Deletion of Synechocystis sp. PCC 6803 Leader Peptidase LepB1 Affects Photosynthetic Complexes and Respiration*

The cyanobacterium Synechocystis sp. PCC 6803 possesses two leader peptidases, LepB1 (Sll0716) and LepB2 (Slr1377), responsible for the processing of signal peptide-containing proteins. Deletion of the gene for LepB1 results in an inability to grow photoautotrophically and an extreme light sensitivity. Here we show, using a combination of Blue Native/SDS-PAGE, Western blotting and iTRAQ analysis, that lack of LepB1 strongly affects the cell's ability to accumulate wild-type levels of both photosystem I (PSI) and cytochrome (Cyt) b6f complexes. The impaired assembly of PSI and Cyt b6f is considered to be caused by the no or slow processing of the integral subunits PsaF and Cyt f respectively. In particular, PsaF, one of the PSI subunits, was found incorporated into PSI in its unprocessed form, which could influence the assembly and/or stability of PSI. In contrast to these results, we found the amount of assembled photosystem II (PSII) unchanged, despite a slower processing of PsbO. Thus, imbalance in the ratios of PSI and Cyt b6f to photosystem II leads to an imbalanced photosynthetic electron flow up- and down-stream of the plastoquinone pool, resulting in the observed light sensitivity of the mutant. We conclude that LepB1 is the natural leader peptidase for PsaF, PsbO, and Cyt f. The maturation of PsbO and Cyt f can be partially performed by LepB2, whereas PsaF processing is completely dependent on LepB1. iTRAQ analysis also revealed a number of indirect effects accompanying the mutation, primarily a strong induction of the CydAB oxidase as well as a significant decrease in phycobiliproteins and chlorophyll/heme biosynthesis enzymes.

plant chloroplasts, they contain three different types of membranes, an outer membrane, a plasma membrane (PM), and a thylakoid membrane (TM). The thylakoid membrane is the site not only for photosynthesis but also the main site for respiration. The ultrastructure and organization of the membranes is however still under debate and mainly two different opinions prevail concerning the organization of plasma and thylakoid membranes. One is that the two membranes are continuous, making the periplasm and lumen a common compartment, the second that the membranes are completely separated (1)(2)(3)(4)(5)(6)(7)(8)(9).
The complex membrane organization within the cyanobacterium implies the existence of a sophisticated system for the sorting and transport of extracytoplasmic proteins to the different membranes and compartments. Targeting of these proteins in eubacteria and archaea occurs through the wellcharacterized Sec and Tat translocon pathways (10). Protein substrates for the Sec and Tat systems were shown to be distinguished by specific N-terminal signal sequences (11). However, most thylakoid and plasma membrane integral proteins do not have N-terminal signal peptides. For insertion into the membrane all TM and PM integral proteins require, in addition to the Sec translocon, a protein insertase of the Alb3/Oxa1 family, YidC (12)(13)(14), as shown for the core photosystem II (PSII) protein D1 (PsbA) (15,16). The Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) genome, as well as all other completely sequenced cyanobacterial genomes, contains only a single set of genes encoding the subunits of the Sec and Tat translocons (17)(18)(19). This suggests that the same translocation machineries are working in plasma and thylakoid membranes and this in turn indicates that information additional to the signal sequence is needed for correct targeting (20,21). The question of where in the intricate Synechocystis membrane system translocation and protein sorting takes place is still subject for intense research.
Sec and Tat signal peptides are both cleaved by Type I signal peptidases (also called leader peptidase, Lep) (11). In contrast to other Gram-negative bacteria, almost all the cyanobacterial genomes analyzed so far contain two lep genes. Although the overall sequence identity of leader peptidases from various species is relatively low, five conserved regions were identified in the catalytic domain, called boxes B, C, D, and E, as well as the membrane anchor domain A (22,23). Both LepB1 and LepB2 in Synechocystis have the conserved A-E boxes (21,23), including the invariant amino acid residues demonstrated to be essential for catalytic activity. No other proteins with these conserved regions are found in the Synechocystis genome.
In subproteomic analyses LepB2 was found in the plasma membrane, whereas LepB1 was found in the thylakoid membrane (7,24,25). Both LepB1 and LepB2 are integral membrane proteins with one transmembrane helix, present in very low amounts in the membrane. The above observations may therefore not implicate completely unique membrane localizations for the two leader peptidases. Knockout mutations of the two genes, however, give significantly different results: whereas LepB2 appears to be totally essential for cell viability, cells with a deletion in LepB1 are still able to grow under heterotrophic conditions in dim light (26). The light sensitivity disappeared when 3-(3,4-dichlorophenyl)-1,1-dimetylurea (DCMU) 1 was present. DCMU inhibits the reduction of the plastoquinone (PQ) pool by photosystem II (PSII) and this indicates that the primary cause for the photosensitivity observed is the impairment in electron flow downstream of PSII. The reported decrease in the amount of cytochrome f (Cyt f), a component of the cytochrome b 6 f (Cyt b 6 f) complex, further strengthens this hypothesis (26).
In the present study, we have analyzed the LepB1 mutant strain by using two-dimensional Blue Native/SDS-PAGE (2D BN/SDS-PAGE), Western blotting with specific antibodies and isobaric tags for relative and absolute quantitation (iTRAQ), with special emphasis on the integrity of the photosynthetic apparatus, to reach a better understanding of the role of LepB1 in protein maturation in Synechocystis. Our studies show that LepB1 has irreplaceable substrate specificity, as in the case of PsaF, an integral membrane subunit of the photosystem I (PSI). The processing of other photosynthetic proteins (PsbO and Cyt f) is also affected although LepB2 is able to partly compensate the loss of LepB1. This defect in processing severely disrupts the ability to maintain proper organization of the thylakoid membrane complexes and also affects the cellular proteome in a far-reaching manner.

