Changes in topography and function of thylakoid membranes following membrane protein phosphorylation

The kinetics of the intracellular redistribution of phytochrome (sequestering) in Avena sativa L. coleoptiles following a brief, saturating actinic pulse of red (R) light have been determined. Immunocytochemical labelling of phytochrome with monoclonal antibodies showed that at 22°C sequestering can occur within 1–2 s from the onset of R irradiation and is dependent upon the continued presence of the far-red-absorbing form of phytochrome (Pfr). The initial rate, but not the final extent, of sequestering is reduced by lowering the temperature of the tissue to 1°C. Sequestering at 22°C appears to involve two distinct stages: (1) a rapid association of Pfr with putative binding sites initiates the sequestered condition, following which (2) these sites of sequestered phytochrome appear to aggregate. Neither of these two processes was affected by the cytoskeletal inhibitors colchicine or cytochalasin B. Phytochrome sequestering therefore resembles R-light-induced phytochrome pelletability with respect to kinetics, temperature sensitivity, and dependence upon the continued presence of Pfr in the cell.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DIC differential interference contrast - FR far-red - Ig immunoglobulin - Pfr, Pr far-red-absorbing and red-absorbing form of phytochrome, respectively - R red     

Regulation of energy transfer by cations and protein phosphorylation in relation to thylakoid membrane organisation

A brief review is given of the state of knowledge which indicates that the State I-State II transition in higher plants and green algae is due to the reversible phosphorylation of the chlorophyll a/b light harvesting complex. The importance of membrane reorganisational changes in this process is discussed in terms of changes in electrostatic parameters as emphasised by the interplay of the effect of phosphorylation and the background levels of cations surrounding the membrane. It is argued that recognition of this interplay is vital when using the bipartite or tripartite models of Butler to obtain quantitative information of energy transfer between the various pigment complexes.     

Light-dependent phosphorylation of thylakoid membrane polypeptides.

The phosphorylation of thylakoid membrane proteins was studied using isolated chloroplasts from $\text{Euglena gracilis}$. We have found, using [³²P] labelling, that this phenomenon was light-driven, reversible in the dark, and completely inhibited by Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Polyacrylamide gel electrophoresis containing SDS has revealed five main bands which have been found to be proteins. Amino acid analysis of the bands has shown that [³²P] is incorporated into phosphothreonine.     

Desiccation enhances phosphorylation of PSII and affects the distribution of protein complexes in the thylakoid membrane

Desiccation has significant effects on photosynthetic processes in intertidal macro‐algae. We studied an intertidal macro‐alga, Ulva sp., which can tolerate desiccation, to investigate changes in photosynthetic performance and the components and structure of thylakoid membrane proteins in response to desiccation. Our results demonstrate that photosystem II (PSII) is more sensitive to desiccation than photosystem I (PSI) in Ulva sp. Comparative proteomics of the thylakoid membrane proteins at different levels of desiccation suggested that there were few changes in the content of proteins involved in photosynthesis during desiccation. Interestingly, we found that both the PSII subunit, PsbS (Photosystem II S subunit) (a four‐helix protein in the LHC superfamily), and light‐harvesting complex stress‐related (LHCSR) proteins, which are required for non‐photochemical quenching in land plants and algae, respectively, were present under both normal and desiccation conditions and both increased slightly during desiccation. In addition, the results of immunoblot analysis suggested that the phosphorylation of PSII and LHCII increases during desiccation. To investigate further, we separated out a supercomplex formed during desiccation by blue native‐polyacrylamide gel electrophoresis and identified the components by mass spectrometry analysis. Our results show that phosphorylation of the complex increases slightly with decreased water content. All the results suggest that during the course of desiccation, few changes occur in the content of thylakoid membrane proteins, but a rearrangement of the protein complex occurs in the intertidal macro‐alga Ulva sp.     

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

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An investigation of the mechanistic aspects of excitation energy redistribution following thylakoid membrane protein phosphorylation

Accompanying thylakoid membrane protein phosphorylation is a redistribution of energy between PS II and PS I; mechanistic aspects of this redistribution have been investigated in both a mature and a developing chloroplast system. Data are presented which suggest that the mechanism of these changes is dependent upon the developmental status and/or morphological characteristics of the chloroplast.     

