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The chloroplast lumen: New insights into thiol redox regulation and functions of lumenal proteins
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Wolfgang Schröder)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In higher plants oxygenic photosynthesis primarily takes place in the chloroplasts of leaves. Within the chloroplasts is an intricate membrane system, the thylakoid membrane, which is the site of light harvesting and photosynthetic electron transport. Enclosed by this membrane is the lumen space, which initially was believed to only contain a few proteins, but now is known to house a distinct set of >50 proteins, many for which there is still no proposed function. The work presented in this thesis is focused on understanding the functions of the proteins in the lumen space. Using proteomic methods, we investigated first the regulation of lumenal proteins by light and secondly by dithiol-disulphide exchange, mediated by the disulphide reductase protein thioredoxin. We furthermore performed structural and functional studies of the lumenal pentapeptide repeat proteins and of the PsbP-domain protein PPD6. When studying the diurnal expression pattern of the lumen proteins, using difference gel electrophoresis, we observed an increased abundance of fifteen lumen protein in light-adapted Arabidopsis thaliana plants. Among these proteins were subunits of the oxygen evolving complex, plastocyanin and proteins of unknown function. In our analysis of putative lumenal targets of thioredoxin, we identified nineteen proteins, constituting more than 40 % of the lumen proteins observable by our methods. A subset of these putative target proteins were selected for further studies, including structure determination by x-ray crystallography. The crystal structure of the pentapeptide repeat protein TL15 was solved to 1.3 Å resolution and further biochemical characterization suggested that it may function as a novel type of redox regulated molecular chaperone in the lumen. PPD6, a member of the PsbP-family of proteins, which is unique in that it possesses a conserved disulphide bond not found in any other PsbP-family protein, was also expressed, purified and crystallized. A preliminary x-ray analysis suggests that PPD6 exists as a dimer in the crystalline state and binds zinc ions. The high representation of targets of thioredoxin among the lumen proteins, along with the characterization of the pentapeptide repeat protein family, implies that dithiol-disulphide exchange reactions play an important role in the thylakoid lumen of higher plants, regulating processes such as photoprotection, protein turnover and protein folding.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2012. , 65 p.
Keyword [en]
Photosynthesis, thylakoid lumen, thioredoxin, pentapeptide repeat, proteomics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-58423ISBN: 978-91-7459-467-6 (print)OAI: oai:DiVA.org:umu-58423DiVA: diva2:548173
Public defence
2012-09-21, KBC huset, KB3B1, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-08-31 Created: 2012-08-29 Last updated: 2012-08-30Bibliographically approved
List of papers
1. Light induced changes in protein expression and uniform regulation of transcription in the thylakoid lumen of Arabidopsis thaliana
Open this publication in new window or tab >>Light induced changes in protein expression and uniform regulation of transcription in the thylakoid lumen of Arabidopsis thaliana
2009 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 4, no 5, e5649- p.Article in journal (Refereed) Published
Abstract [en]

In plants oxygenic photosynthesis is performed by large protein complexes found in the thylakoid membranes of chloroplasts. The soluble thylakoid lumen space is a narrow and compressed region within the thylakoid membrane which contains 80-200 proteins. Because the thylakoid lumen proteins are in close proximity to the protein complexes of photosynthesis, it is reasonable to assume that the lumen proteins are highly influenced by the presence of light. To identify light regulated proteins in the thylakoid lumen of Arabidopsis thaliana we developed a faster thylakoid preparation and combined this with difference gel electrophoresis (DIGE) of dark-adapted and light-adapted lumen proteomes. The DIGE experiments revealed that 19 lumen proteins exhibit increased relative protein levels after eight hour light exposure. Among the proteins showing increased abundance were the PsbP and PsbQ subunits of Photosystem II, major plastocyanin and several other proteins of known or unknown function. In addition, co-expression analysis of publicly available transcriptomic data showed that the co-regulation of lumen protein expression is not limited to light but rather that lumen protein genes exhibit a high uniformity of expression. The large proportion of thylakoid lumen proteins displaying increased abundance in light-adapted plants, taken together with the observed uniform regulation of transcription, implies that the majority of thylakoid lumen proteins have functions that are related to photosynthetic activity. This is the first time that an analysis of the differences in protein level during a normal day/night cycle has been performed and it shows that even a normal cycle of light significantly influences the thylakoid lumen proteome. In this study we also show for the first time, using co-expression analysis, that the prevalent lumenal chloroplast proteins are very similarly regulated at the level of transcription.

