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Functional studies on the Light-harvesting-Like (LiL) Proteins in Cyanobacteria and Cryptophytes
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Christiane Funk)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The light-harvesting like (LiL) proteins are a widely spread group of proteins within photosynthetic organisms. They are membrane proteins composed of one to four transmembrane helices and – in homology to the light-harvesting complexes of algae and higher plants – at least one of these transmembrane helices contains the chlorophyll a/b-binding (CAB) domain. Opposite to the light-harvesting antenna complexes, LiL proteins are stress induced and they have been shown to be involved in protection of the photosynthetic apparatus. The work presented in this thesis is focused on understanding the function of one-helical LiL proteins of the cryptophyte algae Guillardia theta and the cyanobacterium Synechocystis sp. PCC 6803. G. theta contains two genes encoding LiL proteins, one is localized in the plastid (hlipP), the other in the nucleomorph (HlipNm). Both genes are expressed in normal growth condition, but they are not induced by high light. Immunostaining indicated that HlipNm is translated, but not light-induced. These proteins therefore seem not to be involved in photoprotective mechanisms of G. theta. In the cyanobacterium Synechocystis sp. PCC 6803 four one-helical LiL proteins were identified, they are called Small CAB-like Proteins (SCPs); a fifth LiL (ScpA) is fused with the ferrochelatase (FC), an enzyme involved in the heme synthesis. Our analysis revealed that SCPs are involved in the de novo assembly/repair cycle of Photosystem II, stabilizing the chlorophyll pigments at their protein scaffold. The in vitro characterization of the recombinant FC showed that ScpA is involved in the product-release of the catalytic domain of the enzyme, thereby regulating substrate availability for chlorophyll- or heme- biosynthesis. Finally, using a transcriptomic and metabolomic approaches, I was able to show that deletion of all SCP genes has profound impact on the cell organization and metabolism. In SCP-depleted cells, production of reactive oxygen species (ROS) is increased, while the amount of Photosystem II per cell volume is decreased, causing a macronutrient-deficient phenotype. Therefore, SCPs are important for stress protection and help to maintain a metabolic equilibrium within the cell.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2012. , 67 p.
Keyword [en]
Photosynthesis, cyanobacteria, Guillardia theta, photosystem II, chlorophyll-binding proteins, onehelix LiL proteins, photoprotection
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-59801ISBN: 978-91-7459-486-7 (print)OAI: oai:DiVA.org:umu-59801DiVA: diva2:556757
Public defence
2012-10-26, KBC-huset, KB3A9, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2012-10-05 Created: 2012-09-26 Last updated: 2012-09-26Bibliographically approved
List of papers
1. High-light stress and the one-helix LHC-like proteins of the cryptophyte Guillardia theta
Open this publication in new window or tab >>High-light stress and the one-helix LHC-like proteins of the cryptophyte Guillardia theta
2011 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 7, 841-846 p.Article in journal (Refereed) Published
Abstract [en]

Cryptophytes like the cryptomonad Guillardia theta are part of the marine phytoplankton and therefore major players in global carbon and biogeochemical cycles. Despite the importance for the cell in being able to cope with large changes in illumination on a daily basis, very little is known about photoprotection mechanisms in cryptophytes. Here we show that G. theta is able to perform non-photochemical quenching, although none of the usual xanthophyll cycle pigments (e.g. zeaxanthin, diadinoxanthin, diatoxanthin) are present at detectable levels. Instead, acclimation to high light intensity seems to involve an increase of alloxanthin. G. theta has genes for two one-helix "light-harvesting-like" proteins, related to some cyanobacterial genes which are induced in response to high light stress. Both the plastid-encoded gene (hlipP) and the nucleomorph-encoded gene (HlipNm) are expressed, but transcript levels decrease rather than increase during high light exposure, suggesting they are not involved in a high light stress response. The HlipNm protein was detected with a specific antibody; expression was constant, independent of the light exposure.

