoid Membranes—The thylakoid membranes were isolated from 2 g of Arabidopsis leaves harvested either in the daytime (4 h after the light was turned on) or in night (15 h after the light was turned off) essentially according to the protocol (35) in the presence of 10 mM NaF to inhibit protein phosphatases. The isolated thylakoids were washed once with 50 mM Tricine, pH 7.8, 100 mM sorbitol, 5 mM MgCl2, 10 mM NaF, and twice with 25 mM NH4HCO3, 10 mM NaF.

SDS-PAGE and Immunoblotting—Thylakoid membrane proteins were separated by SDS-PAGE (15% acrylamide) and stained with Coomassie Blue. For immunoblotting the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon). The membranes were then blocked with bovine serum albumin or milk, incubated with rabbit anti-phosphothreonine antibodies purchased either from New England Biolabs or from Zymed Laboratories Inc. (36), or with specific antibodies against D1 (amino acids 234-242 in Synechocystis protein) and D2 (amino acids 230-245 in Synechocystis protein), which were kindly provided by Eva-Mari Aro (Turku University). The blots were incubated with horseradish peroxidase-conjugated secondary antibody, developed using ECL system (Amersham Biosciences) and evaluated by chemiluminescence imaging (LAS-1000, Fuji) or using the standard procedure with x-ray films.

Cleavage of the Surface-exposed Peptides from the Thylakoids—The isolated thylakoids were resuspended in 25 mM NH4HCO3, 10 mM NaF to a final concentration of 2.5 mg of chlorophyll/ml and incubated with sequencing grade-modified trypsin (Promega) (5 µg of enzyme/mg of chlorophyll) for 3 h at 22°C. The digestion products were frozen, thawed, and centrifuged at 15,000 x g. The supernatant was collected while the membranes were resuspended in water and centrifuged again. Both supernatants containing released thylakoid peptides were pooled and centrifuged at 100,000 x g.

Preparation of Peptide Methyl Esters—The peptides released by trypsin from the thylakoids isolated from the wild type or stn8 plants were lyophilized and methyl-esterified with 2 N methanolic HCl or DCl. The reagent was prepared by dropwise addition of 80 µl of acetylchloride (Aldrich) to 500 µl of anhydrous d0-methyl alcohol (Aldrich) or d3-methyl d-alcohol (Aldrich) with stirring. 250 µl of the reagent was added to lyophilized peptide digest prepared from thylakoids that contained 50 µg of chlorophyll. The reaction was carried out for 2.5 h at room temperature, and the solvent was then lyophilized. The samples were reconstituted in 20 µl of water, acetonitrile, methanol (1:1:1) and equal aliquots of two differentially methylated samples were mixed as specified under "Results." The solvent was removed by evaporation under vacuum.

Isolation of Phosphopeptides—Phosphopeptides were enriched by IMAC as described (37). The IMAC procedure was modified as follows. The microcolumns containing 7.5 µl of chelating Sepharose (Amersham Biosciences) were prepared in GELoader tips (Eppendorf) and loaded with 80 µl of 0.1 M FeCl3. Unbound iron ions were removed by washing with 2x 20 µl of 0.1% (v/v) acetic acid. The mixture of methylated peptides in 5 µl of methanol, water, and acetonitrile (1:1:1) was loaded onto the column, which was washed with 2x 20 µl of 0.1% acetic acid in water, 2x 20 µl of 0.1% acetic acid in 20% acetonitrile, and 2x 20 µl of 20% acetonitrile. Phosphopeptides were eluted by 40 µl of 20 mM Na2HPO4 with 20% acetonitrile and desalted using C18 ZipTip (Millipore).

Electrospray Ionization Mass Spectrometry—The spectra were acquired on a hybrid mass spectrometer API QSTAR Pulsar i (Applied Biosystem) equipped with a nanoelectrospray ion source (MDS Protana). The nanoelectrospray capillaries were loaded with 2 µl of peptide solution in 50% acetonitrile, 1% formic acid in water. Mass spectra were acquired with instrument settings recommended by Applied Biosystems. Signals for methyl esters of phosphopeptides obtained after IMAC of isotopically labeled peptide mixtures appeared as doublets separated by n(3 Da)/z, were n is the number of carboxylic groups in the peptide and z is the charge on the peptide. The difference in intensity of two signals in each doublet reflected the ratio in phosphorylation of a particular site between the wild type and stn8 plants.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Chloroplast thylakoid protein kinase STN8 (31) is encoded by the At5g01920 gene. To determine the function of this kinase, two independent Arabidopsis lines with T-DNA insertions in the first exon of this gene (Fig. 1A) were obtained from the Salk Institute collection (2). These insertion lines were identical to those described in Ref. 31. In this paper we mark the mutants as stn8-1 (SALK 060869) and stn8-2 (SALK 064913). After self-crosses, homozygous plants for each T-DNA insertion were selected on the basis of PCR on genomic DNA by using corresponding primers, as shown in Fig. 1B. To verify the lack of STN8 transcripts in the mutant plants we made RT-PCR analysis of mRNA from the mutant and wild type plants. Fig. 1C shows that the STN8 transcript was present only in wild type Arabidopsis, whereas 18 S RNA, used as a positive control, was present in both mutants and wild type plants. No PCR products were observed in the negative control reactions with template non-reverse transcribed RNA, which ruled out a possible amplification from contaminating genomic DNA (data not shown). In accordance with Ref. 31 there was no difference in phenotype between mutant and wild type plants under normal growth conditions. To monitor the changes in the endogenous phosphorylation of thylakoid membrane proteins induced by the absence of STN8 we performed immunoblotting analyses using two different anti-phosphothreonine antibodies (36). Thylakoid membranes were isolated in parallel from leaves of the wild type, stn8-1, and stn8-2 mutants harvested after 15 h of dark period or 4 h into light period. Immunoreactivity with two different commercial antibodies differs for various phosphoproteins from the thylakoid membranes, as shown by representative Western blots in Fig. 2, A and B, and as has also been shown previously (12, 36). Disruption of stn8 did not affect the level of LHCII phosphorylation and its changes during growth dark-light transition in Arabidopsis (Fig. 2, A and B). This is in agreement with the fact that STN7, but not the STN8 protein kinase is responsible for LHCII phosphorylation (31). However, we observed that phosphorylation of D1, D2, and CP43 proteins of PSII core was reduced in the stn8 mutants in comparison with the wild type. Phosphorylation of D1 and/or D2 was lower in the mutant leaves harvested both in darkness or light, whereas reduced phosphorylation of CP43 in the mutants, as compared with the wild type, was obvious only in the light-exposed leaves (Fig. 2, A and B). In agreement with the previous reports (12, 36), neither of the two anti-phosphothreonine antibodies (Fig. 2, A and B) recognized phosphorylated PsbH protein of PSII core (7, 9).



