Phosphorylation of Thylakoid Proteins of Oryza sativa: In Vitro Characterization and Effects of Chilling Temperatures

The phosphorylation of thylakoid proteins of rice (Oryza sativa L.) was studied in vitro using [γ-³²P]ATP. Several thylakoid proteins are labeled, including the light-harvesting complex of photosystem II. Protein phosphorylation is sensitive to temperature, pH, and ADP, ATP, and divalent cation concentrations. In the range pH 7 to 8.2, phosphorylation of the light-harvesting polypeptides declines above pH 7.5, whereas labeling of several other thylakoid polypeptides increases. Increasing divalent cation concentration from 3 to 20 millimolar results in a decrease in phosphorylation of the 26 kilodalton light-harvesting complex polypeptide and increased phosphorylation of several other polypeptides. ADP has an inhibitory effect on the phosphorylation of the light-harvesting complex polypeptides. Phosphorylation of the 26 kilodalton light-harvesting polypeptide requires 0.45 millimolar ATP for half-maximal phosphorylation, compared to 0.3 millimolar for the 32 kilodalton phosphoprotein. Low temperature inhibits the phosphorylation of thylakoid proteins in chilling-sensitive rice. However, phosphorylation of histones by thylakoid-bound kinase(s) is independent of temperature in the range of 25 to 5°C, suggesting that the effect of low temperature is on accessibility of the substrate, rather than on the activity of the kinase.     

Protein phosphorylation in plant mitochondria

Protein kinase activity was detected in osmotically lysed mitochondria isolated from etiolated seedlings of corn, pea, soybean, and wheat, as well as from potato tubers. Ther kinase(s) phosphorylated both endogenous polypeptides and exogenous, nonmitochondrial proteins when supplied with ATP and Mg²⁺. Eight to fifteen endogenous mitochondrial polypeptides were phosphorylated. The major mitochondrial polypeptide labeled in all species migrated during denaturing electrophoresis with an apparent monomeric molecular weight of 47,000. Incorporation of phosphate into endogenous proteins appeared to be biphasic, being most rapid during the first 1 to 2 minutes but slower thereafter. The kinase activity was greatest at neutral and alkaline pH values and utilized ATP with a K_m of approximately 200 micromolar. The kinase was markedly inhibited by CaCl₂ but was essentially unaffected by NaF, calmodulin, oligomycin, or cAMP. These data suggest that plant mitochondrial protein phosphorylation may be similar to protein phosphorylation in animal mitochondria.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b₆f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b₆f assembly and heme biogenesis.     

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.     

State transitions at the crossroad of thylakoid signalling pathways

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b ₆ f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.     

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.

     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.     

Chilling Sensitivity in Oryza sativa: The Role of Protein Phosphorylation in Protection against Photoinhibition

The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L.). Brief exposure to chilling temperatures (0-15°C, 10 min) did not cause a significant difference in photosynthetic O₂ evolution capacity in vivo between rice and barley. Analysis of in vivo chlorophyll fluorescence in chilling-sensitive rice suggests that low temperatures cause an increased reduction of the plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction centers. Analysis of ³²P incorporation into thylakoid proteins both in vivo and in vitro demonstrated that chilling temperature inhibited protein phosphorylation in rice, but not in barley. Low temperature (77 K) fluorescence analysis of isolated thylakoid membranes indicated that state I to state II transitions occurred in barley, but not in rice subjected to chilling temperatures. These observations suggest that protein phosphorylation may play an important role in protection against photoinhibition caused by exposure to chilling temperatures.     

Role of Plastid Protein Phosphatase TAP38 in LHCII Dephosphorylation and Thylakoid Electron Flow

Short-term changes in illumination elicit alterations in thylakoid protein phosphorylation and reorganization of the photosynthetic machinery. Phosphorylation of LHCII, the light-harvesting complex of photosystem II, facilitates its relocation to photosystem I and permits excitation energy redistribution between the photosystems (state transitions). The protein kinase STN7 is required for LHCII phosphorylation and state transitions in the flowering plant Arabidopsis thaliana. LHCII phosphorylation is reversible, but extensive efforts to identify the protein phosphatase(s) that dephosphorylate LHCII have been unsuccessful. Here, we show that the thylakoid-associated phosphatase TAP38 is required for LHCII dephosphorylation and for the transition from state 2 to state 1 in A. thaliana. In tap38 mutants, thylakoid electron flow is enhanced, resulting in more rapid growth under constant low-light regimes. TAP38 gene overexpression markedly decreases LHCII phosphorylation and inhibits state 1→2 transition, thus mimicking the stn7 phenotype. Furthermore, the recombinant TAP38 protein is able, in an in vitro assay, to directly dephosphorylate LHCII. The dependence of LHCII dephosphorylation upon TAP38 dosage, together with the in vitro TAP38-mediated dephosphorylation of LHCII, suggests that TAP38 directly acts on LHCII. Although reversible phosphorylation of LHCII and state transitions are crucial for plant fitness under natural light conditions, LHCII hyperphosphorylation associated with an arrest of photosynthesis in state 2 due to inactivation of TAP38 improves photosynthetic performance and plant growth under state 2-favoring light conditions.     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Protein kinase activity in cell free extracts of Streptomyces avermitilis; comparing wild type and overproducing mutant

Summary Protein kinase activity was assayed in cell-free extracts prepared from mycelia of a wild strain (MA) and an overproducing mutant (RX 2) of Streptomyces avermitilis. At least 10 different polypeptides ranging in M _r from 10 000 to 120 000 were found to be phosphorylated on analysis of ³²P labeled cell lysates by gel electrophoresis and phosphorimage. The protein profile and the level of phosphorylation varied depending on the culture stage of the mycelia.     

