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  • 1.
    Aboulaich, Nabila
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ortegren, Unn
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Association and insulin regulated translocation of hormone-sensitive lipase with PTRF2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 350, no 3, p. 657-661Article in journal (Refereed)
    Abstract [en]

    Polymerase I and transcript release factor (PTRF) is in human adipocytes mainly localized at the plasma membrane. This localization was under control of insulin, which translocated PTRF to the cytosol and nucleus, indicating a novel role for PTRF in insulin transcriptional control. In the plasma membrane PTRF was specifically bound to a triacylglycerol-metabolizing subclass of caveolae containing hormone-sensitive lipase (HSL). In response to insulin PTRF was translocated to the cytosol in parallel with HSL. PTRF and HSL were quantitatively immunoprecipitated from the cytosol by antibodies against either PTRF or HSL. The findings indicate also a novel extranuclear function for PTRF in the control of lipolysis.

  • 2.
    Aboulaich, Nabila
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vainonen, Julia P
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes2004In: The Biochemical journal, ISSN 1470-8728, Vol. 383, no Pt 2, p. 237-248Article in journal (Refereed)
    Abstract [en]

    Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

  • 3.
    Aboulaich, Nabila
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 17, p. 11446-11449Article in journal (Refereed)
    Abstract [en]

    In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

  • 4.
    Aro, Eva-Mari
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Rokka, Anne
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Determination of phosphoproteins in higher plant thylakoids2004In: Methods in Molecular Biology / [ed] Walker, John M., Totowa, New Jersey: Humana Press Inc , 2004, p. 271-285Chapter in book (Other academic)
    Abstract [en]

    For almost 30 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.  These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, all protocols from the series are indexed in Pub Med, comprehensive and reliable.

  • 5.
    Bronnikov, Gennady
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Aboulaich, Nabila
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Strålfors, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 367, no 1, p. 201-207Article in journal (Refereed)
    Abstract [en]

    The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing VMAX and KM for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1. © 2007 Elsevier Inc. All rights reserved.

  • 6.
    Carlberg, Inger
    et al.
    Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University.
    Hansson, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kieselbach, Thomas
    Department of Medical Nutrition and Biosciences, Karolinska Institute, Huddinge, Sweden.
    Schröder, Wolfgang P.
    Department of Biochemistry, Umeå University, Umeå, Sweden .
    Andersson, Bertil
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes2003In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 2, p. 757-762Article in journal (Refereed)
    Abstract [en]

    The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.

  • 7.
    Edvardsson, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Eshaghi, Said
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    Vener, Alexander V.
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    Andersson, Bertil
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP202003In: FEBS Letters, ISSN 0014-5793, Vol. 542, no 1-3, p. 137-141Article in journal (Refereed)
    Abstract [en]

    Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl-prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 ( hylakoid umen PIase of kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N-termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.

  • 8.
    Edvardsson, Anna
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Shapiguzov, Alexey
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Petersson, Ulrika A
    Schröder, Wolfgang P
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-22007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 33, p. 9432-9442Article in journal (Refereed)
    Abstract [en]

    The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates. © 2007 American Chemical Society.

  • 9.
    Edvardsson, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Shapiguzov, Alexey
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Petersson, Ulrika A.
    Schröder, Wolfgang P.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Knockout of AtCYP20-2 confirms degeneration of peptidyl-prolyl isomerase activity of immunophilins in the thylakoid lumen of Arabidopsis thaliana2007Article in journal (Refereed)
  • 10.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Carlberg, Inger
    Department of Biochemistry and Biophysics, Stockholm UniVersity, Stockholm, Sweden.
    Zygadlo, Agnieszka
    Department of Plant Biology, UniVersity of Copenhagen, Copenhagen, Denmark.
    Piippo, Mirva
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Nurmi, Markus
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Aro, Eva-Mari
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Vibe Scheller, Henrik
    Department of Plant Biology, UniVersity of Copenhagen, Copenhagen, Denmark.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Intrinsically Unstructured Phosphoprotein TSP9 Regulates Light Harvesting in Arabidopsis thaliana2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 2, p. 499-509Article in journal (Refereed)
    Abstract [en]

    Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

  • 11.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Granath, Pontus
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 6Article in journal (Refereed)
    Abstract [en]

    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.