Changes in the flash-induced oxygen yield pattern by thylakoid membrane phosphorylation

Phosphorylation of thylakoid membrane proteins results in a partial inhibition (approximately 15–20%) of the light-saturated rate of oxygen evolution. The site of inhibition is thought to be located on the acceptor side of photosystem 2 (PS2) between the primary, Q_A, and secondary, Q_B, plastoquinone acceptors (Hodges et al. 1985, 1987). In this paper we report that thylakoid membrane phosphorylation increases the damping of the quaternary oscillation in the flash oxygen yield and increases the extent of the fast component in the deactivation of the S2 oxidation state. These results support the proposal that thylakoid membrane protein phosphorylation decreases the equilibrium constant for the exchange of an electron between Q_A and Q_B. An analysis of the oxygen release patterns using the recurrence matrix model of Lavorel (1976) indicates that thylakoid membrane phosphorylation increases the probability that PS2 miss a S-state transition by 20%. This is equivalent, however, to an insignificant inhibition (approximately 2.4%) of the light-saturated oxygen evolution rate. If a double miss in the S-state transitions is included when the PS2 centres are in S2 the fit between the experimental and theoretical oxygen yield sequences is better, and sufficient to account for the 15–20% inhibition in the steady-state oxygen yield. A double miss in the S-state transition is a consequence of an increased population of PS2 centres retaining Q_A ^–: not only will these PS2 centres fail to catalyse photochemical charge transfer until Q_A ^– is reoxidized, but the re-oxidation reaction will also result in the deactivation of S2 to S1.Abbreviations Chl Chlorophyll - PS2 Photosystem 2 - Si The oxidation states of PS2 (where i can be from 0 to 4) - Q_A ^– and Q_B ^– the anionic semiquinone forms of the primary and secondary plastoquione acceptors of PS2     

ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation

Incubation of pea thylakoid membranes with [32P]-NAD+ in the presence of cholera toxin resulted in the [32P]-ADP-ribosylation of a 60 kDa thylakoid membrane polypeptide. When ATP was included in the incubation mixture, a 29 kDa polypeptide was also labelled. In the absence of electron transfer cofactors or inhibitors, the extent of labelling depended on whether the membranes were preincubated in the light or dark and also on the developmental stage of the leaves used for thylakoid isolation. Irrespective of the latter, the strongest labelling was observed when DCMU was present in the light. After pretreatment of the thylakoid membranes with cholera toxin plus NAD+ under the same conditions, light-stimulated GTPase activity and protein phosphorylation were inhibited. The extent of inhibition for both processes appeared to be correlated with the amount of [32P]-ADP-ribosylation found when [32P]-NAD+ was included in the pretreatment mixture. The data presented are fully consistent with the 60 and 29 kDa polypeptides functioning as thylakoid membrane associated guanine nucleotide binding regulatory proteins.     

Evidence for different kinases in thylakoid protein phosphorylation.

Thylakoid protein phosphorylation was facilitated in darkness by using the ferredoxin-NADPH system. CoCl2 and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) were potent inhibitors of LHCP (light-harvesting chlorophyll-binding protein) phosphorylation, but 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and atrazine had no significant effect. Differential effects on phosphorylation of the 9 kDa polypeptide and LHCP were observed in darkness with DBMIB and certain other inhibitors specific for Photosystem-II electron transport. Similarly, during illumination of intact chloroplasts or of the reconstituted chloroplast system, a differential action of bicarbonate was observed on the relative phosphorylation of the two proteins. The degree of phosphorylation of the 9 kDa polypeptide was increased in the presence of bicarbonate compared with its absence, whereas that of LHCP was relatively unchanged. Changes in the degree of phosphorylation of the 32 kDa polypeptide in these experiments did not correlate consistently with changes in phosphorylation of either LHCP or the 9 kDa polypeptide, although changes in the 32 kDa polypeptide more often paralleled phosphorylation of the 9 kDa polypeptide rather than the phosphorylation of LHCP. These observations suggest that the protein kinase that phosphorylates LHCP is distinct from that which phosphorylates the 9 kDa polypeptide.     

Novel light-regulated chloroplast thylakoid membrane protein

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.     

