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Functional proteomics of protein phosphorylation in algal photosynthetic membranes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery.

The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light.

Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific.

We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon.

This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

Place, publisher, year, edition, pages
Linköping University Electronic Press, 2008. , 40 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1038
Keyword [en]
Protein phosphorylation, mass spectrometry, photosynthesis, proteomics
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-10708ISBN: 978-91-85523-02-3 (print)OAI: oai:DiVA.org:liu-10708DiVA: diva2:17417
Public defence
2008-02-29, Linden, ingång 65, Hälsouniversitetet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2008-02-06 Created: 2008-02-06 Last updated: 2015-11-19
List of papers
1. CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii
Open this publication in new window or tab >>CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii
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2006 (English)In: Proteomics, ISSN 1615-9853, Vol. 6, no 9, 2693-2704 p.Article in journal (Refereed) Published
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.

Keyword
Amino acid sequencing, Chlamydomonas reinhardtii, CO2 limitation, Mass spectrometry, Protein phosphorylation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12927 (URN)10.1002/pmic.200500461 (DOI)
Available from: 2008-02-06 Created: 2008-02-06 Last updated: 2009-06-05
2. The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation
Open this publication in new window or tab >>The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation
2004 (English)In: FEBS letters, ISSN 0014-5793, Vol. 564, no 1-2, 104-108 p.Article in journal (Refereed) Published
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.

Keyword
Transit peptide, Thylakoid membrane, CP29, Protein phosphorylation, Mass spectrometry, Chlamydomonas reinhardtii
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12928 (URN)10.1016/S0014-5793(04)00323-0 (DOI)
Available from: 2008-02-06 Created: 2008-02-06 Last updated: 2009-06-05
3. Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions
Open this publication in new window or tab >>Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions
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2005 (English)In: The FEBS journal, ISSN 1742-464X, Vol. 272, no 18, 4797-4806 p.Article in journal (Refereed) Published
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.

Keyword
Chlamydomonas, CP29, photosynthesis, protein phosphorylation, State transitions
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12929 (URN)10.1111/j.1742-4658.2005.04894.x (DOI)
Available from: 2008-02-06 Created: 2008-02-06
4. Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii
Open this publication in new window or tab >>Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii
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2006 (English)In: Molecular & cellular proteomics, ISSN 1535-9476, Vol. 5, no 8, 1412-1425 p.Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12930 (URN)10.1074/mcp.M600066-MCP200 (DOI)
Available from: 2008-02-06 Created: 2008-02-06 Last updated: 2009-06-05

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