Figure 6. PsbO bound to PSII is protected against degradation by native proteases. PSII membrane fragments from spinach were isolated and incubated for 5 h in the absence (PSII) or the presence (PSIIred) of reduced thioredoxin, either with no protease or with rHhoA, rHhoB, or rHtrA. After SDS-PAGE, the proteins were blotted and immunostained using an antibody directed against PsbO. Small arrows indicate PsbO degradation fragments (F1 to F4). doi:10.1371/journal.pone.0045713.g006 For PsbO from Synechocystis 6803, Figure 1B shows that PsbO in PSII-enriched membrane fragments is essentially stable, but becomes degraded under reducing conditions. The presence of externally added recombinant HhoA clearly enhances the degradation of PsbO under reducing conditions, while for externally added rHhoB and rHtrA no effect was observed. These observations suggest that PsbO of Synechocystis 6803 is a substrate for rHhoA, but not for rHhoB and rHtrA. Subcellular location of the Deg proteases in Synechocystis 6803 The location of the three Deg proteases in Synechocystis 6803 has not previously been determined. In contrast to green plants, protein sorting in cyanobacteria is poorly understood, and reliable methods for separating the periplasm and the thylakoid lumen do not yet exist [69]. Our immunological studies have identified all three proteases attached to the plasma membrane, although HhoA and HtrA also seem to be associated with the thylakoid membrane (Figure 2B). It has been shown previously that all three Deg proteases have overlapping functions [25], which is supported by a common sub-localization within the cell. Components of the reaction centres of PSI and PSII as well as PsbO have previously been identified in the plasma membrane of Synechocystis 6803 [53,70]. Additionally, convergence sites between the thylakoid membranes and the plasma membrane have been observed [71], which allow the Deg proteases located at the plasma membrane access to PSII in vivo. A co-localization of the cyanobacterial Deg proteases within the plasma membrane and the thylakoid membrane (partly close to PSII) is therefore possible, although a localization within PSII is not likely, as the levels of the Deg proteases in the PSII-enriched fraction were very low. In Arabidopsis however, Deg1 has been co-isolated with PSII and has been shown to interact specifically with the reaction-centre protein D2 [17]. In summary, it had been proposed that the cyanobacterial Deg proteases were involved in response to heat- and light-stress Redox-Dependent Degradation by HhoA [22,24,25,72], but physiological substrates of cyanobacterial Deg/ HtrA proteases had not been identified. Here, we were able to show biochemically that PsbO from spinach and from Synechocystis 6803 could be substrates for the cyanobacterial Deg protease HhoA, and that the degradation of PsbO by rHhoA is likely redox- dependent. Further research is needed to determine whether redox-dependent proteolysis of PsbO by Deg proteases plays a role in PSII assembly and repair. Genetic evidence supporting our finding is currently lacking. Our efforts to investigate the possible accumulation of PsbO in a triple knockout mutant (hhoA-, hhoB- and htrA-) in Synechocystis both under high light or high temperature stress were not successful. Further experiments in a suitable homologous system in plants will be needed to address this question. Materials and Methods Lumen, PSII, PsbO, and PsbQ purification from spinach and leaves Spinach leaves were bought on the local market. Arabidopsis plants were grown as described previously [20]. Thylakoid membranes and PSII membrane fragments were isolated from spinach leaves essentially as described previously [73,74]. Chlo- roplast lumen from spinach and Arabidopsis leaves was prepared as described previously [62]. PsbO was released from the PSII membrane fragments by salt-wash treatment after removal of PsbQ and PsbP [75]. PsbQ was purified from this fraction, and obtained in 1 M NaCl, 10 mM MES, pH 6.5. The buffer was switched to 50 mM Tris-HCl, pH 7.5 and loaded onto a Q- Sepharose column equilibrated in the same buffer. PsbQ was recovered in the flow through; the buffer was switched to 50 mM CAPS, pH 10 and loaded onto a second Q-Sepharose column equilibrated in the same buffer. After washing with five bed volumes of starting buffer, the proteins were eluted with 600 mM NaCl, 50 mM CAPS, pH 10. The PsbO fraction obtained in 0.8 M Tris-HCl buffer, pH 8.4 was diluted with Milli-Q water to 50 mM Tris-HCl and concentrated by binding to a small Q- Sepharose column (0.561 cm) equilibrated with 50 mM Tris-HCl buffer, pH 8.4. Bound proteins were eluted with 600 mM NaCl in the same buffer. Fractions were analyzed by SDS-PAGE [76], and those containing PsbQ or PsbO were pooled before concentration and desalting by ultrafiltration with 3-kDa and 10-kDa cutoffs, respectively (Amicon Ultra-15, Millipore). Membrane and PSII purification from Synechocystis 6803 cells Soluble and membrane fractions of Synechocystis 6803 were separated by a combination of sucrose-density centrifugation and aqueous two-phase partitioning [56]. PSII containing His-tagged CP47 was isolated from the HT3 strain of Synechocystis 6803 [51]. Cells were grown in liquid BG-11 medium in the presence of 25 mg ml21 kanamycin under 50 mmol photons m22 s21, with air bubbling at 30uC. His-tagged PSII was purified as described previously [52] except that imidazole was used instead of histidine to elute the proteins bound to the Ni-NTA column. After two washing steps with 15 and 35 mM imidazole in buffer A [50 mM MES-NaOH, pH 6.0; 10 mM MgCl2; 5 mM CaCl2; and 25% (v/ v) glycerol] with 0.04% (w/v) dodecyl maltoside, proteins were eluted with 100 mM imidazole in buffer A with 0.04% (w/v) dodecyl maltoside. The eluted fractions were pooled and the sample was concentrated and equilibrated with buffer A by ultrafiltration (50-kDa cutoff, Amicon Ultra-15, Millipore). PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45713