Redox-Dependent Degradation by HhoA Figure 4. PsbO degradation in the presence of recombinant Deg proteases of Synechocystis sp. PCC 6803. (A) PsbO from spinach was isolated and incubated in the absence or the presence (PsbOred) of the thioredoxin system for 5 h either without addition of recombinant proteases (panel 1) or after addition of rHhoA (panel 2), rHhoB (panel 3), or rHtrA (panel 4) at two different enzyme-to-substrate ratios, 1:50 and 1:10 (56). After SDS-PAGE, the proteins (9 mg) were stained with CBB. Arrows indicate the position of PsbO degradation fragments (F1 to F4). (B) The degradation fragments of reduced spinach PsbO produced by recombinant HhoA were transferred onto PVDF membrane, stained using CBB, and analyzed by Edman sequencing. The cutting sites are indicated by dashed lines in the mature PsbO sequence. doi:10.1371/journal.pone.0045713.g004 ed. The relevance of these observations for the turnover of PsbO under physiological conditions is supported by the recent finding that PsbO levels in the lto1 mutant of Arabidopsis are clearly lower than in wild-type plants [65]. The lto1 gene encodes for a lumen thiol oxidoreductase (LTO1) that catalyzes disulfide formation of thylakoid proteins on the lumen side of the thylakoid membrane. In the lto1 mutant, oxidation of reduced PsbO is inhibited, which would facilitate degradation of this protein and explain the lower levels of PsbO in this mutant than in the wild type [65]. Recombinant HhoA from Synechocystis 6803 cleaves PsbO from spinach in the N-terminal region specifically before (F1) and after (F4) the disulfide bridge, which is consistent with the redox- dependence of this process. This degradation is very specific, and other extrinsic PSII proteins such as PsbQ are not degraded. The cleavage sites of the HhoA-specific fragments F1 (YLEV-KGTG) and F4 (TKLM-TRLT) are new and not in the Merops peptidase database. This suggests that HhoA belongs to a subfamily of the chymotrypsin family of peptidases that have their own unique substrate specificity. Degradation kinetics In purified chloroplast lumen of Arabidopsis, degradation of PsbO has been shown to be complete within 5 h of the onset of reduction by thioredoxin [20]. Similar results were obtained for PsbO from spinach, but the rate of the degradation was slower than for the PsbO proteins from Arabidopsis. This might be due to a more negative redox potential of PsbO in spinach than in PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45713