Stt7-dependent Phosphorylation during State Transitions in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b₆f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser⁵³³ in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.The primary photochemical reactions of photosynthesis are catalyzed by the pigment-protein complexes photosystem II (PSII)¹ and PSI (PSI), which are linked in series through the plastoquinone pool, the cytochrome b₆f complex, and plastocyanin in the thylakoid membranes. Upon light absorption by the antenna systems of PSII and PSI, charge separations occur across the membrane that lead to the oxidation of water by PSII and electron flow to PSI and ultimately to the reduction of NADP⁺. Because the antenna systems of PSII and PSI have different pigment composition, they are differentially sensitized upon changes in light quality and quantity. However, photosynthetic organisms have the ability to adapt to changes in light. They balance energy input and consumption in the short term through dissipation of excess absorbed light energy into heat through non-photochemical quenching and regulate absorption of excitation energy between PSII and PSI through state transitions (supplemental Fig. 1). This reversible redistribution leads to an overall increase in photosynthetic quantum yield. State transitions occur when preferential excitation of PSII reduces the plastoquinone pool. This leads to the activation of a thylakoid protein kinase as a result of the docking of plastoquinol to the Qo site of the cytochrome b₆f complex (1, 2) and to the phosphorylation of the polypeptides of the light-harvesting complex II (LHCII), a part of which migrates to PSI (state 2) (3–5). The process is reversible as preferential excitation of PSI inactivates the kinase and allows for dephosphorylation of LHCII and its return to PSII (state 1) (3, 6). In the green alga Chlamydomonas reinhardtii, the LHCII protein set consists of Type I (Lhcbm3, Lhcbm4, Lhcbm6, Lhcbm8, and Lhcbm9), Type II (Lhcbm5), Type III (Lhcbm2 and Lhcbm7), and Type IV (Lhcbm1 and Lhcbm10) proteins and of Lhcb7, CP26, and CP29 (7). Because of their nearly identical sequences and sizes, several of these Lhcbm proteins cannot be distinguished by SDS-PAGE. Most of them fractionate into four bands called P11 and P13 (Type I), P16 (Type IV), and P17 (Type III). Whereas P16 is not phosphorylated, phosphorylation events occur on P11, P13, and P17 (7, 8).The association of the mobile part of LHCII to PSI during a transition from state 1 to state 2 requires the PsaH subunit (9) and CP29, which also moves to PSI and is essential for docking LHCII to PSI (10–12). The lateral displacement of LHCII from the PSII-rich grana to the PSI-rich lamellar thylakoid regions results in transfer to PSI of about 80% of the excitation energy absorbed by LHCII in C. reinhardtii (13), a considerably higher amount than in land plants in which only 15–20% of LHCII is mobile (3). In C. reinhardtii, state transitions are associated with a reorganization of the photosynthetic electron transfer chain with a switch from linear to cyclic electron flow during a transition from state 1 to state 2 (14, 15). Thus, cells produce ATP and NADPH in state 1 but only ATP in state 2. It appears that the major function of state transitions in this alga is to adjust the level of ATP and the ATP/NADPH ratio to cellular demands (5).Thylakoid membranes contain appressed grana and nonappressed stromal domains in which PSII and PSI are enriched, respectively. Because LHCII is a major stabilizer of the grana structure (16), the movement of LHCII from PSII to PSI is expected to lead to major rearrangements of these membranes during state transitions. Indeed, based on extensive electron microscope studies, it was proposed that fusion and fission events occur at the interface between the grana and stroma lamellar domains that lead to a remodeling of the membranes (17).Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of Chlamydomonas revealed a total of 19 sites corresponding to 15 genes (18). It was shown that the major changes are clustered at the interface between the PSII core and the associated LHCII proteins during state transitions. Phosphorylation of the PSII core subunits D2 and PsbR and multiple phosphorylations of the minor LHCII antenna subunit CP29 were detected as well as phosphorylation of Lhcbm1, which belongs to the major LHCII complex (18).Although the phosphorylation of LHCII was observed many years ago (6), it is only recently that kinases involved in this process were uncovered. Fleischmann et al. (19) and Kruse et al. (20) used a genetic approach in C. reinhardtii with the aim of dissecting the signal transduction chain of state transitions. Two allelic mutants blocked in state 1 were identified that are affected in the Stt7 gene encoding a thylakoid Ser-Thr protein kinase that is required for LHCII phosphorylation during a transition from state 1 to state 2 (21). This Stt7 kinase is conserved in land plants and has an ortholog, STN7, in Arabidopsis (22).The 754-amino acid Stt7 kinase has a catalytic domain characteristic of Ser-Thr kinases (21). It contains a putative 41-amino acid transit peptide at its N-terminal end, and the protein is localized on the thylakoid membrane. Stt7 is associated with photosynthetic complexes including LHCII, PSI, and the cytochrome b₆f complex (23). Stt7 also contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys residues that are critical for its activity and state transitions (23). Moreover, the level of Stt7 decreases considerably under state 1 conditions, and the kinase acts in catalytic amounts (23). However, it is not yet known whether this kinase directly phosphorylates LHCII or whether it is part of a kinase cascade involved in the signaling pathway of state transitions.In this work, we used a mass spectrometry-based approach (24) to map the in vivo Stt7-dependent protein phosphorylation sites within thylakoid membranes isolated from the green alga C. reinhardtii subjected to state 1 and state 2 conditions. In contrast with the earlier studies via direct MS/MS sequencing of the IMAC-enriched phosphorylated peptides from thylakoid proteins (18, 25), we performed additional LC-MS/MS-based analyses using alternating collision-induced dissociation and electron transfer dissociation of peptide ions. This approach revealed novel phosphorylation sites in LHCII polypeptides, in several other membrane and membrane-associated proteins, and in the thylakoid protein kinases Stt7 and Stl1, suggesting the existence of a thylakoid protein kinase cascade. Relative quantification of phosphorylated peptides labeled with stable isotopes determined the specific Stt7-dependent phosphorylation site in CP29 linker protein under state 2. Moreover, we also identified phosphorylation sites that are redox-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent. This mapping provides new insights into the regulatory network of protein phosphorylation in algal photosynthetic membranes during state transitions.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1