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  		FIGURE 1.
Characterization of homozygous stn8 Arabidopsis mutants. A, schematic presentation of the localization of two different T-DNA insertions in the At5g01920 gene (thick lines represent exons; thin lines represent introns), of the primers used for the PCR analysis with genomic DNA (short arrows numbered 1-3) and in RT-PCR (short arrows numbered 4 and 5), and domain organization of the STN8 protein kinase encoded by the At5g01920 gene. B, ethidium bromide-stained gel with the products of PCR with genomic DNA isolated from homozygous mutant lines and wild type as a control. 1, PCR with T-DNA Left Border and the gene-specific primer; 2, PCR with gene-specific primers. C, ethidium bromide-stained gel with RT-PCR products showing no STN8 transcript in homozygous mutant lines and the presence of 18 S rRNA in both mutant lines and wild type (WT).

 



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  		FIGURE 2.
Phosphorylation of thylakoid membrane proteins in the leaves of stn8 mutants and wild type (WT) Arabidopsis. Thylakoids were isolated at the end of dark period (D) or after 4 h of light (L), separated by SDS-PAGE, and immunoblotted with anti-phosphothreonine antibody from Zymed Laboratories Inc. (A) or New England Biolabs (B). C, immunoblot analysis with anti-phosphothreonine antibody (New England Biolabs) of thylakoid membranes before (-) and after (+) trypsin treatment. D, Coomassie-stained gel of the same samples as in C.

 
For further characterization of thylakoid membrane protein phosphorylation in stn8 mutants of Arabidopsis, we used mass spectrometry coupled with differential stable isotopic labeling and affinity chromatography to quantify phosphorylation changes (38, 39). Thylakoid membranes were isolated in parallel from the stn8 mutants and wild type plants in the presence of NaF that inhibits dephosphorylation of thylakoid proteins (40, 41). The isolated thylakoids were resuspended to equal chlorophyll concentration and subjected to proteolytic shaving of the surface-exposed phosphopeptides from the membranes by trypsin (7). The released peptide mixtures were separated from the rest of the membranes by centrifugation. Immunoblotting analysis of the membranes confirmed complete removal of the phosphothreonine-containing domains from the membrane proteins after trypsin treatment of thylakoids from both mutant and wild type plants (Fig. 2C). A Coomassie-stained gel also showed identical patterns of the membrane proteins before and after shaving of the mutant and wild type thylakoids with trypsin (Fig. 2D). Thus, we have obtained the mixtures of peptides released from the surface of stn8-1, stn8-2, and wild type thylakoids that all had the same chlorophyll and protein content. As the next step we performed the differential labeling of the peptides from the mutant and wild type thylakoids by parallel esterification of peptide carboxylic groups either by hydrogen- or deuterium-containing methanol (38, 39). The tryptic peptides from the wild type thylakoids were esterified with d0-methanol (modification of each carboxyl group gives a peptide mass increment of 14 Da), whereas the peptides from the mutant thylakoids were esterified with d3-methanol (modification of each carboxyl group gives a peptide mass increment of 17 Da). Light isotope-labeled peptides from wild type thylakoids were mixed 1:1 with heavy isotope-labeled peptides from stn8-1 thylakoids. The same procedure was done for the peptides from the wild type and stn8-2 thylakoids. In both cases we performed reverse labeling of the wild type and mutant peptides as an internal control. The methyl esters of the phosphorylated peptides were then captured and enriched by IMAC, separately from the four resulting mixtures of peptide methyl esters. The phosphorylated peptides enriched by each round of IMAC were analyzed by electrospray ionization MS in positive ionization mode, which allowed simultaneous measurements of intensities for light and heavy isotope-labeled phosphopeptide pairs and quantitative comparison of the phosphorylation differences in the mutant and wild type proteins. These analyses revealed a specific decrease in PSII core protein phosphorylation because of the loss of the stn8 gene in Arabidopsis.