  • 12.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP292011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9Article in journal (Refereed)
    Abstract [en]

    Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the protein complexes revealed that the high light treatment of the wild type caused migration of CP29 from the PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of the PSII supercomplexes in plants exposed to high light operate via the STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions.

  • 13.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Wasilewska, Wioleta
    Warsaw University, Poland .
    Romanowska, Elzbieta
    Warsaw University, Poland .
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 18, p. 2852-2861Article in journal (Refereed)
    Abstract [en]

    In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns. LC-MS/MS with alternating CID and electron transfer dissociation sequencing of peptide ions mapped 15 protein phosphorylation sites. Phosphorylated D2, CP29, CP26, Lhcb2 proteins, and ATPsynthase were found only in M membranes. A previously unknown phosphorylation site was mapped in phosphoenolpyruvate carboxykinase from the BS cells. Phosphorylation stoichiometry was calculated from the ratios of normalized ion currents for phosphorylated to nonphosphorylated peptide pairs from the D1, D2, CP43, and PbsH proteins of photosystem II (PSII). Every PSII in M thylakoids contained on average 1.5 +/- 0.1 or 2.3 +/- 0.2 phosphoryl groups in plants grown under either low or high light, while in BS membranes the corresponding numbers were 0.25 +/- 0.1 or 0.7 +/- 0.2, respectively. It is suggested that the phosphorylation level, as well as turnover of PSII depend on the structure of thylakoids.

  • 14.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Willig, Adrian
    Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.
    Granath, Pontus
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Crevecoeur, Michele
    Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.
    Rochaix, Jean-David
    Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211 Geneva 4, Switzerland.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis2009In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 21, no 12, p. 3950-3964Article in journal (Refereed)
    Abstract [en]

    Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to highlight. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.

  • 15.
    Hansson, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Dupuis, Tiphaine
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
    Strömquist, Ragna
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
    Andersson, Bertil
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Carlberg, Inger
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
    The mobile thylakoid phosphoprotein TSP9 interacts with the light harvesting complex II and the peripheries of both photosystems2006In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, no 22, p. 16214-16222Article in journal (Refereed)
    Abstract [en]

    The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.

  • 16.
    Hansson, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Identification of three previosly unknown in vivo phosphorylation sites in thylakoid membranes of Arabidopsis thaliana2003In: Molecular and Cellular Proteomics, ISSN 1535-9476, Vol. 2, no 8, p. 550-559Article in journal (Refereed)
    Abstract [en]

    The proteins in plant photosynthetic thylakoid membranes undergo light-induced phosphorylation, but only a few phosphoproteins have been characterized. To access the unknown sites of in vivo protein phosphorylation the thylakoid membranes were isolated from Arabidopsis thaliana grown in normal light, and the surface-exposed peptides were cleaved from the membranes by trypsin. The peptides were methylated and subjected to immobilized metal affinity chromatography, and the enriched phosphopeptides were sequenced using tandem nanospray quadrupole time-of-flight mass spectrometry. Three new phosphopeptides were revealed in addition to the five known phosphorylation sites in photosystem II proteins. All phosphopeptides are found phosphorylated at threonine residues implementing a strict threonine specificity of the thylakoid kinases. For the first time protein phosphorylation is found in photosystem I. The phosphorylation site is localized to the first threonine in the N terminus of PsaD protein that assists in the electron transfer from photosystem I to ferredoxin. A new phosphorylation site is also revealed in the acetylated N terminus of the minor chlorophyll a-binding protein CP29. The third novel phosphopeptide, composed of 25 amino acids, belongs to a nuclear encoded protein annotated as "expressed protein" in the Arabidopsis database. The protein precursor has a chloroplast-targeting peptide followed by the mature protein with two transmembrane helices and a molecular mass of 14 kDa. This previously uncharacterized protein is named thylakoid membrane phosphoprotein of 14 kDa (TMP14). The finding of the novel phosphoproteins extends involvement of the redox-regulated protein phosphorylation in photosynthetic membranes beyond the photosystem II and its light-harvesting antennae.

