Pitter F Huesgen

Helder Miranda

Xuan T Lam

Manuela Perthold

Holger Schuhmann

Iwona Adamska
email: iwona.adamska@uni-konstanz.de

Christiane Funk

Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Keywords: biochemical characterisation, oligomerisation, PDZ domain, serine proteases, substrate specificity, Synechocystis sp. PCC 6803 BSA, bovine serum albumin, Deg, degradation of periplasmic proteins, HtrA, high temperature requirement

Cyanobacteria require efficient protein quality control mechanisms to survive under dynamic, often stressful environmental conditions. It was reported that three serine proteases, HtrA, HhoA and HhoB are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. Here we show that all three proteases can degrade unfolded model substrates, but differ in respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg 2+ and Ca 2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison to each other and to the well-studied Escherichia coli orthologues DegP and DegS. Deletion of the PDZ domain decreased, but not abolished proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterisation of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant stress resistance functions in Synechocystis sp. PCC 6803.

INTRODUCTION

The comparatively self-sufficient lifestyle of photosynthetic organisms comes at a price, a high inherent risk of damage to proteins and pigments by reactive oxygen species (ROS) that are formed as byproducts of oxygenic photosynthesis, particularly under unfavourable environmental conditions [START_REF] Barber | Too much of a good thing: light can be bad for photosynthesis[END_REF]. In the natural environment cyanobacteria, such as Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), which was isolated from a fresh-water lake, are exposed to tremendous changes in light intensities and temperatures. Under these conditions formation of ROS and subsequent damage to proteins is increased [START_REF] Melis | Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage ?[END_REF]. To cope with this problem, Synechocystis 6803 contains a variety of chaperones and proteases that monitor proper fold and function of proteins [START_REF] Sokolenko | The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and Arabidopsis thaliana chloroplasts[END_REF]. Particularly interesting candidates are enzymes of the multifunctional Deg/HtrA family of ATP-independent serine endopeptidases.

Deg/HtrA proteases are defined by the combination of a catalytic domain of the S1B serine proteases, containing histidine-aspartate-serine as a catalytic triad, with one or more C-terminal PDZ domains [START_REF] Clausen | The HtrA family of proteases: implications for protein composition and cell fate[END_REF]. The PDZ domains mediate specific protein-protein interactions by preferentially binding to the C-terminal 3-4 amino acid residues of the target proteins [START_REF] Hasselblatt | Regulation of the σ E stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress[END_REF][START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF][START_REF] Sohn | Allosteric activation of DegS, a stress sensor PDZ protease[END_REF]. In Escherichia coli, DegP/HtrA is a major heat shock protein that acts both as chaperone and as unspecific housekeeping protease on denatured proteins [START_REF] Spiess | A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein[END_REF], whereas its membrane-anchored paralogue DegS/HhoB acts as a sensor for folding stress in the periplasm and initiates the σ E stress response [START_REF] Alba | degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σ E activity[END_REF][START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF][START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF]. Synechocystis 6803 has three genes encoding Deg/HtrA proteases, named htrA/degP (slr1204), hhoA/degQ (sll1679) and hhoB/degS (sll1427) after their E. coli orthologues [START_REF] Kaneko | Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions[END_REF]. However, phylogenetic analysis showed that the three Deg/HtrA paralogues of Synechocystis 6803 are more closely related to each other than to the E. coli orthologues carrying the same name, and vice versa [START_REF] Huesgen | The family of Deg proteases in cyanobacteria and chloroplasts of higher plants[END_REF]. In order to avoid confusion and prevent assumptions purely based on the name, we will hereafter use the designations HtrA, HhoA and HhoB for the Synechocystis 6803 Deg/HtrA proteases, and DegP, DegQ and DegS for the E. coli paralogues.

Crystallographic studies showed that the basic functional unit of Deg/HtrA proteases is a homo-trimer that is stabilised by residues of the protease domains [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF][START_REF] Krojer | Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine[END_REF][START_REF] Li | Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi[END_REF]. The E. coli chaperone/protease DegP further stacks two of the trimers with the catalytic centres facing each other and thus forms a very compact, proteolytic inactive, hexameric complex [START_REF] Krojer | Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine[END_REF]. DegP is activated through reversible conformational changes triggered by binding of peptides to the first of its two PDZ domains [START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF][START_REF] Krojer | HtrA proteases have a conserved activation mechanism that can be triggered by distinct molecular cues[END_REF][START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF][START_REF] Merdanovic | Determinants of structural and functional plasticity of a widely conserved protease chaperone complex[END_REF][START_REF] Meltzer | Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control[END_REF] or by a shift to higher temperature [START_REF] Spiess | A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein[END_REF]. In solution, activated DegP forms large spherical 12-or 24-mers, composed of 4 or 8 homo-trimers, enclosing the substrates [START_REF] Jiang | Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins[END_REF][START_REF] Krojer | Structural basis for the regulated protease and chaperone function of DegP[END_REF]. On lipid membranes, E. coli DegP/HtrA forms bowl-shaped structures with 4-, 5-, or 6-fold symmetry with a trimer as the basic structural unit [START_REF] Shen | Bowlshaped oligomeric structures on membranes as DegP's new functional forms in protein quality control[END_REF]. In contrast, the E. coli stress sensor DegS is a trimer that is anchored in the plasma membrane by its N-terminal transmembrane domain, exposing the protease and PDZ domains to the periplasm in a proteolytically inactive conformation [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF]. Binding of activating peptides, such as the C-termini of unfolded outer membrane porins, to the PDZ domains triggers conformational changes that activate DegS [START_REF] Hasselblatt | Regulation of the σ E stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress[END_REF][START_REF] Sohn | Allosteric activation of DegS, a stress sensor PDZ protease[END_REF][START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF].