Fig. 3A shows a part of the mass spectrum with the doublet signals corresponding to singly protonated ions of N-terminal phosphopeptide Ac-tAILER from the D1 protein (852.4, light form from the wild type, and 858.4, heavy form from stn8-1 mutant) and to N-terminal phosphopeptide Ac-tIALGK from the D2 protein (738.4, light form from the wild type, and 741.4, heavy form from the stn8-1 mutant). These peptides were prepared from the light-exposed leaves of the plants. Fig. 3B shows the spectrum obtained from the reverse labeling experiment for these peptides from the mutant and wild type. Both spectra (Fig. 3, A and B) demonstrate 50 to 60% decrease of D1 and D2 phosphorylation in the stn8-1 mutant. Similar results were obtained in the experiments with stn8-2 mutant (see the summary of the results in TABLE ONE). When the peptides were prepared from the plant leaves harvested at the end of the night period, the quantitative MS measurements also revealed analogous reduction in the phosphorylation of D1 and D2 because of the loss of STN8 (Fig. 3C, for the straight labeling, and Fig. 3D, for the reverse labeling). The phosphorylation level of D1 protein in stn8 plants corresponds to 53% of that for the wild type during the light period and to 63% after the dark period (Fig. 3, TABLE ONE). The phosphorylation level of D2 protein in the mutant plants corresponds to 36 and 34% of wild type under light and dark, respectively (Fig. 3, TABLE ONE). The decrease found in D1 and D2 phosphorylation in stn8 mutants might be an artifact if the expression of these proteins in mutant plants is lower compared to wild type Arabidopsis. To exclude this possibility we performed immunoblotting analysis of thylakoid proteins with protein-specific antibody against D1 and D2. Fig. 3E shows that the level of D1 and D2 in stn8 Arabidopsis is the same as in wild type. Thus, the difference found in the phosphorylation of D1 and D2 is caused by disruption of stn8 protein kinase gene. This conclusion is supported by both MS results (Fig. 3) and the results from Western blot analyses (Fig. 2, A and B).


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  		TABLE ONE
The phosphorylation ratios of PSII core proteins in stn8 mutants and wild type Arabidopsis

The mean values and deviations are calculated from the results of two to three separate MS analyses of each sample obtained in two independent IMAC isolations of the methylated phosphopeptides from each mixture of the stable isotope-labeled peptides.

 
Our MS analysis identified two phosphorylated tryptic peptides that originated from LHCII proteins. One peptide with the sequence Ac-RKtVAKPK, which has been characterized earlier (7), had m/z values of 532.3 and 533.8 for doubly charged ions of light and heavy isotope-methylated forms, respectively. Because of the high sequence similarity of the LHCII proteins this peptide could originate from the products of the following LHCII genes in Arabidopsis: At1g29910, At1g29920, At1g29930, and At2g34420. The second short phosphopeptide with the sequence Ac-RRtVK was found and sequenced from Arabidopsis for the first time and had m/z values of 398.2 and 399.7 for doubly charged ions of light and heavy isotope-methylated forms (data not shown). This peptide could originate from the products of At2g05070, At2g05100, and At3g27700 LHCII genes in Arabidopsis. Notably, we did not find any significant difference in the level of phosphorylation for both LHCII peptides in the light-exposed stn8 plants in comparison with the wild type (data not shown). This is in agreement with the results obtained using anti-phosphothreonine antibodies (Fig. 2, A and B) and with a previous report on phosphorylation of LHCII by STN7 but not by STN8 (31).



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  		FIGURE 3.
Relative quantitation of D1 and D2 protein phosphorylation in stn8 mutants and wild type (WT) plants by MS analysis of (d0/d3) esterified phosphopeptides. A and B, thylakoids were isolated from the leaves after 4 h of light exposure. C and D, thylakoids were isolated from the leaves after 15 h of darkness. The mass spectra show signals of the phosphorylated peptides from D1 and D2 proteins. The peptides were shaved from the surface of isolated thylakoids by trypsin and esterified with d0-methanol or d3-methanol. A and C, light isotope-labeled peptides from the wild type were mixed 1:1 with heavy isotope-labeled peptides from stn8-1. B and D, light isotope-labeled peptides from stn8-1 were mixed 1:1 with heavy isotope-labeled peptides from the wild type. The phosphorylated peptides were enriched by IMAC from the mixtures of the differentially labeled peptides, and mass spectra were acquired. The signals at m/z 852.4 and 858.4 correspond to the singly charged ions of light and heavy isotope-labeled peptides from D1, respectively. The signals at m/z 738.4 and 741.4 correspond to the singly charged ions of light and heavy isotope-labeled peptides from D2, respectively. amu, atomic mass units. E, immunoblot analysis of the thylakoids from wild type and stn8 mutant lines with the antibodies specific to D1 and D2 proteins. The amounts of chlorophyll in the samples loaded onto the gel are indicated.