  • 17.
    Ingelsson, Björn
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Shapiguzov, Alexey
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kieselbach, Thomas
    Umeå University.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    PeptidylProlyl Isomerase Activity in Chloroplast Thylakoid Lumen is a Dispensable Function of Immunophilins in Arabidopsis thaliana2009In: Plant and Cell Physiology, ISSN 0032-0781, E-ISSN 1471-9053, Vol. 50, no 10, p. 1801-1814Article in journal (Refereed)
    Abstract [en]

    Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidylprolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatographymass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2 of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

  • 18.
    Ingelsson, Björn
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC162012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 9, p. 1265-1271Article in journal (Refereed)
    Abstract [en]

    Light-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1–1.3 proteins; and (ii) phosphorylation of Thr-451 in pTAC16 protein was STN7-dependent. pTAC16 was found distributed between thylakoids and nucleoid. Its knockout did not affect the nucleoid protein composition and the Thr-451 phosphorylated protein was excluded from the nucleoid. Thr-451 of pTAC16 is conserved in all studied plants and its phosphorylation may regulate membrane-anchoring functions of the nucleoid.

  • 19.
    Jufvas, Åsa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Sjödin, Simon
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Lundqvist, Kim
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Amin, Risul
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes.2013In: Clinical Epigenetics, E-ISSN 1868-7083, Vol. 5, no 1, article id 15Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies.

    RESULTS: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation compared with cells from normal-weight subjects. In contrast, trimethylation at lysine 4 was 40% higher in adipocytes from overweight diabetic subjects compared with normal-weight and overweight non-diabetic subjects. There was no association between level of modification and age of subjects.

    CONCLUSIONS: The findings define genome-wide molecular modifications of histones in adipocytes that are directly associated with overweight and diabetes, and thus suggest a molecular basis for existing epidemiological evidence of epigenetic inheritance.

  • 20.
    Jufvas, Åsa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 1Article in journal (Refereed)
    Abstract [en]

    Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 mu g of protein with histone content of 60%-70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments.

  • 21.
    Kargul, Joanna
    et al.
    Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK .
    Turkina, Maria V
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Nield, Jon
    Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK .
    Benson, Sam
    Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK .
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Barber, James
    Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK .
    Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions2005In: The FEBS journal, ISSN 1742-464X, Vol. 272, no 18, p. 4797-4806Article in journal (Refereed)
    Abstract [en]

    The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI–PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.

  • 22.
    Khotin, Mikhail
    et al.
    Russian Acadamy of Science.
    Turoverova, Lidia
    Russian Acadamy of Science.
    Aksenova, Vasilisa
    Russian Acadamy of Science.
    Barlev, Nikolai
    Russian Acadamy of Science.
    Borutinskaité, Veronika
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Bajenova, Olga
    St Petersburg State University.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Pinaev, George P.
    Russian Acadamy of Science.
    Tentler, Dmitri
    Russian Acadamy of Science.
    Proteomic analysis of ACTN4-interacting proteins reveals its a putative involvement in mRNA metabolism2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 397, no 2, p. 192-196Article in journal (Refereed)
    Abstract [en]

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  • 23.
    Khrouchtchova, Anastassia
    et al.
    Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
    Hansson, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Paakkarinen, Virpi
    Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland.
    Vainonen, Julia P.
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Zhang, Suping
    Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
    Jensen, Poul Erik
    Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
    Vibe Scheller, Henrik
    Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
    Aro, Eva-Mari
    Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland.
    Haldrup, Anna
    Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
    A previosly found thylakoid membrane protein of 14 kDa (TMP14) is a novel subunit of photosystem I and is designated PSI-P2005In: FEBS Letters, ISSN 0014-5793, Vol. 579, no 21, p. 4808-4812Article in journal (Refereed)
    Abstract [en]

    We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS–PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.

  • 24.
    Klang Årstrand, Hanna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Phosphorylation of ribosomal proteins in cytoplasm and nucleus of Arabidopsis thalianaManuscript (preprint) (Other academic)
    Abstract [en]