Use of zinc ions to study thylakoid protein phosphorylation and the state 1-state 2 transition in vitro

At ATP concentrations less than 0.2 millimolar, zinc ions cause a marked stimulation of endogenous protein phosphorylation in thylakoid membranes isolated from tobacco (Nicotiana tabacum L. cv Turkish Samsun), pea (Pisum sativum L. cv Feltham First) and spinach (Spinacia oleracea L. cv Northland). The greatest stimulatory effect was observed at Zn²⁺ concentrations of 1 to 2 millimolar; higher concentrations were inhibitory. The stimulatory effect of Zn²⁺ was independent of Mg²⁺ concentration from 1 to 5 millimolar and thus does not appear to be due to the formation of a Zn²⁺ -ATP complex. Phosphorylation of histones IIA, an exogenous protein substrate, was inhibited by 2 millimolar Zn²⁺. At low levels of ATP, Zn²⁺ not only stimulates general endogenous protein phosphorylation, but also the phosphorylation of the apoproteins of the light-harvesting chlorophyll a/b-protein complex. However, under these conditions Zn²⁺ inhibits the ATP-induced quenching of photosystem II fluorescence and the increase in the ratio of photosystem I to photosystem II fluorescence which are both characteristic of the State 1-State 2 transition. These results suggest that phosphorylation of the light-harvesting chlorophyll a/b-protein complex may not directly bring about the State 1-State 2 transition.     

Redox-controlled thylakoid protein phosphorylation. News and views

Thylakoid protein phosphorylation regulates state transition and PSII protein turnover under light-dependent redox control via a signal transduction system. The redox-dependent activation/deactivation of the membrane-bound protein kinase(s), mostly localized in the grana partitions, differs for the various phosphoproteins. Reduction of the plastoquinone pool may be sufficient to activate phosphorylation of few of these proteins. Phosphorylation of LHCII, requires the presence of the cytochrome bf complex in an 'activating mode' characterized by the reduction of its high potential path components and ability to interact with a reduced plastoquinol without oxidizing it. Activation and maintenance of this kinase activity is considered to involve alternate interactions with a cytochrome bf in its activating mode and with the substrate PSII(LHCII). The segregation of the thylakoid components into grana and stroma partitions appears to be mandatory for the kinase activation process. The protein substrate specificity and kinetics differs for various kinases. The thylakoid redox-controlled kinase(s) have not yet been isolated. Preparations highly enriched in kinase activity capable to phosphorylate LHCII and PSII core proteins, contain two kinase active bands, resolved by denaturing electrophoresis and renaturation, and having apparent molecular masses of about 53 and 66 kDa. The roughly estimated abundance of these putative kinase(s) in the grana partitions may be compatible with a ratio of kinase(s): PSII(LHCII) dimers:cytochrome bf dimers in the range of 1:60:30 and a ratio of kinase:phosphorylation sites of about 1:2000. Only about 10–20% of these sites are phosphorylated during state transition. The low turnover rate of the LHCII kinase(s) (< 5) may be due to hindrance of the required random lateral migration within the grana domain rich in tightly packed PSII(LHCII) and cytochrome bf complexes.     

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.     

Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.     

A systematic approach to the analysis of protein phosphorylation

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes

Background

The ‘phosphate-binding tag’ (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.

Methodology/Principal Findings

We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a ‘closed system’ since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.

Conclusion/Significance

We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional ‘loading’ or ‘reference’ standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

Chloroplast thylakoid protein phosphatase is a membrane surface-associated activity

Chloroplast thylakoid protein phosphatase activity was measured using ³²P-labeled histone as an exogenous substrate and an assay of the ³²Pi released involving formation of a phosphomolybdate complex and organic extraction. The activity was liberated from wheat (Triticum aestivum) thylakoids by washing the membranes in NaCl-containing solutions followed by centrifugation. The liberated phosphatase activity had a pH optimum of approximately 6.75, was inhibited by addition of 10 millimolar EDTA or EGTA, and was stimulated by addition of millimolar amounts of dithiothreitol, magnesium, manganese, or calcium ions. The rate of thylakoid protein dephosphorylation was decreased following liberation of a portion of the protein phosphatase activity and was increased by addition of salt-liberated phosphatase fraction. These results suggest that at least a portion of wheat thylakoid protein phosphatase is a peripheral, rather than an integral, membrane protein.