Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent Irma N. Roberts¤, Xuan Tam Lam, Helder Miranda, Thomas Kieselbach, Christiane Funk* Department of Chemistry and Umea˚ Plant Science Centre, Umea˚ University, Umea˚, Sweden Abstract The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction. Citation: Roberts IN, Lam XT, Miranda H, Kieselbach T, Funk C (2012) Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent. PLoS ONE 7(9): e45713. doi:10.1371/journal.pone.0045713 Editor: Frederik Bo¨ rnke, Friedrich-Alexander-University Erlangen-Nurenberg, Germany Received May 14, 2012; Accepted August 24, 2012; Published September 19, 2012 Copyright: 2012 Roberts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants of the Swedish Energy Agency (2009-000928 to CF), the Royal Swedish Academy of Sciences (312-3820-06 to CF), the Lawsky Foundation (to XTL) and Umea˚ University (to CF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Christiane.Funk@chem.umu.se € Current address: Instituto de Investigaciones en Biociencias Agr´ıcolas y Ambientales, Universidad de Buenos Aires, Buenos Aires, Argentina Introduction Cells have evolved an extensive system of molecular chaperones, folding catalysts, and proteases; that control protein quality and prevent protein damage. Biochemical and molecular biological studies have successfully identified many plastidic protease families, most of which are homologs of defined bacterial proteases. Deg/HtrA proteases were initially identified because of their essential role in the degradation of abnormal periplasmic proteins and because they are crucial for the survival of E. coli at high temperatures [1,2]. They have since been found in nearly all organisms; including Archae, bacteria and eukaryotes. Deg proteases are ATP-independent serine endopeptidases, containing a trypsin/chymotrypsin-like protease domain, followed by zero to three PDZ or PDZ-like domains [3,4]–PDZ domains being those that mediate complex assembly, substrate binding, and/or regulation of proteolytic activity [5–7]. In Arabidopsis thaliana, 16 genes coding for Deg-like proteases have been identified and at least seven gene products have been predicted to be located in chloroplasts [8–10]. Based on biochemical data, five Deg proteases have been shown to be localized in the chloroplast: Deg1, Deg5, and Deg8 are located in the thylakoid lumen [11,12], and Deg2 [13] and Deg7 [14] are peripherally associated with the stromal side of the thylakoid membrane. Degradation of the Photosystem II (PSII) reaction-center protein D1 under photoinhibition has been linked to the lumen-located Deg1 [15], and to Deg5 and Deg8 [16]. Additionally, Deg1 seems to assist the assembly of PSII via interaction with the PSII reaction-center D2 protein [17]. It has been shown in vitro that recombinant Deg1 of Arabidopsis is able to degrade in vitro–translated plastocyanin and PsbO [18]. So far, no details of the molecular mechanism of Deg function are known, but recently it has been suggested that some Deg proteases might be redox regulated. In one study it was found that the proteolysis of casein by recombinant Deg1 and Deg2 of Arabidopsis was dependent on the redox potential of the surrounding medium; and while the activity of Deg1 was maximal under reducing conditions, the opposite was true for Deg2 [19]. Deg1 and Deg5 have been identified as potential thioredoxin targets from their ability to form mixed disulfides with Trx A (thioredoxin A) of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter: Synechocystis 6803) [20]. In Synechocystis 6803, three Deg/HtrA proteases have been identified. Owing to their relationship to E. coli they have been named DegP/HtrA (slr1204), DegQ/HhoA (sll1679), and DegS/ HhoB (sll1427) [21]. However, they are more closely related to each other than to the E. coli Deg proteases with the same name [8,9]; so they are probably not orthologs of the E. coli Deg proteases. HhoA, HtrA, and HhoB have very high homology with the lumen-located plant Deg proteases Deg1, Deg5, and Deg8 [8]. Single-deletion mutants of HtrA [22] and HhoA [23] have been found to be more sensitive than wild type, towards light and heat stress, respectively. However, these results were controversial [24,25]. While a triple Deg-deletion mutant displayed a significant phenotype towards light- and heat-stress and phototaxis, none of the double mutants did. Therefore it was concluded that the Deg PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45713