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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.

A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS. The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner. Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink. A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.

The Sun is the ultimate energy source on Earth. Photosynthetic organisms are able to catalyze the conversion of solar energy to chemical energy by a reaction called photosynthesis. In plants, this process occurs inside a green organelle called the chloroplast. The protein complexes involved in the photosynthetic light reactions are situated in the thylakoid membrane, which encloses a tiny space called lumen. The Peptidyl-Prolyl cis-trans Isomerase (PPIase) family is the most abundant protein family in the thylakoid lumen. The three PPIase subfamilies, cyclophilins, FKBPs (FK506 binding proteins) and parvulins form a group by their enzymatic activity despite lack of sequence similarity between the subfamilies. Cyclophilins and FKBPs, collectively called immunophilins, were originally discovered as the targets of the immunosuppressive drugs cyclosporine A and FK506, respectively. By suppressing the immune response in humans, these immunophilin-drug complexes revolutionized the field of organ transplantation by preventing graft rejection. Cis-trans isomerization of peptide bonds preceding the amino acid proline is the rate-limiting step of protein folding and several immunophilins have been shown to be important for catalysis of protein folding in vivo. PPIases have been found to be part of large protein complexes as well as in functions such as signalling, protein secretion, RNA processing and cell cycle control. A picture is therefore emerging in which the actual interaction between the PPIase and its target is perhaps more important than the PPIase activity. In the present work, PPIases have been characterized in the chloroplast thylakoid lumen of Spinacia oleracea (spinach) and Arabidopsis thaliana (Arabidopsis). The most active PPIase in the spinach lumen was identified as the cyclophilin TLP20. AtCYP20-2, the Arabidopsis homologue of TLP20, was found to be upregulated at high light and attached to the thylakoid membrane, more precisely to the outer regions of photosystem II supercomplexes. In Arabidopsis, up to 5 cyclophilins and 11 FKBPs were predicted to reside in the lumen. Of these 16 immunophilins, only 2 were identified as active PPIases and significant differences were observed between the two plant species. AtCYP20-2, like TLP20, is an active isomerase although AtFKBP13 is the most active PPIase in the lumen of Arabidopsis. Mutant Arabidopsis plants deficient in AtCYP20-2 displayed no phenothypical changes or decrease in total lumenal PPIase activity. Being the only active PPIase in the mutants, the redox sensitive AtFKBP13 is proposed to compensate for the lack of AtCYP20-2 by oxidative activation. In agreement with the experimental data, the sequence analyses of catalytic domains of lumenal immunophilins demonstrate that only AtCYP20-2 and AtFKBP13 possess the amino acids found essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. It is concluded that with the exception of AtCYP20-2 and AtFKBP13 most immunophilins in the lumen of Arabidopsis lost their PPIase activity on peptide substrates and developed other specialized functions.