In comparison to the detailed studies on E. coli Deg/HtrA proteases, little is known about the physiological function and biochemical mechanism of Synechocystis 6803 orthologues. Bioinformatic analysis of the primary sequences of HtrA, HhoA and HhoB showed that all three proteins contain only a single C-terminal PDZ domain [START_REF] Huesgen | The family of Deg proteases in cyanobacteria and chloroplasts of higher plants[END_REF][START_REF] Jansen | Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases[END_REF][START_REF] Kieselbach | The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis[END_REF], predicted secretory signal peptides of 34 and 25 residues for HhoA and HhoB, respectively, and a N-terminal transmembrane-spanning domain of HtrA [START_REF] Jansen | Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases[END_REF]. Biochemical analysis of the recombinant HhoA (rHhoA) suggested that similar to E. coli DegP [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF] also this protease functions as a general housekeeping enzyme in the removal of misfolded proteins.

Earlier studies investigating the physiological function of cyanobacterial Deg/HtrA proteases focused mainly on their proposed role in the selective degradation of the D1 protein from photosystem II, which is particularly susceptible to photooxidative damage and quickly turned over under high intensity light stress (reviewed in [START_REF] Adir | Photoinhibition-a historical perspective[END_REF][START_REF] Edelman | D1-protein dynamics in photosystem II: the lingering enigma[END_REF][START_REF] Huesgen | Deg/HtrA proteases as components of a network for photosystem II quality control in chloroplasts and cyanobacteria[END_REF]). Analyses of a Synechocystis 6803 triple ΔhtrA/hhoA/hhoB mutant showed that these proteases are not essential for the degradation of the D1 protein when exposed to high intensity light [START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF] or UV-B radiation [START_REF] Cheregi | The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged photosystem II in the cyanobacterium Synechocystis PCC 6803[END_REF]. Nevertheless, Deg/HtrA proteases play vitally important roles in the resistance to heat and high intensity light stresses [START_REF] Sokolenko | The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and Arabidopsis thaliana chloroplasts[END_REF][START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF][START_REF] Silva | Involvement of the HtrA family of proteases in the protection of the cyanobacterium Synechocystis PCC 6803 from light stress and in the repair of photosystem II[END_REF]. The triple ΔhtrA/hhoA/hhoB mutant accumulated more oxidatively damaged proteins than the wild type, did not grow at high intensity light or high temperature, and showed severe deficits in phototaxis [START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF]. This study also suggested an astonishing overlap in function of Deg/HtrA proteases because these severe phenotypes were only observed for the triple ΔhtrA/hhoA/hhoB mutant, but not for any of the double deletion mutants. This is particularly puzzling because proteomic analyses found the Synechocystis 6803 Deg/HtrA proteases in different subcellular compartments. HtrA was found in the outer membrane [START_REF] Huang | Isolation of outer membrane of Synechocystis sp. PCC 6803 and its proteomic characterization[END_REF], HhoA in the periplasm [START_REF] Fulda | Proteomics of Synechocystis sp. strain PCC 6803. Identification of periplasmic proteins in cells grown at low and high salt concentrations[END_REF] and associated with the plasma membrane [START_REF] Huang | Proteomic screening of saltstress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803[END_REF], while no experimental data for the predicted periplasmic localisation of HhoB have been reported.

In this study, we have expressed all three proteases as soluble recombinant enzymes (rHtrA, rHhoA and rHhoB, respectively) in active and inactive variants in order to explore similarities and differences in their enzymatic properties. We show that rHtrA, rHhoA and rHhoB can degrade unfolded model substrates, but with different preferences regarding cleavage site, pH and temperature. Furthermore, accumulation of distinct degradation intermediates and formation of different oligomeric complexes indicate diverging molecular mechanisms, both between the three cyanobacterial Deg/HtrA proteases, as well as in comparison to the well-studied E. coli orthologues.

EXPERIMENTAL

Strains and culture conditions

A glucose-tolerant wild type strain of Synechocystis sp. PCC 6803, was grown at a light intensity of 50 µmol photons m -2 s -1 at 30°C in liquid BG-11 medium [START_REF] Rippka | Generic assignments, strain histories and properties of pure cultures of cyanobacteria[END_REF] buffered with 10 mM TES, pH 8.2.

Plasmid construction

Genomic DNA was isolated from Synechocystis 6803 using a kit (Roche Diagnostics). Cloning of recombinant HhoA constructs has been described previously [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]. The sequences encoding Nterminally truncated active HtrA and HhoB protease constructs (Figure 1A) were amplified by PCR from genomic DNA with gene specific primers (Operon Biotechnologies) and ligated into the pET151-D/TOPO expression vector using the Champion directional cloning kit (Invitrogen) according to manufacturer's instructions. The following primers were used: For rHtrA 5´-CAC CTC CAT TGC TCC TGC CAA TGA G -3` and 5´-GCA TTA TTG GGG AGG GGC GCT GG -3` and for rHhoB 5´-CAC CAG TCT GAC TCC TGC CCC GGT -3` and 5´-GCA TTA TTG ACC CAA ATC TTC GGG A -3`. Plasmids encoding proteolytically inactive variant were obtained by changing the codons for the active site Ser residues to Ala with the Quikchange II point mutagenesis kit (Stratagene). The codon for Ser 296 of rHtrA was mutated to Ala (rHtrA SA ) using the primer pair 5´-CAT TAA CCC TGG TAA CTC TGG TGG TC -3´ and 5´-CAC CAG CGT TAC CCG GGT TAA TGG CC -3´, which also introduce a silent point mutation that generates an additional SmaI restriction site. The codon for Ser 258 of rHhoB was mutated to Ala (rHhoB SA ) using the primers 5´-GGC AAT GCC GGT GGG CCC CTG CTC -3´ and 5´-CAG GGG CCC ACC GGC ATT GCC TGG -3´, which also introduces an ApaI restriction site through silent point mutation. Active and inactive PDZ domain deletion constructs were obtained by the same PCR-based cloning strategy, using the plasmids encoding active or inactive HtrA and HhoB as templates. The following primers were used: For rHtrA SA ΔPDZ, 5´-CAC CTC CAT TGC TCC TGC CAA TGA G -3` and 5´-GC TCA TTT GCC CGT AGC AAT TAA CTG -3`, for rHhoBΔPDZ and rHhoB SA ΔPDZ, 5´-CAC CAG TCT GAC TCC TGC CCC GGT -3` and 5´-GC TCA CAT TTT GCC CTT GGT AAA CAG -3`. All inserted sequences and orientation in the plasmids were confirmed by DNA sequencing (GATC Biotech AG).