Keyword
Guillardia theta, Cryptomonad, High light inducible protein, Non-photochemical quenching, Light-harvesting-like protein
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-42187 (URN)10.1016/j.bbabio.2011.03.011 (DOI)21459077 (PubMedID)
Available from: 2011-04-06 Created: 2011-04-06 Last updated: 2017-12-11Bibliographically approved
2. The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: Their involvement in chlorophyll biogenesis for Photosystem II
Open this publication in new window or tab >>The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: Their involvement in chlorophyll biogenesis for Photosystem II
Show others...
2011 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 9, 1143-1151 p.Article in journal (Refereed) Published
Abstract [en]

The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the family of stress-induced light-harvesting-like proteins, but are constitutively expressed in a mutant deficient of Photosystem I (PSI). Using absorption, fluorescence and thermoluminescence measurements this PSI-less strain was compared with a mutant, in which all SCPs were additionally deleted. Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q(B) site was modified. Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. Although, the excess PBS were functionally disconnected, their fluorescence was quenched under high irradiance by the activated Orange Carotenoid Protein (OCP). Additionally the amount of OCP, but not of the iron-stress induced protein (isiA), was higher in this SCP-depleted mutant compared with the control. As previously described, the lack of SCPs affects the chlorophyll biosynthesis (Vavilin, D., Brune, D. C., Vermaas, W. (2005) Biochim Biophys Acta 1708, 91-101). We demonstrate that chlorophyll synthesis is required for efficient PSII repair and that it is partly impaired in the absence of SCPs. At the same time, the amount of chlorophyll also seems to influence the expression of ScpC and ScpD.

Place, publisher, year, edition, pages
Elsevier, 2011
Keyword
photosystem II, non-photochemical quenching, light-harvesting-like proteins, synechocystis sp. PCC 6803, high-light inducible proteins, chlorophyll
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-44584 (URN)10.1016/j.bbabio.2011.05.002 (DOI)21605542 (PubMedID)
Available from: 2011-06-09 Created: 2011-06-09 Last updated: 2017-12-11Bibliographically approved
3. The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: Transcriptomic, proteomic and metabolomic analyses reveal new insights into their function
Open this publication in new window or tab >>The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: Transcriptomic, proteomic and metabolomic analyses reveal new insights into their function
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 consist of one transmembrane helix and belong to the light-harvesting like family. While in wild type they are induced by various stresses, in cells with Photosystem I (PSI) -less background the SCPs are constitutively expressed. Here we performed a detailed comparison of a PSI-less control strain with a strain depleted of PSI and all SCPs, with the aim to elucidate the function of the SCPs within Photosystem II. Electron microscopical immages showed a drastic alteration of the cellular organization, visible by a rough surface and few thylakoid membranes in the PSI-less/ScpABCDE- strain. Immunoblots pointed to a decreased amount of PSII per cell volume. Deletion of the scp genes also affected the carbon- and nitrogen-balance and thereby the carbohydrate accumulation at the expenses of protein- and chlorophyll-synthesis as judged by transcriptomic and metabolomic analyses. Our results show that SCPs ensure a correct assembly/repair of PSII and therefore are important for the adaptation to environmental stresses  - especially during the generation of reactive oxygen species.

Keyword
Photosystem II, Synechocystis sp. PCC 6803, small CAB-like proteins, carbon/nitrogen balance, stress adaptation
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-59784 (URN)
Available from: 2012-09-25 Created: 2012-09-25 Last updated: 2012-09-27
4. Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803
Open this publication in new window or tab >>Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e55569- p.Article in journal (Refereed) Published
Abstract [en]

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.

National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-59788 (URN)10.1371/journal.pone.0055569 (DOI)
Note

The authors are thankful to the Royal Swedish Academy (to C.F.) and the Kempe foundation (to P.S.) for granting their positions. The work was supported by the Swedish Energy Agency and Umea° University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

 

Available from: 2012-09-25 Created: 2012-09-25 Last updated: 2017-12-07Bibliographically approved

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