National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-23163 (URN)10.1371/journal.pone.0005649 (DOI)19461964 (PubMedID)
Available from: 2009-06-02 Created: 2009-06-02 Last updated: 2017-12-13Bibliographically approved
2. Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function
Open this publication in new window or tab >>Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function
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2010 (English)In: Proteomics, ISSN 1615-9861, Vol. 10, no 5, 987-1001 p.Article in journal (Refereed) Published
Abstract [en]

The light-dependent regulation of stromal enzymes by thioredoxin-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that thioredoxin targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Thioredoxin-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed thioredoxin-linked redox control in the chloroplast lumen of Arabidopsis thaliana.Using complementary proteomics approaches, we identified 19 thioredoxin target proteins, thus covering more than 40 percent of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.

Place, publisher, year, edition, pages
Wiley InterScience, 2010
Keyword
D1-processing, Disulphide, Immunophilin, Pentapeptide protein, Plant proteomics, Violaxanthin de-epoxidase
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-30831 (URN)10.1002/pmic.200900654 (DOI)000275790700009 ()20049866 (PubMedID)
Available from: 2010-02-05 Created: 2010-01-18 Last updated: 2012-08-30Bibliographically approved
3. A novel extended family of stromal thioredoxins
Open this publication in new window or tab >>A novel extended family of stromal thioredoxins
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2009 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 70, no 3, 273-281 p.Article in journal (Refereed) Published
Abstract [en]

Thioredoxins play key regulatory roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. In addition to the established groups of thioredoxins, f, m, x, and y, novel plant thioredoxins were also considered to include WCRKC motif proteins, CDSP32, the APR proteins, the lilium proteins and HCF164. Despite their important roles, the subcellular locations of many novel thioredoxins has remained unknown. Here, we report a study of their subcellular location using the cDNA clone resources of TAIR. In addition to filling all gaps in the subcellular map of the established chloroplast thioredoxins f, m, x and y, we show that the members of the WCRKC family are targeted to the stroma and provide evidence for a stromal location of the lilium proteins. The combined data from this and related studies indicate a consistent stromal location of the known Arabidopsis chloroplast thioredoxins except for thylakoid-bound HCF164.

Place, publisher, year, edition, pages
SpringerLink, 2009
Keyword
Arabidopsis thaliana, Chloroplast, Protein targeting, Subcellular location, Thioredoxin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-19625 (URN)10.1007/s11103-009-9471-4 (DOI)19259774 (PubMedID)
Available from: 2009-03-09 Created: 2009-03-09 Last updated: 2017-12-13Bibliographically approved
4. The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plants
Open this publication in new window or tab >>The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plants
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.

Keyword
Photosynthesis, Chloroplast lumen
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-58364 (URN)
Available from: 2012-08-29 Created: 2012-08-29 Last updated: 2012-08-30Bibliographically approved
5. Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana
Open this publication in new window or tab >>Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana
2012 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 68, no 3, 278-280 p.Article in journal (Refereed) Published
Abstract [en]

The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.

Keyword
photosynthesis, thylakoid lumen, PsbP domain
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-55088 (URN)10.1107/S1744309111042023 (DOI)000301921300007 ()22442221 (PubMedID)
Available from: 2012-05-08 Created: 2012-05-08 Last updated: 2017-12-07Bibliographically approved

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