During state transitions, plants regulate energy distribution between photosystems I and II through reversible phosphorylation and lateral migration of the major light-harvesting complex LHCII. Dephosphorylation of LHCII and the transition from state 2 to state 1 requires a thylakoid membrane-associated phosphatase named TAP38 or PPH1. TAP38/PPH1 specifically targets LHCII but not the core subunits of photosystem II, whereas the underlying molecular mechanism of their mutual recognition is currently unclear. Here, we present the structures of Arabidopsis thaliana TAP38/PPH1 in the substrate-free and substrate-bound states. The protein contains a type 2C serine/threonine protein phosphatase (PP2C) core domain, a Mn²⁺ (or Mg²⁺) binuclear center and two additional motifs contributing to substrate recognition. A 15-mer phosphorylated N-terminal peptide of Lhcb1 binds to TAP38/PPH1 on two surface clefts enclosed by the additional motifs. The first segment of the phosphopeptide is clamped by a pair of tooth-like arginine residues at Cleft 1 site. The binding adopts the lock-and-key mechanism with slight rearrangement of the substrate binding residues on TAP38/PPH1. Meanwhile, a more evident substrate-induced fitting occurs on Cleft 2 harboring the extended part of the phosphopeptide. The results unravel the bases for the specific recognition between TAP38/PPH1 and phosphorylated Lhcb1, a crucial step in state transitions.     

Up-regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir

Overwintering needles of the evergreen conifer Douglas fir exhibited an association between arrest of the xanthophyll cycle in the dissipating state (as zeaxanthin + antheraxanthin; Z + A) with a strongly elevated predawn phosphorylation state of the D1 protein of the photosystem II (PSII) core. Furthermore, the high predawn phosphorylation state of PSII core proteins was associated with strongly increased levels of TLP40, the cyclophilin-like inhibitor of PSII core protein phosphatase, in winter versus summer. In turn, decreases in predawn PSII efficiency, F_v/F_m, in winter were positively correlated with pronounced decreases in the non-phosphorylated form of D1. In contrast to PSII core proteins, the light-harvesting complex of photosystem II (LHCII) did not exhibit any nocturnally sustained phosphorylation. The total level of the D1 protein was found to be the same in summer and winter in Douglas fir when proteins were extracted in a single step from whole needles. In contrast, total D1 protein levels were lower in thylakoid preparations of overwintering needles versus needles collected in summer, indicating that D1 was lost during thylakoid preparation from overwintering Douglas fir needles. In contrast to total D1, the ratio of phosphorylated to non-phosphorylated D1 as well as the levels of the PsbS protein were similar in thylakoid versus whole needle preparations. The level of the PsbS protein, that is required for pH-dependent thermal dissipation, exhibited an increase in winter, whereas LHCII levels remained unchanged.     

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

Background

Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO₂ fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO₂ conditions.

Results/Conclusions

In the present work we demonstrate that after transfer to low CO₂, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO₂ conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO₂ conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO₂ conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO₂ the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.

Significance

This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.     

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.