Expression and purification of recombinant protein

Chemically competent E. coli BL21(DE3)Star cells (Invitrogen) transformed with expression plasmids were grown in 50 to 100 ml Lysogeny Broth (LB) minimal medium containing 100 μg mg -1 ampicilin (LB/Amp100) at 37°C over night. These pre-cultures were harvested by centrifugation (3 000 g, 8 min, 4°C), resuspended and further grown in 1 to 2 l LB/Amp100 at 19°C to OD600 = 0.4 to 0.6. Expression of recombinant protease constructs was induced by the addition of 0.1 mM isopropyl-1-thio-D-galactoside (IPTG). Cultures were further grown over night at 19°C and then harvested by centrifugation (5 000 g, 10 min, 4°C). Cell pellets were resuspended in 10 to 20 ml binding buffer (50 mM HEPES-NaOH, 300 mM NaCl, pH 8.0) and stored at -20°C.

E. coli cells were lysed on ice by 10 to 15 repeats of 10 s sonication with 20 s cooling interval and centrifuged (26 000 g, 1 h, 4°C). For protease activity assays with β-casein and complex formation assays, recombinant protease constructs were purified from the soluble cell lysate by nickel affinity chromatography with HisTrap column connected to an Äkta purifier FPLC system (GE Healthcare) as described [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]. For all protease activity and reconstitution assays, recombinant protease constructs were purified at 5°C from filtered soluble cell lysates (45 μm filter) using His GraviTrap affinity columns (GE Healthcare) according to manufacturer's instructions. Application in binding buffer was followed by a washing step with 10 ml binding buffer containing 60 mM imidazole and an elution step with 5 ml elution buffer (50 mM HEPES-NaOH, 300 mM NaCl, 500 mM imidazole, pH 8.0). Eluate was collected in 1 ml-fractions, then diluted 10-fold with distilled water and concentrated using Amicon Ultra centrifugal filter devices with a molecular weight cut-off of 10 kDa (Amicon) in order to decrease the imidazole concentration and the buffer strength. Protein concentrations were determined by Bradford protein assay (Biorad) with bovine serum albumin (BSA) solutions as standard.

Analysis of protease complex formation

Elution fractions containing approximately 200 μg purified Deg/HtrA protease constructs were incubated either alone or in a 1:3, 1:1 or 3:1 molar ratio with β-casein (Sigma-Aldrich) as model substrate for 10 min at room temperature. Complex sizes were estimated by size-exclusion chromatography using a pre-packed Superdex 200 column (GE Healthcare). The column was repeatedly calibrated with high and mid-weight molecular mass markers (GE Healthcare) according to manufacturer's instructions.

Protease activity assays

For protease activity assays, purified protease constructs were incubated with an excess of βcasein, bovine serum albumin (BSA), or lysozyme in 250 mM Tris, pH 7.0, supplemented with 20 mM CaCl 2 for 2 h at 40°C. Unfolding of lysozyme was induced by incubation with 20 mM DTT for 3 h at 40°C prior to protease activity assays.

Fluorogenic protease activity assays were performed using the Enzchek Protease Assay (Invitrogen). For each reaction, 3 μg purified recombinant protease were incubated with 0.5 μg BODIPY TR-X casein in a total volume of 200 μl in the presence of 0.1 mM NaN 3 . To measure the pH-optima, 10 mM MES (up to pH 6.5) or HEPES (from pH 7.0) were used to buffer the solutions and the measurements were performed at 30°C. Fluorescence signals measured at a wavelength of 617 nm were followed with an Infinite 200 microplate reader (Tecan) using 589 nm as excitation wavelength. For kinetic measurements at different pH, a fluorescence signal increase was followed over time, recording 6 replicates every 10 min, while incubating the sample plate in darkness at 30°C inside the microplate reader. The activity was calculated from the initial rate of fluorescence emission. For determination of the temperature dependence, assays were incubated in 10 mM MES and at the optimal pH of the three Deg/HtrA proteases (pH 5.5 for rHtrA, pH 6.5 for rHhoA and rHhoB) at a temperature range from 10°C to 65°C in a PCR cycler before measuring total fluorescence at room temperature after 40 min incubation.

Sequencing of proteolytic β β-casein fragments

To identify β-casein cleavage sites 200 pmol purified recombinant proteases (active, or inactive variants as negative controls) were incubated in the presence of 10 mM CaCl 2 with 75 μg βcasein at 40°C for several hours. Samples were taken after 0, 30, 90 min and 10 h and analysed by SDS-PAGE (see below). Proteolytic fragments of cleaved β-casein were excised from the gel and sent for N-and C-terminal sequencing to the Protein Analysis Centre (PAC, Karolinska Institute, Stockholm, Sweden).

Protein analysis

Proteins were solubilised in lithium dodecyl sulphate (LDS) buffer and separated by SDS-PAGE as described [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Gels were stained with Coomassie brilliant blue R250 [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

