Redox-Dependent Degradation by HhoA Figure 1. Cyanobacterial PsbO is degraded by rHhoA in a redox-dependent manner. (A) Proteolytic activity of recombinant Synechocystis 6803 Deg/HtrA proteases against naturally unfolded b-casein. Small arrows indicate b-casein degradation fragments. (B) A PSII-enriched fraction was isolated from Synechocystis 6803 using the HT3 mutant with His-tagged CP47. The PSII-enriched fraction was incubated for 5 h in the absence (PSII) or the presence (PSIIred) of the thioredoxin system, either with no protease (panel 1) or with rHhoA (panel 2), rHhoB (panel 3), or rHtrA (panel 4). The proteins (15 mg) were separated by SDS-PAGE and analyzed with CBB staining. Identity of the named bands was confirmed using mass spectrometry. (C) After SDS-PAGE proteins were transferred to PVDF membranes and immunostained using an antibody directed against PsbO. Bars below the gels and blots show the integrated density values of the corresponding bands as quantified by Image J software. The asterisk marks an nonspecific cross- reacting band. doi:10.1371/journal.pone.0045713.g001 immunoblots (Figure 1C), therefore either the antibody did not recognize the degradation products, or PsbO degradation in this experiment was complete. Subcellular location of the Deg proteases of Synechocystis 6803 To investigate if the internal degradation of reduced PsbO observed in the PSII-enriched fraction (Figure 1B and 1C, panel 1) could be attributed to native cyanobacterial Deg proteases, PSII- enriched membranes were isolated from the HT3 strain. After SDS-PAGE, the proteins were immunostained with antibodies directed against HhoA, HhoB, or HtrA. As shown in Figure 2A, all three Deg proteases were detected in the PSII-enriched fraction. However, it is important to note that the PSII-enriched fraction had to be concentrated 10–50 times to achieve immune signals of the same intensity as for the total cell extract. These data suggest that even though all three Deg proteases can be found in the membrane fraction in proximity to PSII, they are present in substoichiometric amounts. Earlier proteomic studies have identified HtrA in the outer membrane of Synechocystis 6803 [53]; and HhoA has been detected in the periplasm, where it has been found both in a soluble form [54] and in a plasma membrane–bound form [55]; while the subcellular location of HhoB is still unknown. To further investigate the subcellular location of the Deg proteases within the membrane system of Synechocystis 6803, the different membrane fractions were isolated in a two-dimensional manner using density- gradient centrifugation and two-phase partitioning [56]. As shown in Figure 2B, all three Deg proteases were immuno-localized in the plasma membrane of Synechocystis 6803. Additionally, HhoA and to a lesser extent HtrA were detected in the thylakoid membrane. The purity of the membrane fractions was controlled immuno- logically by using antibodies directed against the PSII core protein CP47, which is known to be localized in the thylakoid membrane, and against NrtA–a component of the nitrate transporter–, which is localized in the plasma membrane. Native PsbO is degraded in a redox-dependent manner in the thylakoid lumen of spinach To further study the redox-dependent degradation, a pure PsbO fraction was needed that was free of contaminating proteases (native Deg/HtrA, or other proteases within the cell). To our knowledge, no method has been reported that allows the purification of large quantities of PsbO from Synechocystis cells. In addition, an attempt to express recombinant Synechocystis PsbO in two different E. coli strains resulted in the production of insoluble aggregates and a low fraction of apparently misfolded protein, highly prone to precipitation (data not shown). Analysis of the redox state of soluble recombinant PsbO showed that the protein is synthesized mostly in its reduced form, suggesting that E. coli is not able to correctly form the disulfide bridge (Figure S1). As a consequence, studies using purified PsbO from Synechocystis as substrate were not possible, and hence the usefulness of using PsbO purified from other sources was evaluated. Figure 2. Subcellular localization of the Synechocystis Deg proteases. (A) Total wild-type cell extract and a PSII-enriched fraction isolated from the HT3 strain of Synechocystis 6803 were analyzed by SDS-PAGE, and by immunostaining using antibodies directed against recombinant HhoA, HhoB, or HtrA. Of the total cell extract, samples corresponding to 0.1 mg of chlorophyll were loaded for immunostain- ing with anti-HhoA and samples corresponding to 0.5 mg were loaded for anti-HhoB and anti-HtrA. Of the PSII-enriched fraction, samples corresponding to 5 mg of chlorophyll were loaded in each lane. (B) Outer membrane (OM), plasma membrane (PM), thylakoid membrane (TM), and soluble protein fraction (S) were isolated from Synechocystis 6803 by gradient centrifugation and two-phase partitioning, separated by SDS-PAGE, and immunostained using antibodies directed against recombinant HhoA, HhoB, or HtrA. Purity of the membranes was determined by using antibodies directed against the PSII protein CP47 and against NrtA, a component of the nitrate transporter; 15 mg of protein was loaded in each lane. doi:10.1371/journal.pone.0045713.g002 The amino acid sequence and structure of PsbO from prokaryotic and eukaryotic organisms are highly conserved [29]. The PsbO protein of Synechocystis has 43% sequence identity with its spinach and Arabidopsis counterparts (Figure S2), mostly within highly conserved regions [29]. Critical for similarity of protein structures is conservation of the amino acid residues that stabilize the hydrophobic core of a protein [57]. For PsbO from spinach, the molecular interactions of the individual amino acid residues that stabilize the protein core have been calculated [38]. PsbO from spinach has 107 amino acid residues that contribute with ten or more molecular interactions to the stability of the protein fold [38], and 56 of these amino acid residues (52%) are directly conserved in the sequence of PsbO from Synechocystis. In addition, both PsbO from spinach and from Synechocystis can without PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45713