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A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes
Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Department of Medical Nutrition and Biosciences, Karolinska Institute, Huddinge, Sweden.
Department of Biochemistry, Umeå University, Umeå, Sweden .
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2003 (English)In: Proceedings of the National Academy Science of the United States of America, ISSN 1091-6490 (online), 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
2003. Vol. 100, no 2, 757-762 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-13861DOI: 10.1073/pnas.0235452100OAI: diva2:21932
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2009-05-07
In thesis
1. Molecular characterization of protein phosphorylation in plant photosynthetic membranes
Open this publication in new window or tab >>Molecular characterization of protein phosphorylation in plant photosynthetic membranes
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.

Linköping University Medical Dissertations, ISSN 0345-0082 ; 959
protein phosphorylation, photosynthesis, mass spectrometry, protein characterization
National Category
Cell and Molecular Biology
urn:nbn:se:liu:diva-6665 (URN)91-85497-92-4 (ISBN)
Public defence
2006-10-13, Linden, HU, ing 65, Hälsouniversitetet, Linköping, 09:00 (English)
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2009-02-24

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Hansson, MariaAndersson, BertilVener, Alexander V.
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