Bioinformatics

Sequence analysis was performed using SMART [38], TargetP [START_REF] Emanuelsson | Predicting subcellular localization of proteins based on their N-terminal amino acid sequence[END_REF] and SignalP [START_REF] Nielsen | Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites[END_REF] programs. For engineering of mature, soluble protease constructs, the secondary structures of HhoA, HhoB and HtrA were analysed with PsiPred v2.5 [START_REF] Bryson | Protein structure prediction servers at University College London[END_REF] and compared to published structures of E. coli DegP [START_REF] Krojer | Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine[END_REF], DegS [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF], and human HtrA2/Omi [START_REF] Li | Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi[END_REF] and sequence alignments [START_REF] Clausen | The HtrA family of proteases: implications for protein composition and cell fate[END_REF][START_REF] Jansen | Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases[END_REF]. To investigate the enzymatic properties of three cyanobacterial Deg/HtrA proteases in detail, we expressed rHtrA, rHhoA and rHhoB in their active form as recombinant fusion proteins with Cterminal His-tag using the E. coli expression system. For controls, inactive versions of the proteases with mutations in the catalytic site were generated by site-directed point mutagenesis, expressed and purified under similar conditions. Based on earlier studies of membrane-anchored human HtrA2 [START_REF] Li | Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi[END_REF] and E. coli DegS [START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF][START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF], the putative transmembrane-domain of HtrA from Synechocystis 6803 was truncated after the residue Gln 93 (Figure 1A and Supplementary Figure S1). Both active rHtrA, as well as inactive rHtrA SA , with Ser 296 replaced by Ala, were obtained in sufficient quantity and purity after purification by Ni 2+ -affinity chromatography (Figure 1B). rHhoA and its proteolytically inactive version rHhoA SA , both lacking the predicted signal peptide, were expressed and purified as described (Figure 1A andB; [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]). HhoB contains a predicted N-terminal signal sequence of 25 residues and a long additional N-terminal domain of 56 residues (compared to only 23 residues in HhoA) between signal peptide and protease domain. Based on our experience with rHhoA, we first tried to express rHhoB lacking only the predicted signal sequence. However, this construct could not be expressed as soluble protein and its yield was extremely low (data not shown). Therefore, we further truncated HhoB after Gln 50 , placing the introduced His-tag in approximately the same distance from the protease domain as in rHhoA. A third rHhoB construct was truncated after Gln 74 , close to the predicted first helix of the protease domain (Supplementary Figure S1). Of these, only the rHhoB construct truncated after Gln 50 yielded low amounts of active enzyme (Figure 1B, hereafter rHhoB). Also the inactive version rHhoB SA , with Ser 258 replaced by Ala, was obtained in similar yield and purity (Figure 1B).

RESULTS AND DISCUSSION

Engineering and heterologous expression of Deg/HtrA protease constructs of Synechocystis 6803

To investigate the role of the single PDZ domain in each of the three Synechocystis 6803 Deg/HtrA proteases, we further created PDZ domain deletion constructs by truncation within the predicted loops connecting the protease and PDZ domains (Figure 1A and Supplementary Figure S1). rHtrA and rHtrA SA were truncated after Lys 342 to yield rHtrAΔPDZ and rHtrA SA ΔPDZ, respectively. Cloning and expression of rHhoAΔPDZ and its proteolytically inactive variant rHhoA SA ΔPDZ, both truncated after Gly 282 , were expressed and purified as described before (Figure 1A andC; [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]). Similarly, rHhoB and rHhoB SA were truncated after Met 305 , resulting in rHhoBΔPDZ and rHhoB SA ΔPDZ, respectively. All deletion constructs were obtained in good quantity and reasonable purity after Ni 2+ -affinity chromatography (Figure 1C).

All three Deg/HtrA proteases in Synechocystis 6803 are protein quality control factors

We assayed the activity and substrate specificity of the recombinant Deg/HtrA constructs using model substrates that differed with regard to their folding states, such as bovine β-casein, BSA, and lysozyme. All three protease constructs with intact PDZ domain, rHtrA, rHhoA and rHhoB, were able to cleave the naturally unfolded β-casein, but not correctly folded globular BSA or lysozyme (Figure 2A). Reduction of lysozyme with DTT, which induces partial unfolding by breaking disulfide bonds, allowed its cleavage by HhoA and to a lower extent by rHtrA, but not by HhoB (Figure 2A, lower panel). Under the chosen conditions, equal amounts of rHtrA, rHhoA and rHhoB showed very different activity: Little uncleaved β-casein remained in assays with rHtrA, and a prominent degradation fragment appeared. rHhoA completely degraded the excess of β-casein, whereas rHhoB showed only little activity and generated a prominent degradation fragment with a slightly lower molecular mass than intact β-casein (Figure 2A). All substrates tested were not degraded in the presence of inactive protease variants, thus excluding the substrate degradation by active variants was mediated by impurities in the preparation (Figure 2A).

We consistently assayed a lower activity for rHhoB than for rHtrA and rHhoA. We can not exclude that this is merely caused by unfortunate engineering of the rHhoB truncation constructs, but the chosen truncation of HhoB at Gln 50 should have affected neither the fold of the protease domain, nor any other important domain [START_REF] Kieselbach | The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis[END_REF]. Instead, this truncation made rHhoB more similar to rHhoA, which is the most active recombinant protease in our hands. Thus, the low activity of rHhoB might also reflect, similarly to E. coli DegS [START_REF] Alba | degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σ E activity[END_REF], a more stringent substrate specificity, a function as chaperone rather than protease, a requirement for yet unidentified cofactors, such as allosteric activators [START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF][START_REF] Merdanovic | Determinants of structural and functional plasticity of a widely conserved protease chaperone complex[END_REF][START_REF] Meltzer | Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control[END_REF], or a need for the formation of a hetero-oligomeric complex. A formation of hetero-hexameric complex was reported for chloroplast-located Deg5 and Deg8 in Arabidopsis thaliana [START_REF] Sun | Formation of DEG5 and DEG8 complexes and their involvement in the degradation of photodamaged photosystem II reaction center D1 protein in Arabidopsis[END_REF].

Taken together, this provides evidence that all three proteases, HtrA, HhoA and HhoB, might act as protein quality control factors degrading denatured and damaged proteins [START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF]. This further suggests remarkable mechanistic differences to Deg/HtrA proteases in E. coli, where DegP and DegQ with two PDZ domains [START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF][START_REF] Kolmar | The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation[END_REF] act as quality control factors, whereas DegS, containing a transmembrane domain and a single PDZ domain, acts as a stress sensor [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF][START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF].