    Ribosome assembly is a complicated process that takes place in both the cell cytosol and nucleus. Shuttling between these compartments of both ribosomal proteins and ribosome subunits is necessary for the formation of active ribosomes. The goal of this work was to make a comparison of ribosomal proteins from nucleus and cytosol of Arabidopsis thaliana. We made separate preparations of ribosomes from cytosol and nucleus. Our proteomic analysis of ribosomes isolated from Arabidopsis thaliana leaves detected 146 ribosomal proteins from cytosol and 135 ribosomal proteins from the cell nucleus. Phosphopeptides from these preparations were enriched using TiO2 and analyzed using nanoLC-MS/MS. This method allowed us to identify 13 phosphopeptides from 11 ribosomal proteins: S2-3, S6-1, S6-2, L13-1, L13-3, L29-1, P0-2, P0-3, P1 (P1-1, P1-2, P1-3), P2 (2-1, 2-2, 2-3), P3 (3-1, 3-2, 3-3) and additionally two phosphopeptides from two ribosomal associated proteins: Nascent polypeptide-associated complex subunit alpha-like protein 1 and 3.

  • 25.
    Lemeille, Sylvain
    et al.
    University of Geneva.
    Turkina, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Rochaix, Jean-David
    University of Geneva.
    Stt7-dependent Phosphorylation during State Transitions in the Green Alga Chlamydomonas reinhardtii2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 6, p. 1281-1295Article in journal (Refereed)
    Abstract [en]

    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 b(6)f 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 Ser(533) 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.

  • 26.
    Lundin, Björn
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hansson, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Schoefs, Benoít
    Dynamique Vacuolaire et Réponses aux Stress de l'Environnement, Université de Bourgogne, Dijon cedex, France.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Spetea (Wiklund), Cornelia
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein2007In: The Plant Journal, ISSN 0960-7412, Vol. 49, no 3, p. 528-539Article in journal (Refereed)
    Abstract [en]

    The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.

  • 27. Rokka, Anne
    et al.
    Aro, Eva-Mari
    Herrmann, Reinhold
    Andersson, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature.2000In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 123, p. 1525-1535Article in journal (Refereed)
  • 28.
    Rokka, Anne
    et al.
    University of Turku, Turku, Finland.
    Aro, Eva-Mari
    University of Turku, Turku, Finland.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Thylakoid phosphoproteins: identification of phosphorylation sites.2011In: Photosynthesis Research Protocols, Humana Press, 2011, Vol. 684, p. 171-186Chapter in book (Other academic)
    Abstract [en]

    Redox-dependent thylakoid protein phosphorylation regulates both the short- and long-term acclimation of the photosynthetic apparatus to changes in environmental conditions. The major thylakoid phosphoproteins belong to photosystem II (D1, D2, CP43, PsbH) and its light-harvesting antenna (Lhcb1, Lhcb2, CP29), but a number of minor phosphoproteins have also been identified. The detection methods traditionally include the radiolabeling techniques, electrophoretic separation of the phosphorylated and unphosphorylated forms of the protein, and the use of phosphoamino acid antibodies or phosphoprotein-specific dyes. The recent progress in mass spectrometry techniques and methods of proteomics allow for the successful identification and analyses of protein phosphorylation. In mass spectrometry approaches no exogenous tracer is needed and natural phosphorylation of proteins can be characterized with high sensitivity yielding the mapping of exact phosphorylation sites in the proteins as well. Various methods for the detection of thylakoid phosphoproteins, including the preparation of phosphopeptides for mass spectrometric analyses and techniques for phosphopeptide identification by electrospray ionization mass spectrometry (ESI-MS) are described. The experimental protocols for simultaneous identification of multiple phosphopeptides in complex peptide mixtures, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC), and for their sequencing by tandem spectrometry are outlined.

  • 29.
    Romano, Patrick G.N.
    et al.
    Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom.
    Edvardsson, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ruban, Alexander V.
    Andersson, Bertil
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Gray, Julie E.
    Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom.
    Horton, Peter
    Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom.
    Arabidopsis AtCYP20-2 is a light-regulated cyclophilin-type peptidyl-prolyl cis-trans isomerase associated with the photosynthetic membranes2004In: Plant Physiology, ISSN 0032-0889, Vol. 134, no 4, p. 1244-1247Article in journal (Refereed)
  • 30.
    Romanowska, Elzbieta
    et al.
    Warsaw University.
    Wasilewska, Wioleta
    Warsaw University.
    Fristedt, Rikard
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Zienkiewicz, Maksymilian
    Warsaw University.
    Phosphorylation of PSII proteins in maize thylakoids in the presence of Pb ions2012In: Journal of plant physiology (Print), ISSN 0176-1617, E-ISSN 1618-1328, Vol. 169, no 4, p. 345-352Article in journal (Refereed)
    Abstract [en]

    Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream. The photochemical efficiency of PSII, measured as an Fv/Fm ratio, in the maize leaves treated with Pb was only 10% lower than in control leaves. The PSII activity was not affected by Pb ions in mesophyll thylakoids, whereas in bundle sheath it was reduced. Protein phosphorylation in mesophyll and bundle sheath thylakoids was analyzed using mass spectrometry and protein blotting before and after lead treatment. Both methods clearly demonstrated increase in phosphorylation of the PSII proteins upon treatment with Pb2+, however, the extent of D1, D2 and CP43 phosphorylation in the mesophyll chloroplasts was clearly higher than in bundle sheath cells. We found that in the presence of Pb ions there was no detectable dephosphorylation of the strongly phosphorylated D1 and PsbH proteins of PSII complex in darkness or under far red light. These results suggest that Pb2+ stimulates phosphorylation of PSII core proteins, which can affect stability of the PSII complexes and the rate of D1 protein degradation. Increased phosphorylation of the PSII core proteins induced by Pb ions may be a crucial protection mechanism stabilizing optimal composition of the PSII complexes under metal stress conditions. Our results show that acclimation to Pb ions was achieved in both types of maize chloroplasts in the same way. However, these processes are obviously more complex because of different metabolic status in mesophyll and bundle sheath chloroplasts.

  • 31.
    Samol, Iga
    et al.
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Shapiguzov, Alexey
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Ingelsson, Björn
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Fucile, Geoffrey
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Crèvecoeur, Michèle
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Rochaix, Jean-David
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Goldschmidt-Clermont, Michel
    Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai E. Ansermet, 1211 Genève 4, Switzerland.
    Identification of a Photosystem II Phosphatase Involved in Light Acclimation in Arabidopsis2012In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 24, no 6, p. 2596-2609Article in journal (Refereed)
    Abstract [en]

    Reversible protein phosphorylation plays a major role in the rapid acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STN7 phosphorylates LHCII, the light harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8 which is mainly involved in phosphorylation of PSII influences folding of the thylakoid membranes and repair of PSII after photo-damage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases.

    In a reverse genetic screen we have identified the chloroplast PP2C phosphatase, PBCP (PHOTOSYSTEM II CORE PHOSPHATASE), which is required for efficient dephosphorylation of PSII. Its targets identified by immunoblotting and mass spectrometry largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding and degradation of D1 after photo-damage are affected in the absence of PBCP, while its over-expression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological function are distinct from those of STN7 and the counteracting phosphatase PPH1 (TAP38), but their activities may overlap to some degree.

  • 32.
    Shapiguzov, Alexey
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Edvardsson, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen2006In: FEBS Letters, ISSN 0014-5793, Vol. 580, no 15, p. 3671-3676Article in journal (Refereed)
    Abstract [en]

    Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.

  • 33.
    Shapiguzov, Alexey
    et al.
    University of Geneva, Switzerland.
    Ingelsson, Björn
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Samol, Iga
    University of Geneva, Switzerland.
    Andres, Charles
    University of Neuchatel, Switzerland.
    Kessler, Felix
    University of Neuchatel, Switzerland.
    Rochaix, Jean-David
    University of Geneva, Switzerland.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Goldschmidt-Clermont, Michel
    University of Geneva, Switzerland.
    The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 10, p. 4782-4787Article in journal (Refereed)
    Abstract [en]

    The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear tobe specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.

  • 34.
    Sirpio, Sari
    et al.
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Turku, Finland.
    Khrouchtchova, Anastassia
    Department of Plant Biology University of Copenhagen, Frederiksberg, Denmark.
    Allahverdiyeva, Yagut
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Turku, Finland.
    Hansson, Maria
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Fristedt, Rikard
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Scheller, Henrik Vibe
    Department of Plant Biology University of Copenhagen, Frederiksberg, Denmark.
    Jensen, Poul Erik
    Department of Plant Biology University of Copenhagen, Frederiksberg, Denmark.
    Haldrup, Anna
    Department of Plant Biology University of Copenhagen, Frederiksberg, Denmark.
    Aro, Eva-Mari
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Turku, Finland.
    AtCYP38 ensures early biogenesis, correct assembly and sustenance of photosystem II2008In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 55, no 4, p. 639-651Article in journal (Refereed)
    Abstract [en]

    AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn 4-Ca cluster even with high turnover of PSII. © 2008 The Authors.