 
The N-terminal tryptic phosphopeptide Ac-tLFNGTLALAGR (mass 1354.6 Da) from the CP43 protein of the PSII core undergoes partial deamidation, which produces peptide Ac-tLFDGTLALAGR (mass 1355.6 Da) (7). We found that during methyl esterification of the tryptic peptides from the stn8 and wild type plants additional deamidation of the peptide occurred, and the deamidated asparagines were not completely converted to methyl esters. This resulted in a mixture of peptides containing the same phosphorylation site but the m/z values for doubly charged ions corresponding to 685.3, 685.8, and 692.8 for the light isotope-labeled peptides and 686.8, 687.3, and 695.8 for heavy isotope-labeled peptides. Because of the overlapping of the signals for isotopic clusters of each of these peptide forms we were not able to make accurate comparative quantification of CP43 phosphorylation in the mutant and wild type plants (data not shown). Nevertheless, the tendency corresponded to decrease of CP43 phosphorylation in stn8 leaves exposed to light in comparison to that in the leaves from the wild type Arabidopsis. This tendency was in agreement with the reduced CP43 phosphorylation in the light-exposed leaves from the mutants, as determined by Western blotting (Fig. 2, A and B).

Two phosphorylation sites have been identified in the PsbH polypeptide: one at Thr-2 and another at Thr-4 (7). Phosphorylation of Thr-2 was found in both light- and dark-exposed leaves of Arabidopsis plants, whereas phosphorylation of the PsbH protein at Thr-4 was strictly light-dependent (7, 9). This site is also known to undergo rapid dephosphorylation in darkness (7). None of the phosphorylation sites in PsbH is recognized by anti-phosphothreonine antibodies (this work and Refs. 12 and 36). However, using mass spectrometry we characterized the difference between phosphorylation of these sites in PsbH from the wild type and stn8 leaves exposed to light. Fig. 4, A and B, show the signal doublet of doubly charged ions at m/z 608.3 (light labeled peptide) and 612.8 (heavy labeled peptide) corresponding to the phosphopeptide AtQTVEDSSR from PsbH with one phosphate group at Thr-2. Calculated signal ratios (TABLE ONE) indicate that phosphorylation of PsbH at Thr-2 is slightly decreased in the mutant lines. Doubly charged molecular ions at m/z 648.3 (light-labeled peptides) and 652.8 (heavy labeled peptides) corresponding to the phosphopeptide AtQtVEDSSR with an additional phosphate group at Thr-4 was detected only from the wild type thylakoids (Fig. 4, A and B, TABLE ONE). These results demonstrate that Thr-4 in PsbH is not phosphorylated in stn8 Arabidopsis during the light period. We also performed quantification of phosphopeptides released from the thylakoids isolated from the dark-exposed leaves. Thr-4 was not phosphorylated in either wild type or mutant thylakoids (Fig. 4, C and D), whereas the level of PsbH phosphorylation at Thr-2 in stn8 Arabidopsis was similar in the mutant and wild type leaves (Fig. 4, C and D, TABLE ONE). To prove that phosphorylation of PsbH in the mutants occurs only at Thr-2 we sequenced the doubly charged phosphorylated peptide with m/z 608.3 obtained from stn8 Arabidopsis. Fig. 5 shows a representative tandem mass spectrum of light isotope-labeled phosphopeptide AtQTVEDSSR from light-exposed leaves of a stn8 mutant plant. The clear fragment ions y8 and y7in the spectrum indicate that Thr-4 is not phosphorylated. The presence of the b3* fragment ion at m/z 283.1 corresponding to b3-H3PO4 indicates phosphorylation of the peptide at Thr-2. Thus we made two conclusions: first STN8 is essential for the phosphorylation of PsbH at Thr-4 and, second, STN8 is a light-dependent protein kinase because phosphorylation at the Thr-4 occurs only in light-exposed leaves of Arabidopsis.

The complementary data from the Western blotting analyses (Fig. 2, A and B) and mass spectrometric measurements (TABLE ONE) show that STN8 protein kinase in Arabidopsis is involved in phosphorylation of the very N-terminal threonine residues of D1, D2, and CP43 core proteins of PSII and is absolutely required for light-induced phosphorylation of the PsbH protein at Thr-4. Importantly, the latter phosphorylation can occur only after prior phosphorylation of PsbH at Thr-2 (7), obviously by the other protein kinase. The in vivo substrate specificity of STN8 differs completely from that of the highly homologous STN7, which phosphorylates only LHCII (31). To get insight into the reason for the distinct substrate specificity of STN8 we modeled the structure of its catalytic domain using the servers for automated protein modeling on the basis of the known protein kinase structures (www.embl-heidelberg.de/predictprotein, www.compbio.dundee.ac.uk/~www-jpred). The sequence of STN8 fits well into the conserved structures of protein kinases with two lobes embracing the active site: the N-terminal lobe dominated by {beta}-strands and the C-terminal lobe dominated by {alpha}-helixes. Importantly, we revealed that the major sequence differences between STN8 and STN7 catalytic domains in their N-terminal parts (Fig. 6) correspond to the extra long loops connecting {beta}-strands and oriented toward the entrance into the active site of STN8. Moreover, the BLAST data base search (42) shows that this part of the STN8 sequence is homologous to the N-terminal consensus of the protein-tyrosine kinase subfamily (Fig. 6). In the protein-tyrosine kinases the homologous long loops oriented toward the entrance into the active site restrict access of serine and threonine residues, which are shorter than tyrosine. Thus, the restriction of the STN8 substrate specificity toward only very N-terminal residues in the D1, D2, and CP43 proteins and toward already pre-phosphorylated at Thr-2 N terminus of PsbH could be explained by the limiting accessibility of the kinase active site, which is discussed further below.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work we determined that in vivo substrate specificity of STN8 protein kinase in A. thaliana is directed toward the PSII core proteins. This information has been obtained by comparison of endogenous thylakoid protein phosphorylation levels in STN8 knock-out mutants with that in the wild type plants. All results were similar for two independent Arabidopsis lines with T-DNA insertions in the stn8 gene, ensuring that the observed effects on thylakoid protein phosphorylation were because of the stn8 disruption in the mutant plants. To compare the differences in protein phosphorylation levels in the leaves of mutant and wild type plants we used two techniques: immunological, based on anti-phosphothreonine specific antibodies (12, 36); and quantitative MS method, based on stable isotope labeling of phosphorylated peptides, their enrichment by IMAC and mass spectrometric analysis (38, 39). The use of anti-phosphothreonine antibodies is limited to the number of detectable proteins and inability to detect which site undergoes differential phosphorylation in the case of multiple phosphorylations within the protein. Nevertheless, by this method we determined reduction in phosphorylation of D1, D2, and CP43 proteins in the leaves of the mutants, when compared with the wild type. Phosphorylation of D1 and D2 was reduced in both dark- and light-exposed mutant leaves, whereas CP43 phosphorylation was lower only in the light-exposed mutant leaves. MS-based methodology determined that stn8 disruption caused a 50-60% decrease in phosphorylation of D1 and D2 at their very N-terminal threonine residues and completely abolished phosphorylation of PsbH at Thr-4. We also confirmed that phosphorylation of PsbH at Thr-4 in the wild type plants (by STN8) is strictly light-dependent (7). Both immunological and MS techniques confirmed the recent report, which stated that STN8 deficiency did not affect phosphorylation of LHCII proteins (31). Our data conclude that: (i) STN8 is specific in phosphorylation of D1, D2, and CP43 proteins at their N-terminal threonines; (ii) STN8 is essential for phosphorylation of PsbH at Thr-4; (iii) STN8 is the protein kinase activated by light; (iv) there are other protein kinases, besides STN8, in Arabidopsis that phosphorylate N-terminal threonines of D1, D2, and CP43 as well as Thr-2 in PsbH; (v) there is a cross-talk between thylakoid protein kinases at the substrate level: the light-stimulated phosphorylation of Thr-4 by STN8 requires prior phosphorylation of Thr-2 by another kinase.