Deletion of the PDZ domain severely impaired proteolytic activity rHtrAΔPDZ showed only residual activity against β-casein and reduced lysozyme (Figure 2B). Also rHhoAΔPDZ degraded both unfolded substrates less efficiently than its variant with the intact PDZ domain, and larger degradation fragments accumulated after proteolysis of reduced lysozyme (Figure 2B). rHhoBΔPDZ showed only minute activity against β-casein and no activity was detected against reduced lysozyme (Figure 2B). As expected, no degradation of substrates tested was assayed in the presence of inactive protease variants lacking PDZ domains (Figure 2B). Taken together, this data suggests that the PDZ domains are not absolute requirements for proteolytic activity of all three proteases but they are important for the regulation of proteolytic activity and/or for presenting the substrate to the protease domain.

rHtrA, rHhoA and rHhoB generate distinct β β-casein degradation products Proteolysis of the model substrate β-casein by recombinant Deg/HtrA proteases with intact PDZ domains was further investigated in more detail, and different enzymatic kinetics and distinct degradation intermediates were observed (Figure 3A). In the presence of rHtrA only traces of βcasein could be detected after 30 min of incubation, but proteolytic fragments 1 and 2 remained stable and were not further degraded during 90 min of incubation (Figure 3A, left panel). Similarly, most of the β-casein was cleaved by rHhoA within 30 min and the generated proteolytic fragment 3, but not fragments 4 and 5, disappeared within 90 min (Figure 3A, middle panel). Longer incubation times lead to a complete degradation of all fragments (Figure 2A, [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]). In contrast, a very low proteolytic activity was assayed for rHhoB. Significant amounts of β-casein were detected even after 10 h of incubation with this protease and the proteolytic fragment 6 accumulated progressively with the time of incubation (Figure 3A, right panel). This suggests that Synechocystis 6803 Deg/HtrA proteases release their substrates after defined cleavage sites, similar to E. coli DegS [START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF], A. thaliana Deg2 [START_REF] Haußühl | A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II[END_REF] and Deg15 [START_REF] Helm | Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants[END_REF][START_REF] Schuhmann | The DEG15 serine protease cleaves peroxisomal targeting signal 2-containing proteins in Arabidopsis[END_REF]. This is a remarkable contrast to E. coli DegP, which digests unfolded substrate proteins progressively without accumulation of intermediates [START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF][START_REF] Krojer | Structural basis for the regulated protease and chaperone function of DegP[END_REF].

Cleavage site specificity

The cleavage specificity of proteases is defined by the amino acid composition of the substrate around the cleavage site, with non-prime site residues P3, P2, P1 preceding the cleavage site, and prime-site residues P1', P2', P3' following the cleavage site [START_REF] Schechter | On the size of the active site in proteases. I. Papain[END_REF]. To gain insight into the cleavage site specificity of rHtrA, rHhoA and rHhoB, we analysed the most abundant proteolytic β-casein fragments generated by each protease by N-and C-terminal sequencing. The majority of the sequenced fragments contained the N-terminus of mature β-casein, while cleavage occurred at the C-terminal part of this substrate (Figure 3B and Table 1). Three C-terminal cleavage sites, Val 162 , Gln 141 and Val 130 , were identified for rHtrA (Figure 3B and Table 1). A common cleavage site at Leu 165 was found for rHhoA and rHhoB, however, rHhoA additionally cleaved β-casein at Ala 101 generating two proteolytic fragments, the N-terminal 1-101 as well as the C-terminal 102-199 amino acid fragments (Figure 3B and Table 1).

E. coli DegP showed a strong preference for small hydrophobic residues (valine, alanine, threonine and isoleucine) in the P1 position, whereas P2 and P3 positions are less defined [START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF]. Our data show a similar trend for rHtrA and rHhoA (Figure 2B). It was further demonstrated that PDZ1, the first of the two PDZ domains of E. coli DegP, recognised the hydrophobic C-terminus of substrates and presented the substrate for cleavage [START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF]. Since DegP cleavage occurred at a hydrophobic site approximately 12 to 16 amino acids further towards the N-terminus, the resulting cleavage fragment could again be bound by a PDZ1 domain of the trimer, resulting in processive cleavage of the substrate into peptides of 10 to 20 residues [START_REF] Krojer | Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins[END_REF]. Similarly, the PDZ domains of Synechocystis 6803 Deg/HtrA proteases might preferentially bind the hydrophobic residues at the C-terminus of β-casein (-PFPIIV) and present the substrate to the catalytic site for cleavage, explaining the absence of larger C-terminal fragments. However, all three Synechocystis Deg/HtrA proteases are not as processive as E. coli DegP, possibly due to the lack of a second PDZ domain.

Inactive Synechocystis Deg/HtrA proteases form different oligomers Earlier structural studies have shown that the basic unit of Deg/HtrA proteases is a homo-trimer [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF][START_REF] Krojer | Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine[END_REF][START_REF] Li | Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi[END_REF]. Two trimers of E. coli DegP are stacked to a cage-like hexamer by both PDZ domains of the subunits [START_REF] Krojer | Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine[END_REF][START_REF] Iwanczyk | Role of the PDZ domains in Escherichia coli DegP protein[END_REF][START_REF] Jomaa | The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity[END_REF]. Homology modelling predicted all three Synechocystis 6803 Deg/HtrA proteases to form trimers [START_REF] Jansen | Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases[END_REF]. Surprisingly, we found earlier that also rHhoA assembles into a hexamer in solution [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF], despite the fact that it only contains one PDZ domain. Here we assayed the oligomerisation state of rHtrA SA and rHhoB SA by size-exclusion chromatography. rHtrA SA eluted with a volume of 13.2 ml, corresponding to an apparent molecular mass of 140 kDa (Figure 4A), which is consistent with a trimer formation. Published crystal structures show that Deg/HtrA protease trimers from Homo sapiens and E. coli are disk-shaped and wider than tall if viewed from the side [START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF][START_REF] Li | Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi[END_REF]. Therefore, such trimers have a larger hydrodynamic radius than globular proteins of the same molecular mass, resulting in a larger-than-expected apparent molecular mass when estimated by the elution volume in a size-exclusion chromatography calibrated with globular standard marker proteins. However, the rHtrA SA complexes were unstable, and we frequently observed monomers eluting at approximately 14.6 ml (apparent molecular mass of ca. 60 kDa). In agreement with our earlier experiments [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF], rHhoA SA formed a hexamer eluting with a volume of 11.5 ml (apparent molecular mass 320 kDa) (Figure 4B, [START_REF] Shen | Bowlshaped oligomeric structures on membranes as DegP's new functional forms in protein quality control[END_REF]). rHhoB SA eluted at 13.1 ml, indicating a trimeric complex with an apparent molecular mass of approximately 160 kDa (Figure 4C). For rHtrA SA and rHhoB SA additional peaks were observed at the void volume of the Superdex 200 size-exclusion columns, but very little or no protein was detected in these fractions after SDS-PAGE and Coomassie-blue staining (Figs 4A and C, lower panels), indicating that these peaks might represent SDS-insoluble aggregates.

