proteases of Synechocystis 6803 have at least partially overlapping functions [25]. PSII, which catalyzes light-dependent water oxidation and plastoquinone reduction in plants and cyanobacteria, consists of more than 30 proteins located in the thylakoid membrane. The catalytic reaction center as well as the chlorophyll (Chl)-binding proteins are membrane integral, but they are stabilized by several extrinsic proteins bound to the lumenal surface of PSII [26,27], called the oxygen-evolving complex (OEC). While the extrinsic protein PsbO is present in all organisms that perform oxygenic photosynthesis [28,29], the higher-plant PsbP and PsbQ differ from the corresponding proteins PsbU and PsbV present in the cyanobacterial water-oxidizing complex. Although none of its amino acid residues are likely ligands to the Mn4Ca cluster, PsbO has been found to play an important role in the stabilization of the oxygen-evolving complex; and after removal of PsbO the manganese ions are released. However, oxygen-evolving activity is maintained in the presence of high concentrations of Cl2 and Ca2+ [30]. Besides being important for the stabilization of the manganese cluster, PsbO has also been shown to be involved in many other aspects of PSII structure and function [31–34]. PsbO has not yet been crystallized owing to its natively unfolded nature [35]. The only experimental three-dimensional structure of PsbO was derived from cyanobacterial PsbO bound to PSII [26,36] and has served as a template for the construction of homologous models for plant PsbO [29]. It has been found that pH values of 5.7 and 7.2–which are typical for the light and dark conditions in the thylakoid lumen–change the conformation of PsbO [37]. PsbO has two conserved cysteine residues, which correspond to Cys19 and Cys44 in the cyanobacterium T. elongatus [28] and to Cys28 and Cys51 in spinach. These cysteines form a disulfide bridge between the N-terminal loop and the b1 strand [38]. The role of this disulfide bond is controversial; it has been observed to be involved in accumulation of PsbO at the thylakoid membrane [39], and in its rebinding to PSII [40]. However, after deletion of the disulfide bond, a PsbO Cys28Ala/Cys51Ala double mutant was still able to assemble PsbO to PSII and to restore oxygen evolution up to 40% of the control level [41,42]. Unlike unassembled intrinsic subunits of PSII, which are rapidly degraded; a pool of free, assembly-competent, extrinsic OEC proteins has been shown to exist in the thylakoid lumen [43,44]. The availability of soluble OEC proteins is thought to be important for the rapid reassembly of functional oxygen-evolving PSII complexes during the PSII repair cycle. PsbO has been shown to have a long lifetime even in its free form [44], but it can be oxidatively damaged under light stress [45]. Recent in vitro studies have shown that the PsbO proteins of Arabidopsis and spinach are targets of thioredoxin [20,46–48]. Notably, the disulfide bridge of PsbO was not only reduced by thioredoxin, but the redox state of the disulfide was integral to the degradation of PsbO1 and PsbO2 of Arabidopsis [20]. The protease activities responsible for the redox-dependent proteolysis have not yet been identified. Here we demonstrate that recombinant HhoA of the cyanobacterium Synechocystis 6803 is able to degrade PsbO from spinach in a redox-dependent manner, and we present the corresponding cleavage sites. In agreement with earlier observations in Arabidopsis [20], we observed that PsbO degradation was induced after reduction of the disulfide bond in both spinach and Synechocystis 6803. We provide evidence that the redox-dependent degradation of PsbO in cyanobacterial thylakoid membranes is performed by Deg proteases. Finally, we demonstrate the subcellular localization of the three Deg proteases to be at the thylakoid membrane and/or in the periplasmic space. Redox-Dependent Degradation by HhoA Results rHhoA is able to degrade cyanobacterial PsbO in a redox- dependent manner It was recently shown by means of proteomics that the redox state of the cysteine thiols is important for the stability of both PsbO1 and PsbO2 in Arabidopsis [20]. The proteases known to be located in the plant chloroplast lumen are the D1-processing proteases and the Deg proteases Deg1, Deg5, and Deg8. Deg1 is the most abundant protease in the soluble lumen content and it is therefore a reasonable assumption that it would be involved in the redox-dependent degradation of PsbO. However, Deg1 of Arabidopsis was recently reported to be redox regulated itself [19]. In addition, Deg1 and Deg5 of Arabidopsis have been shown to form mixed disulfides with TrxA of Synechocystis 6803 [20], suggesting that the lumenal Deg proteases of green plants might be redox regulated. To unambiguously investigate the redox-de- pendent degradation of the substrate PsbO–and not of the protease itself–we designed in vitro degradation experiments using cyanobacterial recombinant Deg proteases. All three Synechocystis 6803 Deg proteases are highly homologous to the three lumen- located Deg proteases of Arabidopsis, but not to the other Deg proteases of this plant [8]. While HhoA and HhoB are free of cysteines, HtrA contains one N-terminal cysteine, which, however, is not conserved among HtrA of other organisms, suggesting that HtrA is not regulated via redox-active thiols. In addition, no Deg protease was detected among the thioredoxin targets of Synechocystis 6803 [49]. Therefore, the activities of HtrA and the other Deg proteases of Synechocystis 6803 are not controlled by thioredoxin. Activity of the three purified recombinant Deg proteases of Synechocystis 6803–rHhoA, rHhoB, and rHtrA–was confirmed using b-casein as a substrate (Figure 1A). All three recombinant proteases exhibited proteolytic activity, as had previously been demonstrated [50]. To investigate if redox-dependent degradation of PsbO could be observed in Synechocystis, we performed an in vitro proteolytic assay using a PSII-enriched fraction isolated from the HT3 strain as the source of PsbO. The Synechocystis HT3 strain expresses a His-tagged CP47 protein; which allows purification of a highly enriched PSII fraction by nickel-affinity chromatography [51,52]. In agreement with previous reports [52], mass spectro- metric analysis of major bands in this fraction allowed identifica- tion of several PSII subunits (Figure 1B). The isolated PSII- enriched fraction was incubated with each of the three recombinant Deg proteases for 5 h under reduced conditions. The reduced conditions were conferred using the complete E. coli thioredoxin system, consisting of thioredoxin (Trx), thioredoxin reductase (TrxR), and b-NADPH. Figure 1B shows the CBB R- 250 stained gels and Figure 1C shows the immunoblots using an anti-PsbO antibody. As seen in Figures 1B and C, PsbO in the PSII complex was moderately degraded in the presence of reduced thioredoxin (PSIIred), while no degradation was detected when reduced thioredoxin was absent (panel 1). Quantification analysis of the signals shows that 63% of PsbO initial amount remained after 5 h (PSIIred). We believe that this degradation can be attributed to low amounts of Synechocystis proteases co-isolating with the PSII-containing membrane fractions. Nonetheless, addition of recombinant HhoA clearly increased the degradation of PsbO in the presence of thioredoxin, as can be observed both in the CBB-stained gels (Figure 1B) and in the immunoblots (Figure 1C) where about 30 and 35% of the initial amount of PsbO remained after 5 h, respectively. On the other hand, addition of rHhoB or rHtrA did not enhance the degradation of reduced PsbO bound to PSII. It is noteworthy that no additional PsbO fragments were observed in the CBB-stained gels or in the PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45713