  • 35. Song, Jikiu
    et al.
    Lee, Min S
    Carlberg, Inger
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Markley, John L
    Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 51, p. 15633-15643Article in journal (Refereed)
    Abstract [en]

    Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal α-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal α-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function. © 2006 American Chemical Society.

  • 36. Ståhl, Annelie
    et al.
    Nilsson, Stefan
    Lundberg, Pontus
    Bhushan, Shashi
    Biverståhl, Henrik
    Moberg, Per
    Morisset, Magali
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Mäler, Lena
    Langel, Ulo
    Glaser, Elzbieta
    Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 4, p. 847-860Article in journal (Refereed)
    Abstract [en]

    Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F 1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P′1 position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent. © 2005 Elsevier Ltd. All rights reserved.

  • 37. Tikkanen, Mikko
    et al.
    Piippo, Mirva
    Suorsa, Marjaana
    Sirpiö, Sari
    Mulo, Paula
    Vainonen, Julia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Allahverdiyeva, Yagut
    Aro, Eva-Mari
    State transitions revisited - A buffering system for dynamic low light acclimation of Arabidopsis2006In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 62, no 4-5, p. 779-793Article in journal (Refereed)
    Abstract [en]

    The mobile part of the light-harvesting chlorophyll (chl) a/b protein complex (LHCII), composed of the Lhcb1 and Lhcb2 proteins, is the basic unit of chloroplast state transitions-the short term tuning system in balancing the excitation energy between Photosystem (PS) II and PSI. State transitions are catalysed by the thylakoid associated STN7 kinase, and we show here that besides the phosphorylation of the Lhcb1 and Lhcb2 proteins, also the phosphorylation of Lhcb4.2 (CP29) is under the control of the STN7 kinase. Upon growth of Arabidopsis WT and stn7 mutant plants under low and moderate light conditions, the WT plants favoured state 2 whereas stn7 was locked in state 1. The lack of the STN7 kinase and state transitions in stn7 also modified the thylakoid protein contents upon long-term low light acclimation resulting, for example, in low Lhcb1 and in elevated Lhca1 and Lhca2 protein amounts as compared to WT. Adjustments of thylakoid protein contents probably occurred at post-transcriptional level since the DNA microarray experiments from each growth condition did not reveal any significant differences between stn7 and WT transcriptomes. The resulting high Lhcb2/Lhcb1 ratio in stn7 upon growth at low light was accompanied by lower capacity for NPQ than in WT. On the contrary, higher amounts of PsbS in stn7 under moderate and high light growth conditions resulted in higher NPQ compared to WT and consequently also in a protection of PSII against photoinhibition. STN7 kinase and the state transitions are suggested to have a physiological significance for dynamic acclimation to low but fluctuating growth light conditions. They are shown to function as a buffering system upon short high light illumination peaks by shifting the thylakoids from state 2 to state 1 and thereby down regulating the induction of stress-responsive genes, a likely result from transient over-reduction of PSI acceptors. © 2006 Springer Science+Business Media B.V.

  • 38.
    Turkina, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Klang, Hanna
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Differential Phosphorylation of Ribosomal Proteins in Arabidopsis thaliana Plants during Day and Night2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12Article in journal (Refereed)
    Abstract [en]

    Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night. Proteomic analyses of preparations corresponding to both phases of the photoperiod detected phosphorylation at eight serine residues in the C-termini of six ribosomal proteins: S2-3, S6-1, S6-2, P0-2, P1 and L29-1. This included previously unknown phosphorylation of the 40S ribosomal protein S6 at Ser-231. Relative quantification of the phosphorylated peptides using stable isotope labeling and mass spectrometry revealed a 2.2 times increase in the day/night phosphorylation ratio at this site. Phosphorylation of the S6-1 and S6-2 variants of the same protein at Ser-240 increased by the factors of 4.2 and 1.8, respectively. The 1.6 increase in phosphorylation during the day was also found at Ser-58 of the 60S ribosomal protein L29-1. It is suggested that differential phosphorylation of the ribosomal proteins S6-1, S6-2 and L29-1 may contribute to modulation of the diurnal protein synthesis in plants.

