Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants

Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O₂-evolving PSII reaction center complex.     

Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of Arabidopsis thaliana

The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

Role of phosphorylation in the repair cycle and oligomeric structure of photosystem II

The role of PSII protein phosphorylation in the oligomeric structure of the complex and in the repair of photodamaged PSII centers was studied with intact thylakoids and thylakoid membrane subfractions isolated from differentially light-treated pumpkin (Cucurbita pepo L.) leaves. A combination of sucrose gradient fractionation of thylakoid protein complexes and immunodetection with phosphothreonine and protein-specific antibodies was used. We report in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity. Phosphorylated PSII monomers migrate to the stroma-exposed thylakoids, probably following damage of the D1 protein and the dissociation of the light-harvesting complex of PSII. Once in the stroma lamellae, monomers are gradually dephosphorylated to allow the reparation of the complex. First, CP43 is dephosphorylated and as a consequence of this modification it detaches from the PSII core. In addition to D1, D2 is also thereafter dephosphorylated. Phosphorylation of PSII core polypeptides probably ensures the integrity of the monomers until repair can proceed. Dephosphorylation, on the other hand, might serve the need for opening the complex and coordinating D1 proteolysis and the attachment of ribosomes.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II.

It is known that the reaction-center binding protein D1 in photosystem (PS) II is degraded significantly during photoinhibition. The D1 protein also cross-links covalently or aggregates non-covalently with the nearby polypeptides in PS II complexes by illumination. In the present study, we detected the adducts between the D1 protein and the other reaction-center binding protein D2 (D1/D2), the alpha-subunit of cyt b(559) (D1/cyt b(559)), and the antenna chlorophyll-binding protein CP43 (D1/CP43) by SDS/urea-polyacrylamide gel electrophoresis and Western blotting with specific antibodies. The adducts were observed by weak and strong illumination (light intensity: 50-5000 microE m(-2) s(-1)) of PS II membranes, thylakoids and intact chloroplasts from spinach, under aerobic conditions. These results indicate that the cross-linking or aggregation of the D1 protein is a general phenomenon which occurs in vivo as well as in vitro with photodamaged D1 proteins. We found that the formation of the D1/D2, D1/cyt b(559) and D1/CP43 adducts is differently dependent on the light intensity; the D1/D2 heterodimers and D1/cyt b(559) were formed even by illumination with weak light, whereas generation of the D1/CP43 aggregates required strong illumination. We also detected that these D1 adducts were efficiently removed by the addition of stromal components, which may contain proteases, molecular chaperones and the associated proteins. By two-dimensional SDS/urea-polyacrylamide gel electrophoresis, we found that several stromal proteins, including a 15-kDa protein are effective in removing the D1/CP43 aggregates, and that their activity is resistant to SDS.     

Quality control of photosystem II: impact of light and heat stresses

Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.     

Detection of transiently phosphorylated membrane proteins by protein blotting through a nonionic detergent layer

A rapid approach for detecting tentative membrane proteins which are transiently phosphorylated/dephosphorylated is described. Cell fractionation is unnecessary, as are other manipulations of sample preparation during which artifactual modifications or sample loss might occur. The method is shown to be useful for the detection of such phosphorylation during cellular response to the binding of specific ligand. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed successively through gels of different sieving sizes. These "primary" gels were then subjected to "detergent blotting," a variation of electroblotting in which polyacrylamide gel containing the nonionic detergent Nonidet-P40 (secondary gel) was inserted between the primary gel and a Zeta-Probe membrane. Phosphorylated interleukin 2 receptors were selectively retained in the secondary gel. Upon stimulation of human platelets with thrombin, at least 11 polypeptides were found to be rapidly phosphorylated/dephosphorylated using the method. Among them, five phosphorylated polypeptides were trapped in the secondary gel, suggesting that they might be membrane proteins. This technique should be useful to rapidly screen transiently phosphorylated/dephosphorylated membrane proteins which might be involved in membrane transductional signaling.     

Changes of absorption spectra during heat-induced denaturation of Photosystem II core antenna complexes CP43 and CP47: revealing the binding states of chlorophyll molecules in these two complexes

The Photosystem II (PSII) core antenna complexes, CP43 and CP47, were prepared from spinach (Spinacia oleracea L.). The absorption spectra in the red region at room temperature were recorded for the PSII core antenna samples after increased temperature treatment (up to 80 degrees C). Derivative and difference spectra revealed the existence of two groups of chlorophyll a (Chl a) molecules in both CP43 and CP47. The one with the absorption peak in the shorter wavelength region was designated as CP43-669 and CP47-669, while the other with the absorption peak in the longer wavelength region was designated as CP43-682 and CP47-680. The results of the thermal treatment experiment demonstrated that CP43-669 and CP47-669 may exist as monomers of Chl a and that their binding sites on the polypeptides are insensitive to thermal treatment, whereas CP43-682 and CP47-680 may exist as dimers or multimers of Chl a and their binding regions in the polypeptide chains are more sensitive to heat treatment. The excitation energy transfer mechanism between these two different groups of Chl a molecules is also analyzed.     