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  		FIGURE 4.
Relative quantitation of PsbH protein phosphorylation in stn8 mutants and wild type plants by MS analysis of (d0/d3) esterified phosphopeptides. A and B, thylakoids were isolated from the leaves after 4 h of light exposure. C and D, thylakoids were isolated from the leaves after 15 h of darkness. The mass spectra show signals of the doubly charged ions of the monophosphorylated peptide AtQTVEDSSR (608.3, light isotope-labeled; 612.8, heavy isotope-labeled) and doubly phosphorylated peptide AtQtVEDSSR (648.3, light isotope-labeled; 652.8, heavy isotope-labeled). A and C, light isotope-labeled peptides from wild type were mixed 1:1 with the heavy isotope-labeled peptides from stn8-2 before IMAC. B and D, light isotope-labeled peptides from stn8-2 were mixed 1:1 with the heavy isotope-labeled peptides from wild type before IMAC.

 



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  		FIGURE 5.
Collision-induced fragmentation product ion spectrum showing that the PsbH protein in the stn8 mutant is phosphorylated only at Thr-2. The selected doubly charged peptide ion with m/z 608.3, which has been subjected to collision-induced fragmentation, is labeled in the spectrum along with the fragment ion at m/z 559.3 produced after characteristic neutral loss of phosphoric acid (98 Da). The detected y (C-terminal) and b (N-terminal) ions are indicated in the spectrum as well as in the corresponding amino acid sequence. The ions marked with an asterisk indicate that the fragments underwent neutral loss of 98 Da (H3PO4). The lowercase t indicates a phosphorylated threonine residue at position 2 in the peptide.

 
The fact that there are a few protein kinases involved in phosphorylation of PSII core proteins (does not concern Thr-4 in PsbH) is not surprising and also explains the absence of the obvious phenotype in the stn8 mutant plants grown at normal conditions (31). Reversible phosphorylation of PSII proteins has a crucial importance for the turnover and sustained functioning of this photosystem (4, 6, 27, 43). Existence of at least two protein kinases involved in phosphorylation of PSII core proteins should assure the robust regulatory mechanism controlling this photosystem and required for plant survival. The other kinase phosphorylating PSII in A. thaliana is TAK1, which has been shown by antisense TAK1 expression (33). All five protein kinases found in thylakoid membranes of Arabidopsis, three TAKs (32, 33), STN7 and STN8 (10, 31), are probably differentially regulated. Some of them are regulated by light and redox conditions (31-33). It has also been shown that phosphorylation of the D1 protein in higher plant Spirodela oligorrhiza is under control of an endogenous circadian rhythm (20). We found that STN8 is responsible for the daylight-induced phosphorylation of Thr-4 in PsbH protein. It should be noted that phosphorylation of PsbH at Thr-4 is the only dramatic change in phosphorylation of PSII in A. thaliana during normal night-day transitions, because phosphorylation of D1, D2, and CP43 proteins in the daytime is increased by only 5-10% (7). However, we observed the decrease in D1, D2, and CP43 phosphorylation from the daylight-exposed leaves of the stn8 mutants, as compared with the wild type. These data together with a clear-cut abortion of the light-induced phosphorylation of Thr-4 in PsbH in the stn8 mutants show that STN8 is responsible for the daylight-induced phosphorylation of PSII core proteins in A. thaliana.