  • 39.
    Turkina, Maria V
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Blanco-Rivero, Amaya
    Department of Plant Physiology, Umeå University, Umeå, Sweden.
    Vainonen, Julia P
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Villarejo, Arsenio
    Department of Plant Physiology, Umeå University, Umeå, Sweden.
    CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii2006In: Proteomics, ISSN 1615-9853, Vol. 6, no 9, p. 2693-2704Article in journal (Refereed)
    Abstract [en]

    Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.

  • 40.
    Turkina, Maria V
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kargul, Joanna
    Wolfson Laboratories, Division of Molecular Biosciences, Faculty of Life Sciences, Imperial College London.
    Blanco-Rivero, Amaya
    Department of Plant Physiology, Umeå University, Umeå, Sweden.
    Villarejo, Arsenio
    Department of Plant Physiology, Umeå University, Umeå, Sweden.
    Barber, James
    Wolfson Laboratories, Division of Molecular Biosciences, Faculty of Life Sciences, Imperial College London.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii2006In: Molecular & cellular proteomics, ISSN 1535-9476, Vol. 5, no 8, p. 1412-1425Article in journal (Refereed)
    Abstract [en]

    Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.

  • 41.
    Turkina, Maria V
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Identification of phosphorylated proteins.2007In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 355, p. 305-316Article in journal (Refereed)
    Abstract [en]

    Reversible protein phosphorylation is crucially involved in all aspects of plant cell physiology. The highly challenging task of revealing and characterizing the dynamic protein phosphorylation networks in plants has only recently begun to become feasible, owing to application of dedicated proteomics and mass spectrometry techniques. The experimental methodology that identified most of the presently known proteins phosphorylated in vivo is based on protein cleavage with trypsin, following chromatographic enrichment of phosphorylated peptides and mass spectrometric fragmentation and sequencing of these phosphopeptides. This procedure is most efficient when it is limited to the tryptic digestion of proteins in distinct isolated fractions or compartments of plant cells. Immobilized metal affinity chromatography (IMAC) is most useful for phosphopeptide enrichment after methylation of the peptides in the complex protein digests. The following tandem mass spectrometry of the isolated phosphopeptides results in both identification of phosphorylated proteins and mapping of the in vivo phosphorylation sites. The relative quantitation of the extent of phosphorylation at individual protein modification sites may be accomplished by either stable isotope labeling technique or dedicated liquid chromatography-mass spectrometry measurements.

  • 42.
    Turkina, Maria V
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Villarejo, Arsenio
    Umeå Plant Science Center, Department of Plant Physiology, University of Umeå, Umeå, Sweden.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation2004In: FEBS letters, ISSN 0014-5793, Vol. 564, no 1-2, p. 104-108Article in journal (Refereed)
    Abstract [en]

    The surface-exposed peptides were cleaved by trypsin from the photosynthetic thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. Two phosphorylated peptides, enriched from the peptide mixture and sequenced by nanospray quadrupole time-of-flight mass spectrometry, revealed overlapping sequences corresponding to the N-terminus of a nuclear-encoded chlorophyll a/b-binding protein CP29. In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the mature algal CP29 was not removed but processed by methionine excision, N-terminal acetylation and phosphorylation on threonine 6. The importance of this phosphorylation site is proposed as the reason of the unique transit peptide retention.

  • 43.
    Vainonen, Julia P
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Aboulaich, Nabila
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Turkina, Maria V
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    N-terminal processing and modifications of caveolin-1 in caveolae from human adipocytes2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 320, no 2, p. 480-486Article in journal (Refereed)
    Abstract [en]

    Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.

  • 44.
    Vainonen, Julia P.
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hansson, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins2005In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 280, no 39, p. 33679-33686Article in journal (Refereed)
    Abstract [en]

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

  • 45.
    Vainonen, Julia P.
    et al.
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Sakuragi, Yumiko
    Department of Plant Biology, Faculty of Life Sciences University of Copenhagen, Denmark.
    Stael, Simon
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Tikkanen, Mikko
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Allahverdiyeva, Yagut
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Paakkarinen, Virpi
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Aro, Eveliina
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Suorsa, Marjaana
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Scheller, Henrik V.
    Department of Plant Biology, Faculty of Life Sciences University of Copenhagen, Denmark.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Aro, Eva-Mari
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 8, p. 1767-1777Article in journal (Refereed)
    Abstract [en]

    Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development. © 2008 The Authors.

