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Identification of three previosly unknown in vivo phosphorylation sites in thylakoid membranes of Arabidopsis thaliana
Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cellbiology. Linköping University, Faculty of Health Sciences.
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.

Place, publisher, year, edition, pages
2003. Vol. 2, no 8, 550-559 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-13859DOI: 10.1074/mcp.M300050-MCP200OAI: diva2:21930
Available from: 2006-09-07 Created: 2006-09-07
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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