Phosphatase activities in spinach thylakoid membranes-effectors, regulation and location

The dephosphorylation of seven phosphoproteins associated with Photosystem II or its chlorophyll a/b antenna in spinach thylakoids, was characterised. The rates were found to fall into two distinct groups. One, rapidly dephosphorylated, consisted of the two subunits (25 and 27 kD) of the major light harvesting complex of Photosystem II (LHC II) and a 12 kD polypeptide of unknown identity. A marked correlation between the dephosphorylation of these three phosphoproteins, strongly suggested that they were all dephosphorylated by the same enzyme. Within this group, the 25 kD subunit was consistently dephosphorylated most rapidly, probably reflecting its exclusive location in the peripheral pool of LHC II. The other group, only slowly dephosphorylated, included several PS II proteins such as the D1 and D2 reaction centre proteins, the chlorophyll-a binding protein CP43 and the 9 kD PS II-H phosphoprotein. No dephosphorylation was observed in either of the two groups in the absence of Mg²⁺-ions. Dephosphorylation of the two LHC II subunits took place in both grana and stroma-exposed regions of the thylakoid membrane. However, deposphorylation in the latter region was significantly more rapid, indicating a preferential dephosphorylation of the peripheral (or mobile) LHC II. Dephosphorylation of LHC II was found to be markedly affected by the redox state of thiol-groups, which may suggest a possible regulation of LHC II dephosphorylation involving the ferredoxin-thioredoxin system.Abbreviations CP 43 43 kD chlorophyll a- binding protein - D1 and D2 reaction centre proteins of PS II - LHC II light-harvesting complex of PS II - LHC II-25 25 kD subunit of LHC II - LHC II-27 27 kD subunit of LHC II - NEM N-ethylmaleimide - PP2C protein phosphatase 2C - PS II-H psb H gene product     

Dephosphorylation of photosystem II core proteins is light-regulated in vivo.

A number of photosystem II (PSII)-associated proteins, including D1, D2, CP43 and LHCII, are phosphorylated post-translationally by a membrane-bound, redox-regulated kinase activity. In vitro studies have demonstrated that these proteins can be dephosphorylated by membrane-bound phosphatase activity, reportedly insensitive to light or redox control. We demonstrate here that the PSII core proteins, D1, D2 and CP43, undergo light-stimulated, linear electron-transport-independent dephosphorylation in vivo. The in vivo dephosphorylation of D1 was characterized further and shown to depend upon light intensity, and to occur throughout the visible light spectrum with characteristics most consistent with light absorption by chlorophyll. PSII core protein dephosphorylation in vivo was stimulated by photosystem I (PSI)-specific far-red light, and inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of plastoquinol oxidation by the cytochrome b6f complex. Based on these findings, we propose that PSI excitation is involved in regulating dephosphorylation of PSII core proteins in vivo.     

Relationship of multiple forms of chromogranin

Chromogranin polypeptides of Mr 100,000, Mr 85,000, Mr 75,000, and Mr 65,000 have been detected in adrenal medulla chromaffin granules using anti-chromogranin antiserum. Monoclonal antibodies to this protein also detect the multiple molecular weight chromogranin polypeptides. Analysis of phosphorylated amino acids gives a value of 5 phosphoserine residues/mol of Mr 75,000 chromogranin polypeptide. Immunological analysis of dephosphorylated chromogranin shows that the anti-chromogranin serum reacts with both the phosphorylated and unphosphorylated forms of the protein. Each of the chromogranin polypeptides has been isolated using a combination of DEAE-cellulose chromatography and reverse phase high pressure liquid chromatography. Sequencer analysis of each protein revealed a high degree of amino acid identities at the amino terminal of these proteins. Amino-terminal Sequencer analysis of chromogranin fragments also provides evidence for a gene duplication event. Preliminary studies also show that chromogranin may be degraded in the chromaffin granules by a calcium-dependent mechanism.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

Evolution of the Inner Light-Harvesting Antenna Protein Family of Cyanobacteria,Algae, and Plants

Two hypotheses account for the evolution of the inner antenna light-harvesting proteins of oxygenic photosynthesis in cyanobacteria, algae, and plants: one in which the CP43 protein of photosytem II gave rise to the extrinsic CP43-like antennas of cyanobacteria (i.e. IsiA and Pcb proteins), as a late development, and the other in which CP43 and CP43-like proteins derive from an ancestral protein. In order to determine which of these hypotheses is most likely, we analyzed the family of antenna proteins by a variety of phylogenetic techniques, using alignments of the six common membrane-spanning helices, constructed using information on the antenna proteins’ three-dimensional structure, and surveyed for evidence of factors that might confound inference of a correct phylogeny. The first hypothesis was strongly supported. As a consequence, we conclude that the ancestral photosynthetic apparatus, with 11 membrane-spanning helices, split at an early stage during evolution to form, on the one hand, the reaction center of photosystem II and, on the other hand, the ancestor of inner antenna proteins, CP43 (PsbC) and CP47 (PsbB). Only much later in evolution did the CP43 lineage give rise to the CP43’ proteins (IsiA and Pcb) of cyanobacteria. [Reviewing Editor: Dr. Patrick Keeling]     

Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.     

The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene

The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins. We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed. The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively. The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes. The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.     

Evidence that the PsbK polypeptide is associated with the photosystem II core antenna complex CP43

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.