Redox-Dependent Degradation by HhoA Figure 5. PsbQ is not degraded by the recombinant Deg proteases. PsbQ was isolated from spinach and incubated for 5 h in the absence or the presence (PsbQred) of the thioredoxin system, either with no protease (panel 1) or with rHhoA (panel 2), rHhoB (panel 3), or rHtrA (panel 4). After SDS-PAGE, the gel was either stained with CBB (upper panels, 6 mg of protein loaded) or blotted and immunostained using an antibody directed against PsbQ (lower panels, 1.5 mg of protein loaded). doi:10.1371/journal.pone.0045713.g005 Arabidopsis. In addition, the native protease activity responsible for PsbO degradation might need additional factors/proteases to enhance the redox-dependent degradation. PsbO degradation by Deg proteases Various chloroplast-located Deg proteases are known to be involved in the degradation of photosynthetic proteins under stress conditions, and the main target seems to be the reaction-center protein D1 [13–17]. Recombinant Arabidopsis Deg1 was also to some extent able to degrade recombinant PsbO [18]. These results appear to be contradictory to ours, as unreduced PsbO was degraded by Deg1. However, recombinant PsbO is, at least partially, produced in its reduced form. We observed degradation of recombinant PsbO from spinach and Synechocystis 6803 without addition of the thioredoxin system (not shown). However, a control using thiol labeling with monobromobimane showed that recombinant PsbO from Synechocystis 6803 mainly contained reduced cysteine thiols (Figure S1), which indicated that E. coli is not able to correctly form the disulfide bridge of PsbO. Consistent with these observations, a recent study in Arabidopsis showed that Deg1 is not responsible for degradation of mature PsbO in vivo, but acts downstream by degrading its fragments [66]. Is PsbO degraded in its free form or bound to PSII? The conformations of PsbO in solution differ from those when it is bound to PSII [42,67,68]. Conformational changes of PsbO under different light conditions and heat treatments affect its binding ability [37], which might trigger its degradation. It has been shown that the disulfide bridge is important for folding and rebinding of PsbO to PSII [40]; and binding of PsbO to PSII seems to protect it from degradation in spinach (Figure 6). In PSII isolated from Synechocystis 6803, a moderate degradation of PsbO was visible, which was probably due to co-isolated HhoA or other proteases (Figure 1B and 1C). We believe that by degrading free PsbO present in the PSII fractions, the equilibrium between bound and free PsbO is changed, so that more PsbO dissociates from PSII and therefore more PsbO can be degraded by the (recombinant or native) Deg proteases. Free, unassembled PsbO appears to be destabilized after reduction of its disulfide bridge and becomes accessible to proteolysis. While rHhoA was able to degrade PsbO from spinach in a redox-dependent manner, it is still unclear what roles the lumenal Deg proteases Deg1, Deg5, and Deg 8 play in the degradation of PsbO. However, Deg1 does not appear to be involved in the primary cleavage of PsbO [66]. PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45713