Substrate binding to rHtrA SA and rHhoA SA changes their oligomerisation states

In the presence of substrates E. coli DegP massively re-arranges and assembles several trimers into large oligomeric complexes, mediated by interactions of both PDZ domains [START_REF] Krojer | HtrA proteases have a conserved activation mechanism that can be triggered by distinct molecular cues[END_REF][START_REF] Jiang | Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins[END_REF][START_REF] Krojer | Structural basis for the regulated protease and chaperone function of DegP[END_REF]. To test the effect of substrate binding on the oligomerisation state of cyanobacterial Deg/HtrA proteases, we incubated rHtrA SA , rHhoA SA and rHhoB SA with β-casein as model substrate prior to size-exclusion chromatography. In the presence of β-casein, the elution diagram of rHtrA SA displayed an additional peak at ca. 10.5 ml, indicating the formation of a complex with an apparent molecular mass of approximately 580 kDa (Figure 4A). β-casein alone elutes at 13.5 ml (data not shown, compare with Figure 4C), but co-elutes with rHtrA SA in the 10.5 ml-peak, clearly demonstrating that this complex is the result of β-casein binding by rHtrA SA (Figure 4A). As for rHtrA SA alone, an additional prominent peak is detected at the void volume of the column, but only little protein is observed in the sample aliquot of this fraction after SDS-PAGE analysis (Figure 4A). Due to steric constraints, the formation of such higher-order oligomeric HtrAsubstrate complexes appears unlikely under physiological conditions since HtrA is anchored in the outer membrane [START_REF] Cheregi | The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged photosystem II in the cyanobacterium Synechocystis PCC 6803[END_REF]. This suggests that HtrA may function similar to human HtrA2/Omi, which is anchored as an inactive zymogen to the inner mitochondrial membrane [START_REF] Iwanczyk | Role of the PDZ domains in Escherichia coli DegP protein[END_REF]. A soluble, active form of HtrA2/Omi is released by cleavage of the N-terminal 133 amino acids that contain the transmembrane anchor (Figure 1A). This soluble form translocates from the mitochondria to the cytosol and contributes to apoptosis [START_REF] Vande Walle | The mitochondrial serine protease HtrA2/Omi: an overview[END_REF]. A similar release of the cyanobacterial HtrA protease from the outer membrane would also help to explain the overlap in its function with HhoA and HhoB under certain conditions.

Similar to rHtrA SA , also rHhoA SA changed its complex size dramatically in the presence of βcasein, featuring prominent additional peaks at 8.3 ml and at 9.5 ml (Figure 4B). Both rHhoA SA and β-casein co-eluted in the peak at 9.5 ml, indicating the formation of a large rHhoA SA /βcasein complex of approximately 670 kDa. Hardly any protein was observed in the fraction containing the early peak at 8.3 ml, which is close to the column void volume and might therefore contain insoluble aggregates. In contrast, the elution profile of rHhoB SA in the presence of β-casein showed no additional peaks and the elution profiles of rHhoB SA and β-casein alone were very similar, indicating that these two proteins did not form a stable complex under conditions tested (Figure 4C). This observation is consistent with the low activity of rHhoB observed in proteolytic assays (Figure 2C) and may indicate either a problem of the rHhoB constructs that impairs formation of higher-oligomeric complexes or a lack of required cofactors, such as specific peptides [START_REF] Krojer | HtrA proteases have a conserved activation mechanism that can be triggered by distinct molecular cues[END_REF][START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF][START_REF] Merdanovic | Determinants of structural and functional plasticity of a widely conserved protease chaperone complex[END_REF][START_REF] Meltzer | Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control[END_REF]. It was reported that binding of specific effector-peptides to the PDZ domain-1 of E. coli DegP in a resting conformation causes a local rearrangement that is allosterically transmitted to the substrate-binding pocket of the protease domain and such activated protease state assembles into larger oligomeric particles [START_REF] Merdanovic | Determinants of structural and functional plasticity of a widely conserved protease chaperone complex[END_REF][START_REF] Meltzer | Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control[END_REF][START_REF] Jiang | Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins[END_REF][START_REF] Krojer | Structural basis for the regulated protease and chaperone function of DegP[END_REF]. Also E. coli DegS is activated by the binding of allosteric effectors to the PDZ domain, but in contrast to DegP, protease activation and substrate proteolysis are separated events [START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF][START_REF] Wilken | Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease[END_REF].

Our studies on cyanobacterial Deg/HtrA proteases indicate that the formation of large oligomeric complexes in the presence of a substrate might be a common feature for this enzyme family (Figure 4). Unfortunately, size-exclusion chromatography is not suitable for a reliable determination of the exact oligomerisation state of rHtrA SA /β-casein and rHhoA SA /β-casein complexes due to the unknown number of bound β-casein molecules and its unknown effect on the hydrodynamic radius of the complexes. However, in comparison to published data by Krojer and colleagues [START_REF] Krojer | Structural basis for the regulated protease and chaperone function of DegP[END_REF] and Jiang and colleagues [START_REF] Jiang | Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins[END_REF], the formation of 12-or 24-meric complexes for rHhoA SA , and 9-to 12-mers for rHtrA SA is estimated.