TAK kinases in Arabidopsis and Stt7 kinase (the ortholog of STN7 and STN8) in C. reinhardtii have been found to undergo reversible phosphorylation, and the existence of a cascade of kinases in thylakoid protein phosphorylation has been proposed (10, 32). The contemporary techniques did not allow mapping of the phosphorylated sites in these kinases and revealing of the enzymes responsible for these modification. Accordingly, there is a possibility that involvement of the STN8 kinase in phosphorylation of PSII proteins could be mediated by other kinases, as well as there could be STN8 substrates that have not been found in the present work. We found that there is a cross-talk of kinases in Arabidopsis because of sequential phosphorylation of PsbH at Thr-2 and Thr-4 by STN8 and another kinase. In the case of D1 and D2 phosphorylation the kinase cross-talk is obviously more complex, because the sustained reduction in phosphorylation of these proteins in stn8 mutant plants, as compared with the wild type, was found during day and night cycles despite the presence of the other protein kinase able to phosphorylate the same sites in D1 and D2. The cross-talk of the enzymes may also include protein phosphatases, which together with protein kinases control phosphorylation levels of PSII proteins (18, 41, 44).



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  		FIGURE 6.
Sequence comparison of the catalytic domains of STN8 and STN7 protein kinases and N-terminal consensus sequence for protein-tyrosine kinase. The alignment of STN7 and STN8 was made by the ClustalW program (50), the N-terminal consensus sequence for tyrosine kinases TyrKc (gnl|CDD|24220 smart00219 in NCBI Conserved Domain Search (51)) was aligned according to the results of the BLAST search (42). The amino acids in the sequences of STN7 and STN8 are numbered corresponding to the translation products of stn7 and stn8. Black and gray boxes highlight identical and similar amino acids, respectively. The amino acids in the active sites of STN7 and STN8 are boxed. The stretches of "e" and "h" above the sequences designate {beta}-strands and {alpha}-helixes predicted for STN8 at the PredictProtein server (www.embl-heidelberg.de/predictprotein). The arrows point to the additional basic amino acids around the active site of STN8, as compared with STN7.

 
STN7 and STN8 are two highly homologous protein kinases in A. thaliana (10). Both STN7 (31) and STN8 (present work) are activated in thylakoid membranes by light. However, the molecular mechanism of this activation is not presently understood. The light and redox activation of thylakoid protein phosphorylation is controlled by the binding of reduced plastoquinole in the quinol-oxidizing site of the cytochrome bf complex at the luminal side of the thylakoid membrane (13-15, 45, 46). Both STN7 and STN8 have protein kinase domains exposed to the stroma of chloroplast, single transmembrane span, and N-terminal stretch exposed into the thylakoid lumen. The luminal extensions of STN7 and homologous Stt7 protein kinase in C. reinhardtii contain two cysteine residues that were implied in a possible redox regulation of these kinases (10). The luminal extension of STN8 does not contain a similar couple of cysteine residues. Interestingly, the NCBI-BLAST protein data base search (42) finds that 40 N-terminal amino acid residues of mature STN8 exposed to the thylakoid lumen are homologous to the stretch in Arabidopsis oxidoreductase from the 2OG-Fe(II) oxygenase family (accession NP_850613 [GenBank] ). Unfortunately, the structure of this oxidoreductase is presently unknown, as well as the role of the amino acid sequence, homologous to STN8, in the interaction with the enzyme redox components. We can speculate that the lumen-exposed part of STN8 can be involved in sensing of the occupancy of the quinol-oxidizing site of the cytochrome bf complex. This hypothesis can be verified in the future by the deletion/mutation of the corresponding N-terminal part of STN8 and monitoring of the ability of the enzyme to perform light-induced phosphorylation of PsbH at Thr-4.

STN7 protein kinase in Arabidopsis is responsible for phosphorylation of LHCII (31). We found two phosphorylated peptides, Ac-RKtVAKPK and Ac-RRtVK, that could correspond to the N termini of LHCII proteins encoded by seven different genes in Arabidopsis. The phosphorylated threonine residues in these peptides are surrounded by the basic lysine and arginine residues, which could be a signature for the substrate specificity of STN7. On the contrary, STN8 does not phosphorylate these sites in LHCII proteins. We found that STN8 has rather peculiar substrate specificity restricted to the very N-terminal threonines of D1, D2, and CP43, as well as to the Thr-4 in PsbH, but only if this protein has already been phosphorylated by the other protein kinase at Thr-2. The difference in the substrate specificity between STN7 and STN8 should be in the structure of their protein kinase domains. The most significant difference between STN7 and STN8 is in the ATP-binding N-terminal part of their catalytic domains, which are aligned as shown in Fig. 6. Surprisingly, this part of the STN8 sequence has a high homology to a consensus of a subfamily of protein-tyrosine kinases (TyrKc) with the known structure (47). The structural basis for selectivity of tyrosine kinases is in the peptide loops surrounding the active site and obstructing access of the shorter serine and threonine residues of the substrate proteins (48). According to the prediction of the STN8 structure, the N-terminal insertions in STN8 and TyrKc, as compared with STN7, correspond to the loops oriented toward the entrance into the active site of the protein kinase. This may explain the specificity of STN8 toward the very N-terminal threonines of D1, D2, and CP43: the N-terminal residues should have easier access into the kinase active site than the residues inside the protein sequences. The other peculiarity in the specificity of STN8 is phosphorylation of Thr-4 in PsbH already pre-phosphorylated at Thr-2. Similar phosphorylation-dependent substrate specificity has earlier been found and structurally explained for casein kinase-1 (49). The phosphorylation-dependent specificity of this enzyme was assigned to the additional basic residues in the loops C-terminal adjacent to the active site of the casein kinase-1, as was compared with cAMP-dependent protein kinase and cyclin-dependent kinase 2 (49). The alignment in Fig. 6 shows that STN8 has seven additional basic residues in the loops around the active site, as compared with STN7. Importantly, four of these basic residues in STN8 correspond to the acidic residues in STN7, which probably may be a reason for STN7 substrate specificity toward the threonines surrounded by basic residues in LHCII. These structural considerations based on our experimental determination of the in vivo substrate specificity of STN8 can explain the drastic difference in the substrate selectivity of two highly homologous light-activated protein kinases, STN7 and STN8, in the photosynthetic membranes of A. thaliana.


