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Molecular characterization of protein phosphorylation in plant photosynthetic membranes
Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Higher plants cannot move to a more favorable place when the environmental conditions are changing. To adapt to changes in light, temperature and access to water the plants had to evolve special mechanisms at the molecular level. Post-translational modifications of proteins, like phosphorylation, often serve as “on-and-off” switches in regulation of cellular activity and may affect protein-protein interactions. Photosynthesis in higher plants is regulated by reversible protein phosphorylation events, in a unique light- and redox-controlled system. Several biochemical methods are effectively used for characterization of phosphorylated proteins in photosynthetic membranes. Nevertheless, mass spectrometry is the most effective technique when it comes to identification of exact phosphorylation site(s) in the protein sequence, which is the ultimate evidence of protein phosphorylation. The same tandem mass spectrometry analysis identifies other in vivo post-translational modifications as well, such as acetylation of the N-terminus of mature protein. To study membrane proteins is a challenging project. In the present work the “shaving” of surface-exposed part of the membrane proteins, where phosphorylation occur, is used. In combination with mass spectrometry, this technique does not require the use of radioactive labeling or antibodies. The present work in spinach and Arabidopsis thaliana has identified and characterized several known phosphoproteins, new phosphorylation sites in well-known photosynthetic proteins, as well as two phosphoproteins previously unknown to be present in the photosynthetic membrane. Several photosystem II (PSII) core proteins become phosphorylated in their N-termini (D1, D2, CP43, PsbH), process involved in the regulation of the repair cycle of photo-damaged PSII complexes. The protein-protein interactions between PSII and its light harvesting complex (LHCII) seem to be affected by phosphorylation events in the interface area. In higher plants, phosphorylation sites have been identified in LHCII polypeptides, in one of the proteins (CP29) present in the interface area, as well as in the peripheral TSP9 protein. The TSP9 protein is unique among photosynthetic phosphoproteins, since it is a plant-specific soluble protein that becomes triple-phosphorylated in the middle part of the protein. It is also shown that photosystem I (PSI) is subjected to protein phosphorylation. The extrinsic PSI subunit PsaD becomes phosphorylated in its N-terminus. In addition, the latest characterized subunit of PSI, PsaP, is identified as a phosphoprotein. PsaP is an intrinsic protein assembled on the same side of the PSI complex as LHCII attaches. Several kinases are involved in phosphorylation of photosynthetic proteins, some more specific to PSII core proteins whereas others recognize LHCII proteins better. The STN8 kinase does not phosphorylate LHCII proteins, but is involved in the phosphorylation of the PSII core proteins D1, D2, CP43 and PsbH. STN8 is light-activated and is also specific in phosphorylation of threonine-4 (Thr-4) in the PsbH protein, but only after another kinase has phosphorylated Thr-2 first. A common feature of all kinases in plant photosynthetic membranes is the specificity for Thr residues and that the phosphorylation reactions occur in the N-terminal sequence of the proteins, except for the TSP9 protein. Nowadays, research is on the way to solve the complex network of regulation of photosynthetic activity via protein phosphorylation, but far more efforts are needed to get a complete view of the importance of all phosphorylation events and enzymatic specificity.

Place, publisher, year, edition, pages
2006.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 959
Keyword [en]
protein phosphorylation, photosynthesis, mass spectrometry, protein characterization
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-6665ISBN: 91-85497-92-4 (print)OAI: oai:DiVA.org:liu-6665DiVA: diva2:21935
Public defence
2006-10-13, Linden, HU, ing 65, Hälsouniversitetet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2009-02-24
List of papers
1. Identification of three previosly unknown in vivo phosphorylation sites in thylakoid membranes of Arabidopsis thaliana
Open this publication in new window or tab >>Identification of three previosly unknown in vivo phosphorylation sites in thylakoid membranes of Arabidopsis thaliana
2003 (English)In: Molecular and Cellular Proteomics, ISSN 1535-9476, Vol. 2, no 8, 550-559 p.Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13859 (URN)10.1074/mcp.M300050-MCP200 (DOI)
Available from: 2006-09-07 Created: 2006-09-07
2. A previosly found thylakoid membrane protein of 14 kDa (TMP14) is a novel subunit of photosystem I and is designated PSI-P
Open this publication in new window or tab >>A previosly found thylakoid membrane protein of 14 kDa (TMP14) is a novel subunit of photosystem I and is designated PSI-P
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2005 (English)In: FEBS Letters, ISSN 0014-5793, Vol. 579, no 21, 4808-4812 p.Article in journal (Refereed) Published
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.

Keyword
Photosystem I; Protein phosphorylation; State transitions; Thylakoids; Arabidopsis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13860 (URN)10.1016/j.febslet.2005.07.061 (DOI)
Available from: 2006-09-07 Created: 2006-09-07
3. A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes
Open this publication in new window or tab >>A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes
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2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 2, 757-762 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
National Academy of Sciences, 2003
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13861 (URN)10.1073/pnas.0235452100 (DOI)
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2017-01-03Bibliographically approved
4. The mobile thylakoid phosphoprotein TSP9 interacts with the light harvesting complex II and the peripheries of both photosystems
Open this publication in new window or tab >>The mobile thylakoid phosphoprotein TSP9 interacts with the light harvesting complex II and the peripheries of both photosystems
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, no 22, 16214-16222 p.Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13862 (URN)10.1074/jbc.M605833200 (DOI)
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2010-05-24
5. STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins
Open this publication in new window or tab >>STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins
2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 280, no 39, 33679-33686 p.Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13863 (URN)10.1074/jbc.M505729200 (DOI)
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2009-06-08

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