Deletion of PDZ domains abolishes formation of oligomers beyond a trimer

We had shown earlier [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF] that the deletion the PDZ domain of rHhoA prevents formation of a hexamer. Instead, rHhoAΔPDZ assembled into a trimer, which is consistent with the homology model that attributed trimerisation to the N-terminus of the protease domain [START_REF] Jansen | Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases[END_REF]. Similarly, rHtrAΔPDZ and rHhoBΔPDZ were detected as trimers (data not shown). However, the trimers of all three constructs were not very stable and monomers were frequently observed. The equilibrium of trimers and monomers appeared concentration-dependent, with monomers prevailing at low protein concentrations. All three PDZ-domain deletion constructs, rHtrAΔPDZ, rHhoAΔPDZ and rHhoBΔPDZ, were not able to assemble to larger complexes in the presence of β-casein (summarised in Figure 5). This is in agreement with reports showing that peptide binding to the PDZ domain of E. coli DegP [START_REF] Krojer | HtrA proteases have a conserved activation mechanism that can be triggered by distinct molecular cues[END_REF][START_REF] Meltzer | Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli[END_REF][START_REF] Merdanovic | Determinants of structural and functional plasticity of a widely conserved protease chaperone complex[END_REF] promotes formation of higher-order-particles.

Mg 2+ and Ca 2+ increase activity of rHtrA, rHhoA and rHhoB

We characterised the proteolytic activity of rHtrA, rHhoA and rHhoB in biochemical terms using BODIPY TR-X-labelled casein as a fluorogenic substrate. The effect of increasing concentrations of divalent ions on the proteolytic activity of three Deg/HtrA proteases was tested. The values were normalised to the protease activity without the addition of divalent cations to display the actual increase of activity in the presence of divalent ions. While the activity of rHtrA and rHhoA increased almost 4-to 6-times in the presence of 5 mM CaCl 2 and remained constant at higher CaCl 2 concentrations, the activity of rHhoB increased more gradually up to 14-times reaching the maximal level at 10 mM CaCl 2 (Figure 6A). Similar results were obtained after the addition of MgCl 2 (Supplementary Figure S2A). The reason for this activation by Mg 2+ or Ca 2+ is not clear at the moment, but either: (i) divalent cations could interact with the substrate and make it more susceptible for degradation, and/or (ii) binding of divalent cations could affect the structure and thus activates the Deg/HtrA protease. The much stronger effect of Mg 2+ or Ca 2+ ions on rHhoB activity (Figure 6A) can be explained by the almost negligible activity in the absence of divalent ions (Figure 6B).

Overlapping, but distinct pH and temperature profiles of rHtrA, rHhoA and rHhoB

The pH preference profiles were measured for all three Deg/HtrA proteases in the absence (Figure 6B) and the presence (Figure 6C) of CaCl 2 at a range from pH 4.5 to 8.0. In the absence of CaCl 2 rHtrA showed a maximal proteolytic activity between pH 5.5 and 6.0, whereas rHhoA had its optimum at pH 6.5 (Figure 6B). Almost no proteolytic activity was detected for rHhoB (Figure 6B). However, the addition of 10 mM CaCl 2 , resulted in strong changes in the pH optima leading to their broadening and a shift towards a basic pH (Figure 6C). For rHtrA the highest activity was detected at pH 7.0, whereas the activity of rHhoA progressively increased with higher pH (Figure 6C, please note that the fluorescence detector gain was decreased after the addition of divalent cations to avoid saturation). This striking shift in the pH optimum of HhoA in the presence of Ca 2+ is in agreement with earlier observations [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]. Similar pH preference profiles were also observed after the addition of MgCl 2 , ruling out a Ca 2+ -specific effect (data not shown). The activity of rHhoB in the presence of Ca 2+ was constantly high at pH between 5.5 and 6.5 and decreased slightly at pH 6.5 to 8.0 (Figure 6C).

In the absence of Ca 2+ , rHtrA, rHhoA and rHhoB were all most active at slightly acidic conditions. In the presence of 10 mM Ca 2+ , both rHtrA and rHhoB showed a broader pH optimum with a maximum between pH 5.5 and 7.0. In contrast, rHhoA showed a striking shift of its pH optimum to basic conditions. To our knowledge, such a strong shift in the pH preference has not been reported for any other protease.

The effect of temperature on the proteolytic activity of Deg/HtrA proteases was assessed by measuring total fluorescence at one fixed time point after 40 min incubation at the indicated temperature (Figure 6D). To ensure that neither the substrate was limiting at the chosen time point, nor saturation of the detector occurred at/or near the fluorescence maxima, control experiments were performed, in which the fluorescence emission generated by degradation of BODIPY TR-X-labelled casein was measured over time at 30°C (Supplementary Figure S2B). Only low proteolytic activity was recorded for the three active Deg/HtrA proteases at a temperature ranging between 10 to 25°C (Figure 6D). While rHtrA had its temperature optimum between 30°C and 40°C the activity of rHhoA increased with rising temperature reaching its maximum at around 50 to 55°C and decreased rapidly at higher temperatures, probably due to denaturation of the enzyme (Figure 6D). A similar temperature curve has also been observed previously for rHhoA using Resorufin-labelled casein as substrate [START_REF] Huesgen | The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain[END_REF]. The temperature dependency of rHhoB was comparable to rHhoA, however, its optimum was shifted to slightly lower temperatures and the maximal activity was reached already at 45 to 50°C (Figure 6D).

Implications for the physiological function of HtrA, HhoA and HhoB in Synechocystis 6803

All three Synechocystis 6803 Deg/HtrA proteases were able to act on unfolded protein substrates, suggesting functions as general protein quality control proteases consistent with the stresssensitive phenotype of the triple mutant ΔhtrA/hhoA/hhoB [START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF]. The overlapping biochemical properties of HtrA, HhoA and HhoB may allow any two of the enzymes to compensate for the lack of the third, which could explain the striking lack of stress-sensitive phenotypes of the double deletion mutants in comparison to the triple mutant ΔhtrA/hhoA/hhoB [START_REF] Barker | The Deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle[END_REF]. However, our data also suggests that each of the three enzymes could contribute differently to the overall proteolytic potential with varying environmental conditions. For instance, Synechocystis 6803 cells prefer to grow at alkaline conditions [START_REF] Kurian | Proteomic characterization of acid stress response in Synechocystis sp[END_REF] and are commonly grown in BG11-media, which contain approximately 0.5 mM of combined Mg 2+ and Ca 2+ ions. Thus, HhoA would be the most active of the three Synechocystis 6803 Deg/HtrA proteases under optimal physiological conditions.