   FOOTNOTES
 
* This work was supported by grants from the Swedish Research Council for Environment, Agriculture and Space Planning (Formas), Nordiskt Kontaktorgan för Jordbruksforskning, and the Swedish Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Biology, University of Turku, FIN-20014 Turku, Finland. Back

2 To whom correspondence should be addressed: Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden. Tel.: 46-13-224050; Fax: 46-13-224314; E-mail: aleve{at}ibk.liu.se.

3 The abbreviations used are: PSII, photosystem II; Ac-, acetyl in the N terminus of the peptide; IMAC, immobilized metal affinity chromatography; t, phosphorylated threonine residue in the peptide sequences; LHCII, light harvesting chlorophyll a/b-binding proteins of photosystem II; MS, mass spectrometry; RT, reverse transcriptase; TAK, thylakoid-associated kinases; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back


   ACKNOWLEDGMENTS
 
We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and Eva-Mari Aro for antibodies against D1 and D2 proteins.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. The Arabidopsis Genome Initiative (2000) Nature 408, 796-815[CrossRef][Medline] [Order article via Infotrieve]
  2. Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C. C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D. E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W. L., Berry, C. C., and Ecker, J. R. (2003) Science 301, 653-657[Abstract/Free Full Text]
  3. Loyet, K. M., Stults, J. T., and Arnott, D. (2005) Mol. Cell. Proteomics 4, 235-245[Abstract/Free Full Text]
  4. Aro, E.-M., and Ohad, I. (2003) Antioxid. Redox Signal. 5, 55-67[CrossRef][Medline] [Order article via Infotrieve]
  5. Allen, J. F. (2003) Science 299, 1530-1532[Abstract/Free Full Text]
  6. Vener, A. V. (2005) in Photoprotection, Photoinhibition, Gene Regulation, and Environment (Demmig-Adams, B., Adams, W., and Mattoo, A. K., eds) pp. 107-126, Springer, The Netherlands
  7. Vener, A. V., Harms, A., Sussman, M. R., and Vierstra, R. D. (2001) J. Biol. Chem. 276, 6959-6966[Abstract/Free Full Text]
  8. Gomez, S. M., Nishio, J. N., Faull, K. F., and Whitelegge, J. P. (2002) Mol. Cell. Proteomics 1, 46-59[Abstract/Free Full Text]
  9. Hansson, M., and Vener, A. V. (2003) Mol. Cell. Proteomics 2, 550-559[Abstract/Free Full Text]
  10. Depege, N., Bellafiore, S., and Rochaix, J. D. (2003) Science 299, 1572-1575[Abstract/Free Full Text]
  11. Turkina, M. V., Villarejo, A., and Vener, A. V. (2004) FEBS Lett. 564, 104-108[CrossRef][Medline] [Order article via Infotrieve]
  12. Rintamäki, E., Salonen, M., Suoranta, U. M., Carlberg, I., Andersson, B., and Aro, E.-M. (1997) J. Biol. Chem. 272, 30476-30482[Abstract/Free Full Text]
  13. Vener, A. V., Van Kan, P. J., Gal, A., Andersson, B., and Ohad, I. (1995) J. Biol. Chem. 270, 25225-25232[Abstract/Free Full Text]
  14. Vener, A. V., Van Kan, P. J. M., Rich, P. R., Ohad, I., and Andersson, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1585-1590[Abstract/Free Full Text]
  15. Zito, F., Finazzi, G., Delosme, R., Nitschke, W., Picot, D., and Wollman, F. A. (1999) EMBO J. 18, 2961-2969[CrossRef][Medline] [Order article via Infotrieve]
  16. Carlberg, I., Rintamäki, E., Aro, E.-M., and Andersson, B. (1999) Biochemistry 38, 3197-3204[CrossRef][Medline] [Order article via Infotrieve]
  17. Rintamäki, E., Martinsuo, P., Pursiheimo, S., and Aro, E.-M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11644-11649[Abstract/Free Full Text]
  18. Rokka, A., Aro, E.-M., Herrmann, R. G., Andersson, B., and Vener, A. V. (2000) Plant Physiol. 123, 1525-1536[Abstract/Free Full Text]
  19. Adams, W. W., Demmig-Adams, B., Rosenstiel, T. N., and Ebbert, V. (2001) Photosynth. Res. 67, 51-62[CrossRef][Medline] [Order article via Infotrieve]
  20. Booij-James, I. S., Swegle, W. M., Edelman, M., and Mattoo, A. K. (2002) Plant Physiol. 130, 2069-2075[Abstract/Free Full Text]
  21. Greer, D. H., Berry, J. A., and Björkman, O. (1986) Planta 168, 253-260
