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.     

Role of the PSII-H subunit in photoprotection: novel aspects of D1 turnover in Synechocystis 6803

Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

     

Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli

In this work, we featured an expression system that enables the production of sufficient quantities ( approximately mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies. PsbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione-S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells. A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism. As a result, the majority of fusion protein was obtained in a soluble state and could be purified from crude bacterial lysate by affinity chromatography on immobilized glutathione under non-denaturing conditions. The PsbH protein was cleaved from the carrier protein with Factor Xa protease and purified on DEAE-cellulose column with yields of up to 2.1 microg protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies.     

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.     

Phosphorylation of thylakoid proteins by a purified kinase

A simplified method is given for the purification of a 64-kilodalton protein kinase from spinach or pea thylakoid membranes (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385). In a heterogeneous reconstitution system comprised of purified kinase and washed thylakoids (having their intrinsic kinase inactivated or removed), endogenous light-harvesting pigment protein of photosystem II could serve as a substrate. Its phosphorylation did not require rebinding of kinase to the thylakoid membrane and, like the phosphorylation of solubilized pigment protein, was not under redox control. No reconstitution was observed upon replacing 64-kilodalton protein kinase with 25-kilodalton protein kinase (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 14062-14068). Tryptic digestion of phosphorylated membranes removed the site of phosphorylation; the phosphorylated amino acid present in light-harvesting pigment protein and its tryptic peptide was threonine. Immunoglobulin from a polyclonal antiserum, raised against the purified enzyme, fully inhibited kinase activity toward solubilized and endogenous pigment protein. At higher titers, the antibody was effective in totally inhibiting the redox-sensitive phosphorylation of thylakoid proteins by endogenous kinase; inhibition profiles for phosphorylation of pigment protein and thylakoid proteins of 32, 16, and 9 kilodaltons were essentially identical. The 64-kilodalton protein kinase would thus appear to be responsible for all of the observed phosphorylation of thylakoid phosphoproteins.     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.     

Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.     

Phosphorylation and nitration levels of photosynthetic proteins are conversely regulated by light stress

Using a label-free mass spectrometric approach, we investigated light-induced changes in the distribution of phosphorylated and nitrated proteins within subpopulations of native photosynthetic complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to growth light (GL) and subsequently exposed to high light (HL). Eight protein phosphorylation sites were identified in photosystem II (PSII) and the phosphorylation level of seven was regulated by HL as determined based on peak areas from ion chromatograms of phosphorylated and non-phosphorylated peptides. Although the phosphorylation of PSII proteins was reported in the past, we demonstrated for the first time that two minor antenna LHCB4 isoforms are alternately phosphorylated under GL and HL conditions in PSII monomers, dimers and supercomplexes. A role of LHCB4 phosphorylation in state transition and monomerization of PSII under HL conditions is proposed. We determined changes in the nitration level of 23 tyrosine residues in five photosystem I (PSI) and nine PSII proteins and demonstrated for the majority of them a lower nitration level in PSI and PSII complexes and supercomplexes under HL conditions, as compared to GL. In contrast, the nitration level significantly increased in assembled/disassembled PSI and PSII subcomplexes under HL conditions. A possible role of nitration in (1) monomerization of LHCB1-3 trimers under HL conditions (2) binding properties of ferredoxin-NADP+ oxidoreductase to photosystem I, and (3) PSII photodamage and repair cycle, is discussed. Based on these data, we propose that the conversely regulated phosphorylation and nitration levels regulate the stability and turnover of photosynthetic complexes under HL conditions.     

龙须菜叶绿素- 蛋白复合物的分离及鉴定

以海洋经济海藻——龙须菜（Gracilaria lemaneiformis）为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物。P700差示光谱鉴定分离的光系统Ⅰ（PSⅠ）颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ（PSⅡ）颗粒。结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒。     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and activeoxygen species with harmful effects on photosystem II (PSII).Such photodamage occurs at all light intensities. Damaged PSIIcentres, however, do not usually accumulate in the thylakoidmembrane due to a rapid and efficient repair mechanism. Theexcellent design of PSII gives protection to most of the proteincomponents and the damage is most often targeted only to thereaction centre D1 protein. Repair of PSII via turnover of thedamaged protein subunits is a complex process involving (i)highly regulated reversible phosphorylation of several PSIIcore subunits, (ii) monomerization and migration of the PSIIcore from the grana to the stroma lamellae, (iii) partial disassemblyof the PSII core monomer, (iv) highly specific proteolysis ofthe damaged proteins, and finally (v) a multi-step replacementof the damaged proteins with de novo synthesized copies followedby (vi) the reassembly, dimerization, and photoactivation ofthe PSII complexes. These processes will shortly be reviewedpaying particular attention to the damage, turnover, and assemblyof the PSII complex in grana and stroma thylakoids during thephotoinhibition–repair cycle of PSII. Moreover, a two-dimensionalBlue-native gel map of thylakoid membrane protein complexes,and their modification in the grana and stroma lamellae duringa high-light treatment, is presented. Key words: Arabidopsis thylakoid membrane proteome, assembly of photosystem II, D1 protein, light stress, photosystem II photoinhibition, repair of photosystem II     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Formation of cross-linking between photosystem 2 proteins during irradiation of thylakoid membranes at high temperature

Irradiation of thylakoid membranes at 40 °C resulted in complete inhibition of photosystem (PS) 2 activity measured as 2,6-dichlorophenol indophenol (DCIP) photoreduction either in the absence or presence of 1,5-diphenylcarbazide (DPC). Concomitant with the inactivation of PS2 activity, several thylakoid proteins were lost and high molecular mass cross-linking products appeared that cross-reacted with antibodies against proteins of PS2 but not with antibodies against proteins of other three complexes PS1, ATP synthase, and cytochrome b₆f. Irradiation of thylakoid membranes suspended in buffer of basic pH or high concentration of Tris at 25 °C resulted in the formation of cross-linking products similar to those in thylakoid membranes irradiated at 40 °C. Presence of radical scavengers and DPC during the high temperature treatment prevented the formation of cross-linking products. These results suggest the involvement of oxygen evolving co mplex (OEC) in the formation of cross-linking between PS2 proteins in thylakoid membrane irradiated at high temperature. This revised version was published online in September 2006 with corrections to the Cover Date.     

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo‐adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C‐terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co‐localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light‐induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two‐dimensional gel electrophoresis discriminated, for the first time, the 58‐kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC‐MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.     

Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography.

Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution.     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Determination of photosystem II subunits by matrix-assisted laser desorption/ionization mass spectrometry

Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes.