Validación de resultados

Functional analysis of ∆sll0408showed that the absence of cTLP40 decreased the performance of the strain in particular after adaptation to high light. Under these conditions the mutant possesses a longer duplication time, a different set of pigments and a lower oxygen evolution rate. Studies on the assembly and stability of PSII by immunological and in vivo labelling experiments showed that mutant had a lower amount of PSII dimer. In addition, the mutant had a higher synthesis rate of PSII proteins (evaluated through the D1 content) but higher degradation rate which resulted in a similar steady state level of PSII proteins. We tried to analyze a possible reason for the lower amount of PSII dimer focusing on two possible mechanisms: inefficient repair of PSII or instability of the complex.

The repair of PSII complex (the so-called repair cycle of PSII) comprises the removal of damaged protein (D1) from PSII and the insertion of a newly synthesised one into the complex. Damaged D1 is degraded after its removal. Damage of D1 occurs after photoinhibition of PSII which is caused by high light, extreme temperature regimes or nutrient deficiency. It is well known that the photoinhibition of PSII is caused by formation of reactive oxygen species which are generated in PSII during oxygen evolution (Vass et al., 1992; Okada et al., 1996; Kerne et al., 1997). However, recent studies challenged this idea and showed that oxidative stress inhibited the repair of PSII by blocking the translation of D1 protein but do not inactivate PSII directly (Nishiyama et al., 2004). In addition it has been suggested that the photoinhibition of PSII occurs only after the complete inactivation of the oxygen-evolving complex (Ohnishi et al., 2005).

Considering the enzymatic activity of cTLP40, we suggested that cTLP40 could be involved in the isomerisation of proline residues of PSII subunits. The isomerisation of the proline bond can significantly change the structure of a protein exposing or hiding possible protein-protein interaction site. The absence of

cTLP40 could cause the presence of structurally modified proteins interfering with the normal PSII cycle. The effect of absence of cTLP40 is stronger at high light conditions, where, due to higher photoinhibition rate, PSII should be faster repaired and assembled. Since cTLP40 is located in the thylakoid lumen, our candidate/s should have one or more proline residues facing the lumenal side of thylakoids. In addition, we considered that during PSII assembly studies most of D1 was distributed between D1/D2/CP47 complex and PSII monomer. This raised the idea that the incorporation of CP43 into D1/D2/CP47 complex could be impaired in mutant ∆sll0408. CP43 is a six-helix transmembrane protein with three connecting loops located on the lumenal side (see also Section 1.3.1.4). In particular, the last connecting loop was intensively studied because of its large lumenal extension (reviewed in Bricker and Frankel, 2002; Anderson et al., 2002). One of these studies showed that CP43 in Synechocystis can have a posttraslational modification (different level of oxidation) on the tryptophan 352 (Anderson et al., 2002). This tryptophan residue is located in the E-loop in the region A350_PWELPLRGPN360 _{which was shown to be highly conserved in different}

organisms. Considering that mutation analysis in this region impaired severely the assembly and functionality of PSII (Kuhn and Vermaas, 1993; Anderson et al., 2002), the authors suggested a possible role of tryptophan modification as a signal in PSII turnover. Computer modelling analysis of this region, suggested an important role of proline 351 and 355 for the orientation of that tryptophan residue (Anderson et al., 2002). Since PPIase activity can be tested in an in vitro assay (Fischer et al., 1989; Kofron et al., 1991), we chose two peptides containing proline 351 and proline 355 which could be important for tryptophan orientation (Fig. 35). Full-length cTLP40 containing a His-tag was overexpressed in E. coli

and purified by metal affinity chromatography. One possible contaminant in this procedure could be the bacterial protein SlyD which is a rotamase (Mukherjee et al., 2003). We used a modifed version of Ni2+-metal affinity chromatography which excludes copurification of SlyD (Mcmurry and Macnab, 2004). However, our experiment showed a possible residual rotamase activity in the bacterial lysate which could depend from another minor rotamase activity present in E. coli (for example CypA and CypB, Hayano et al., 1991). This rotamase can efficiently isomerise the two CP43-derivedpeptides. Thus, the result then of our experiment

was not conclusive and should be repeated employing a new purification procedure for cTLP40 to remove all contaminating rotamase activities.

To test whether the absence of cTLP40 can interfere with the dimerisation of PSII, we analyzed small subunits of PSII which are involved in the dimerisation and stabilisation of the complex (reviewed in Shi and Schröder, 2004). The best candidate was the PsbI protein which has one transmembrane domain and contains a lumenal extension with a single proline residue. In addition, PSII crystallographic analysis localised PsbI at the interface of the dimerisation site, suggesting a role in the stabilisation of PSII dimer (Ferreira et al., 2003). The

Synechocystis strain deficient in PsbI was previously described (Ikeuchi et al., 1995). The mutant could still grow autotrophically and was more sensible to light than the wild-type. In addition, analysis of the mutant revealed that the absence of PsbI protein resulted in a loss of PSII activity of 25 - 30%. Since this phenotype was not very strong and resembled the phenotype of ∆sll0408, it was checked whether PsbI could be a substrate for cTLP40. However, the data showed that the PsbI-peptide-containing proline could not be isomerased from cTLP40.