  • 46.
    Vainonen, Julia P
    et al.
    University of Turku, Turku, Finland.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Aro, Eva-Mari
    University of Turku, Turku, Finland.
    Determination of in vivo Protein Phosphorylation in Photosynthetic Membranes2009In: Plant Signal Transduction: Methods and Protocols / [ed] Thomas Pfannschmidt, New York, NY, United States: Humana Press, 2009, Vol. 479, p. 133-146Chapter in book (Other academic)
    Abstract [en]

    Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic separation of proteins and detection of phosphoproteins with a phosphoaminoacid-specific antibody or phosphoprotein-specific dye. The use of mass spectrometry allows the identification of exact phosphorylation site(s) in the proteins. Various methods for detection of phosphoproteins in thylakoids are outlined including phosphopeptide preparation for mass spectrometric analyses and quantitative analysis of protein phosphorylation.

  • 47.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 6, p. 449-457Article in journal (Refereed)
    Abstract [en]

    Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO2 availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes. © 2006 Elsevier B.V. All rights reserved.

  • 48.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Peptidyl-prolyl isomerases and regulation of photosynthetic functions.2001In: Regulation of photosynthesis / [ed] Eva-Mari Aro and Bertil Andersson, Linköping: Linköpings universitet , 2001, p. 177-193Chapter in book (Other academic)
    Abstract [en]

    The book covers the expression of photosynthesis related genes including regulation both at transcriptional and translational levels. Biogenesis, turnover and senescence of thylakoid pigment protein complexes are reviewed and some crucial regulatory steps in carbon metabolism are highlighted. The stress and acclimation responses in chloroplasts are examined at molecular level. The book also provides examples of novel methods for studies of photosynthetic regulation.

  • 49.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Phosphorylation of thylakoid proteins2008In: Photoprotection, photoinhibition, gene regulation, and environment / [ed] Barbara Demmig-Adams, William W. Adams, Autar Mattoo., Holland: Springer , 2008, p. 107-126Chapter in book (Other academic)
    Abstract [en]

    Photoprotection, Photoinhibition, Gene Regulation, and Environment examines the processes whereby plants monitor environmental conditions and orchestrate their response to change, an ability paramount to the life of all plants. "Excess light", absorbed by the light-harvesting systems of photosynthetic organisms, is an integrative indicator of the environment, communicating the presence of intense light and any conditions unfavorable for growth and photosynthesis. Key plant responses are photoprotection and photoinhibition. In this volume, the dual role of photoprotective responses in the preservation of leaf integrity and in redox signaling networks modulating stress acclimation, growth, and development is addressed. In addition, the still unresolved impact of photoinhibition on plant survival and productivity is discussed. Specific topics include dissipation of excess energy via thermal and other pathways, scavenging of reactive oxygen by antioxidants, proteins key to photoprotection and photoinhibition, peroxidation of lipids, as well as signaling by reactive oxygen, lipid-derived messengers, and other messengers that modulate gene expression. Approaches include biochemical, physiological, genetic, molecular, and field studies, addressing intense visible and ultraviolet light, winter conditions, nutrient deficiency, drought, and salinity

  • 50.
    Vener, Alexander
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Harms, A.
    Univ. of Wisconsin Biotech. Center, University of Wisconsin, Madison, WI 53706, United States.
    Sussman, M.R.
    Cell. and Molecular Biology Program, Department of Horticulture, University of Wisconsin, Madison, WI 53706, United States, Univ. of Wisconsin Biotech. Center, University of Wisconsin, Madison, WI 53706, United States.
    Vierstra, R.D.
    Cell. and Molecular Biology Program, Department of Horticulture, University of Wisconsin, Madison, WI 53706, United States, Dept. of Horticulture, University of Wisconsin, 1575 Linden Dr., Madison, WI 53706, United States.
    Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes of Arabidopsis thaliana2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 10, p. 6959-6966Article in journal (Refereed)
    Abstract [en]

    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 Dl, 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 Dl, 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.

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