  22. Cleland, R. E., Ramage, R. T., and Critchley, C. (1990) Aust. J. Plant Physiol. 17, 641-651
  23. Tyystjarvi, E., and Aro, E. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2213-2218[Abstract/Free Full Text]
  24. Ohad, I., Kyle, D. J., and Arntzen, C. J. (1984) J. Cell Biol. 99, 481-485[Abstract/Free Full Text]
  25. Mattoo, A. K., Pick, U., Hoffman-Falk, H., and Edelman, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 1572-1576[Abstract/Free Full Text]
  26. Mattoo, A. K., and Edelman, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1497-1501[Abstract/Free Full Text]
  27. Ebbert, V., and Godde, D. (1996) Photosynth. Res. 50, 257-269
  28. Koivuniemi, A., Aro, E.-M., and Andersson, B. (1995) Biochemistry 34, 16022-16029[CrossRef][Medline] [Order article via Infotrieve]
  29. Rintamäki, E., Salo, R., Lehtonen, E., and Aro, E.-M. (1995) Planta 195, 379-386
  30. Baena-Gonzalez, E., Barbato, R., and Aro, E.-M. (1999) Planta 208, 196-204[CrossRef]
  31. Bellafiore, S., Barneche, F., Peltier, G., and Rochaix, J. D. (2005) Nature 433, 892-895[CrossRef][Medline] [Order article via Infotrieve]
  32. Snyders, S., and Kohorn, B. D. (1999) J. Biol. Chem. 274, 9137-9140[Abstract/Free Full Text]
  33. Snyders, S., and Kohorn, B. D. (2001) J. Biol. Chem. 276, 32169-32176[Abstract/Free Full Text]
  34. Noren, H., Svensson, P., and Andersson, B. (1999) Biosci. Rep. 19, 499-509[CrossRef][Medline] [Order article via Infotrieve]
  35. Kieselbach, T., Hagman, Å., Andersson, B., and Schröder, W. P. (1998) J. Biol. Chem. 273, 6710-6716[Abstract/Free Full Text]
  36. Aro, E. M., Rokka, A., and Vener, A. V. (2004) Methods Mol. Biol. 274, 271-286[Medline] [Order article via Infotrieve]
  37. Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Nat. Biotechnol. 20, 301-305[CrossRef][Medline] [Order article via Infotrieve]
  38. Ficarro, S., Chertihin, O., Westbrook, V. A., White, F., Jayes, F., Kalab, P., Marto, J. A., Shabanowitz, J., Herr, J. C., Hunt, D. F., and Visconti, P. E. (2003) J. Biol. Chem. 278, 11579-11589[Abstract/Free Full Text]
  39. He, T., Alving, K., Feild, B., Norton, J., Joseloff, E. G., Patterson, S. D., and Domon, B. (2004) J. Am. Soc. Mass Spectrom. 15, 363-373[CrossRef][Medline] [Order article via Infotrieve]
  40. Bennett, J. (1980) Eur. J. Biochem. 104, 85-89[Medline] [Order article via Infotrieve]
  41. Vener, A. V., Rokka, A., Fulgosi, H., Andersson, B., and Herrmann, R. G. (1999) Biochemistry 38, 14955-14965[CrossRef][Medline] [Order article via Infotrieve]
  42. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
  43. Rintamäki, E., Kettunen, R., and Aro, E.-M. (1996) J. Biol. Chem. 271, 14870-14875[Abstract/Free Full Text]
  44. Carlberg, I., and Andersson, B. (1996) Photosynth. Res. 47, 145-156
  45. Finazzi, G., Zito, F., Barbagallo, R. P., and Wollman, F. A. (2001) J. Biol. Chem. 276, 9770-9774[Abstract/Free Full Text]
  46. Vener, A. V., Ohad, I., and Andersson, B. (1998) Curr. Opin. Plant Biol. 1, 217-223[CrossRef][Medline] [Order article via Infotrieve]
  47. Till, J. H., Becerra, M., Watty, A., Lu, Y., Ma, Y., Neubert, T. A., Burden, S. J., and Hubbard, S. R. (2002) Structure (Camb.) 10, 1187-1196[Medline] [Order article via Infotrieve]
  48. Hubbard, S. R., and Till, J. H. (2000) Annu. Rev. Biochem. 69, 373-398[CrossRef][Medline] [Order article via Infotrieve]
  49. Xu, R. M., Carmel, G., Sweet, R. M., Kuret, J., and Cheng, X. (1995) EMBO J. 14, 1015-1023[Medline] [Order article via Infotrieve]
  50. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract/Free Full Text]
  51. Marchler-Bauer, A., Anderson, J. B., Cherukuri, P. F., DeWeese-Scott, C., Geer, L. Y., Gwadz, M., He, S., Hurwitz, D. I., Jackson, J. D., Ke, Z., Lanczycki, C. J., Liebert, C. A., Liu, C., Lu, F., Marchler, G. H., Mullokandov, M., Shoemaker, B. A., Simonyan, V., Song, J. S., Thiessen, P. A., Yamashita, R. A., Yin, J. J., Zhang, D., and Bryant, S. H. (2005) Nucleic Acids Res. 33, D192-D196[Abstract/Free Full Text]

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