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The mobile thylakoid phosphoprotein TSP9 interacts with the light harvesting complex II and the peripheries of both photosystems
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden .
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
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2006 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, nr 22, s. 16214-16222Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2006. Vol. 282, nr 22, s. 16214-16222
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-13862DOI: 10.1074/jbc.M605833200OAI: oai:DiVA.org:liu-13862DiVA, id: diva2:21933
Tillgänglig från: 2006-09-07 Skapad: 2006-09-07 Senast uppdaterad: 2010-05-24
Ingår i avhandling
1. Molecular characterization of protein phosphorylation in plant photosynthetic membranes
Öppna denna publikation i ny flik eller fönster >>Molecular characterization of protein phosphorylation in plant photosynthetic membranes
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 959
Nyckelord
protein phosphorylation, photosynthesis, mass spectrometry, protein characterization
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:liu:diva-6665 (URN)91-85497-92-4 (ISBN)
Disputation
2006-10-13, Linden, HU, ing 65, Hälsouniversitetet, Linköping, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2006-09-07 Skapad: 2006-09-07 Senast uppdaterad: 2018-01-13

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Hansson, MariaAndersson, BertilVener, Alexander V.

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