Redox-Dependent Degradation by HhoA Figure 3. PsbO degradation in the thylakoid lumen of spinach and Arabidopsis is redox dependent. (A) Thylakoid lumen of spinach and Arabidopsis was isolated and incubated for 3 h in the presence of the complete thioredoxin system. Controls lacked either b-NADPH or thioredoxin (Trx) and thioredoxin-reductase (TrxR). After SDS-PAGE, the proteins (20 mg) were stained with CBB. Arrows indicate the position of the PsbO protein band in spinach, and the positions of PsbO1 and PsbO2 in Arabidopsis samples and of other prominent proteins identified by mass spectrometry. The remaining PsbO amount after 3 h of incubation was quantified using Image J software and was as it follows: 61, 46 and 41% in the presence of the complete thioredoxin system; 84, 81 and 98% in the controls lacking Trx and TrxR; and 82, 76 and 83% in the controls lacking b-NADPH for spinach PsbO, PsbO1 and PsbO2, respectively. (B) Thylakoid lumen of spinach was isolated and incubated with the complete thioredoxin system (Trx/TrxR/b- NADPH) or with thioredoxin and thioredoxin reductase (Trx/TrxR) as a control. Sulfhydryl groups of the lumenal proteins were labeled with mBBr. After SDS-PAGE, proteins were visualized with UV. Identity of the named bands was confirmed using mass spectrometry. doi:10.1371/journal.pone.0045713.g003 thioredoxin. Addition of recombinant HhoA, HhoB, or HtrA did not result in any degradation of PsbQ, neither in the presence nor in the absence of the thioredoxin system (panels 2–4). As spinach PsbQ lacks any cysteine residue, a thiol-dependent conformational change that facilitates degradation of this protein would not be expected, and the absence of any redox-effect in the control assay using PsbQ supports our hypothesis that the activities of rHhoA, rHhoB, and rHtrA are not regulated by thioredoxin. PsbO bound to PSII is protected against degradation in reducing conditions PsbO is assembled into PSII. However, a pool of free PsbO has been shown to exist in the thylakoid lumen [43]. Under reducing conditions, unassembled PsbO was degraded without addition of recombinant proteases (Figure 3A), which is consistent with the presence of Deg proteases in the thylakoid lumen. To investigate if PsbO attached to PSII can also be degraded, PSII membrane fragments were isolated from spinach leaves and incubated for 5 h in the presence or absence of reduced thioredoxin. After separation of the proteins by SDS-PAGE, PsbO was immunos- tained (Figure 6). No degradation of PsbO was observed in the PSII fraction of spinach; neither in the absence nor in the presence of reduced thioredoxin (Figure 6). Addition of recombinant rHhoB or rHtrA to non-reduced or reduced PSII did not lead to any degradation. However, addition of rHhoA resulted in slight, redox-dependent degradation of PsbO; evidenced by the appear- ance of the previously described fragments after 5 h of incubation (Figure 6). However, this degradation was slower and weaker than for isolated PsbO (Figure 4A), suggesting that the protein is protected against degradation when it is bound to PSII. We believe that the PSII preparation contained some unassembled PsbO that served as a substrate for rHhoA. Discussion Degradation of PsbO is thioredoxin-dependent The PsbO protein of plants [29] and cyanobacteria [63] is a protein of elongated shape containing two major domains: The very stable domain I is composed of eight antiparallel b strands which form a cylinder with hydrophobic amino acid residues in its central part. Domain II mainly consists of random coils and turns and a distinct a helix. The hydrophilic loops are stabilized by interaction with other PSII proteins and are flexible in the non- assembled protein. PsbO contains two conserved cysteine residues, Cys28 and Cys51 in spinach [38], that form an S-S bridge between the N-terminal loop and the b1 strand. While the function of these two cysteins is controversial, it was recently found that they are redox sensitive and might be relevant for the function of PsbO [20,46–48]. The PsbO protein has been found to be remarkably stable when exposed to different temperatures and pHs [44,64]. However, in Arabidopsis both PsbO proteins–PsbO1 and PsbO2–become unstable after reduction by thioredoxin, and are degraded [20]. In this study, we have confirmed the previous results for Arabidopsis and shown that also the PsbO of spinach and of the cyanobacterium Synechocystis 6803 are redox-dependently degrad- PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45713