The observed activation of all three proteases by Ca 2+ and Mg 2+ ions and the slightly acidic pH optimum of HtrA and HhoB may be particularly important for an effective stress response to high intensity light. Sufficiently high intensity light saturates photosynthesis rates, causing acidification of the thylakoid lumen and increased formation of ROS [START_REF] Barber | Too much of a good thing: light can be bad for photosynthesis[END_REF]. This further leads to oxidative damage of photosynthetic protein complexes and release of Ca 2+ from the thylakoid membranes to the lumen [START_REF] Adir | Photoinhibition-a historical perspective[END_REF]. These conditions then would activate any of the three Deg/HtrA proteases. Since no reliable localisation studies exist for Deg/HtrA proteases in cyanobacteria it is not clear whether these enzymes are present in the periplasmic space, in the thylakoid lumen or in both compartments. Unfortunately, purification of the cyanobacterial thylakoid lumen proteome poses massive technical challenges [START_REF] Spence | Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803[END_REF] and thus neither a comprehensive proteomic analysis has been reported, nor is it possible to probe this compartment for the presence of Deg/HtrA proteases with antibodies. However, three Deg/HtrA proteases have been detected in the thylakoid lumen of higher plants, where no other general proteases were reported [START_REF] Peltier | Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction[END_REF][START_REF] Schubert | Proteome map of the chloroplast lumen of Arabidopsis thaliana[END_REF]. Furthermore, out of the reported 16 paralogues of A. thaliana, the three cyanobacterial proteases show the highest similarity to the three thylakoid lumen-located Deg/HtrA enzymes [START_REF] Kieselbach | The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis[END_REF]. Hence it is very likely that at least one of the Synechocystis 6803 Deg/HtrA proteases, such as e.g. HhoB, is located in the thylakoid lumen.

The temperature profiles of rHtrA, rHhoA and rHhoB give an indication how these enzymes may contribute to high temperature resistance. While rHtrA activity decreased at temperatures higher than 30°C, which is the preferred growth temperature of Synechocystis 6803, the activity of rHhoA and rHhoB increased to well above lethal temperatures. The soluble enzymes HhoA and HhoB should, therefore, play a more important role in protein quality control at high temperature than HtrA. Interestingly, htrA is the only gene regulated by the two-component system Hik10-Rre3 under salt and hyperosmotic stresses, which suggests an additional, more specific function of HtrA [START_REF] Murata | Exploitation of genomic sequences in a systematic analysis to access how cyanobacteria sense environmental stress[END_REF].

Conclusions

In summary, this family-wide biochemical characterisation of the Synechocystis 6803 Deg/HtrA proteases suggests complementary physiological functions of the three enzymes and highlights considerable mechanistic differences of HtrA, HhoA and HhoB, as compared to their wellstudied E. coli orthologues. We have shown that the three Deg/HtrA proteases of Synechocystis 6803, expressed as soluble recombinant His-tagged fusion proteins, can act as protein quality control proteases. Distinct pH-and temperature-optima of the three enzymes, different cleavage sites and oligomerisation status speak in favour of partially overlapping but not completely redundant physiological functions.

AUTHOR CONTRIBUTION

Pitter Huesgen engineered Deg/HtrA constructs, performed oligomerisation studies together with Helder Miranda, Manuela Perthold and Holger Schuhmann, analysed the data and wrote draft of the paper. Helder Miranda and Lam Xuan Tam expressed and purified Deg/HtrA constructs and performed proteolytic assays. Holger Schuhmann performed bioinformatic analysis. Christiane Funk and Iwona Adamska co-conceived the research and edited the manuscript.

Table 1

Cleavage sites within β β-casein by rHtrA, rHhoA and rHhoB assayed by Nand C-terminal sequencing. a The mature secreted version of β-casein (processed at the N-terminal Ala 15 ) was used for these studies (N1-199 amino acids). Our results demonstrated that in contrast to trimeric rHhoB, trimeric rHtrA and hexameric rHhoA form higher-order oligomers in the presence of β-casein. Deletion of the PDZ domain locks all three proteases in the trimer state. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Protease

Biochemical

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  Biochemical Journal Immediate Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  that

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figure 1

 1 Figure 1 Purification of recombinant active and inactive variants of Synechocystis 6803 Deg/HtrA proteases

Figure 2

 2 Figure 2Proteolytic activity of Synechocystis 6803 Deg/HtrA proteases against substrates with different folding states

Figure 3

 3 Figure 3Cleavages site specificity of Synechocystis 6803 Deg/HtrA-proteases

Figure 4

 4 Figure 4 Complex formation by inactive variants of Synechocystis 6803 Deg/HtrA proteases in the absence and the presence of substrate

Figure 5

 5 Figure 5Schematic overview of the oligomerisation data

Figure 6

 6 Figure 6Biochemical properties of rHtrA, rHhoA and rHhoB assayed with a fluorogenic casein substrate

  A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTT PVVVPPFLQPEVMGVSKVKEA MAPKHKEMPFPKYPVEPFTESQSLTLTDV ENLHLPLPLLQ SWMHQPHQPLPPTVM Journal Immediate Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

  Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Biochemical Journal Immediate Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Publication. Published on 21 Feb 2011 as manuscript BJ20102131 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102131 A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

FUNDING

This work was supported by grants of the Deutsche Forschungsgemeinschaft (AD92/8-2, AD92/8-3 to I.A.), the University of Konstanz (to I.A.), the Swedish Energy Agency (2009-000928 to C.F.), the Royal Swedish Academy of Sciences (312-3820-06 to C.F.), the Lawsky Foundation (to L.X.T.) and Umeå University (to C.F.).