EXPERIMENTAL PROCEDURES
Cell Culture and Growth Conditions-Synechocystis sp. PCC 6803 glucose-sensitive strain (Wild type, WT GS) and the sll0716 gene inactivation mutant strain (⌬LepB1) were grown under heterotrophic conditions in BG-11 medium in the presence of 5 mM glucose (an amount well tolerated by the WT GS strain under the conditions used in this study) (27). Independent cultures, 300 ml for each strain, were maintained at 32°C, under dim light (Ͻ1 E m Ϫ2 s Ϫ1 of warm white light) and vigorous bubbling with filtered air (0.45 M, Millipore Millex). ⌬LepB1 was routinely grown in the presence of 25 g/ml kanamycin (26). Cell growth was monitored by measuring OD 730 with a spectrophotometer (BioSpec-Mini, Shimadzu).
Isolation of the Total Membrane Fraction-The membrane fractions were prepared as described elsewhere (28), with slight modifications. Three independent cultures for each strain were harvested in midlogarithmic phase (OD 730 ϭ 1) by centrifugation at 6000 ϫ g, 4°C, 10 min. Cultures from each strain were resuspended in washing buffer (50 mM HEPES-NaOH, pH 7.5, and 30 mM CaCl 2 ), mixed in equal proportion and recentrifuged in the same conditions to have a pooled sample for each condition, containing cells from three independent cultures. Sample pooling was previously shown to be an effective mean to reduce the impact of biological variation even though it may hinder detection of differential expression when biological variability is small (29). Cells were broken by vigorous vortexing for 4 ϫ 2 min with glass beads (0.15-0.212 mm in diameter) in isolation buffer (50 mM HEPES-NaOH, pH 7.5, 30 mM CaCl 2 , 800 mM sorbitol, and 1 mM -amino-n-caproic acid), supplemented with 1 mM phenylmethylsulfonyl fluoride (30). The glass beads were washed five times with isolation buffer. Whole cell lysates obtained from each wash were collected together and centrifuged at 4500 ϫ g, 4°C, 10 min, to remove unbroken cells and cell debris. Total membrane and soluble fractions were separated by centrifugation at 230,000 ϫ g, 4°C for 1 h. The pellet (total membrane fraction) was washed three times with washing buffer, and UV-visible spectra of each supernatant wash were recorded (using a Varian CARY 300 Bio; Agilent, Santa Clara, CA) to monitor the content of phycobiliproteins until no obvious phycocyanin peak could be observed (31). The washed membrane samples were resuspended in storage buffer (50 mM HEPES-NaOH, pH 7.5, 600 mM sucrose, 30 mM CaCl 2 , and 1 M glycinebetaine, 1 mM PMSF) and equal protein amounts (20 g) were used to record UV-Vis spectra, to check the pigment content prior to freezing at Ϫ80°C.
Sample Preparation for 2D BN/SDS-PAGE-2D BN/SDS-PAGE was performed as previously described (28), with some modifications. Isolated total membranes corresponding to 150 g protein (quantified using the Peterson method (32)) were washed once with 10 volumes of washing buffer (300 mM sorbitol, 50 mM Bis-Tris, pH 7.0, and 250 g/ml of Pefabloc [Roche Applied Science]) and resuspended in 7 l resuspension buffer, consisting of 20% glycerol (w/v), 25 mM Bis-Tris (pH 7.0), 10 mM MgCl 2 , and 0.1 unit/l DNaseI (Sigma-Aldrich). The samples were then gently mixed with an equal volume of resuspension buffer supplemented with 4% n-dodecyl-␤-D-maltoside (DDM, Sigma-Aldrich). Solubilization was performed for 50 min on ice, following by incubation at room temperature for 10 min. Insoluble material was removed by centrifugation at 18,000 ϫ g for 15 min (4°C) and the solubilized membrane protein complexes were mixed with one-tenth volume of Coomassie blue solution, consisting of 100 mM Bis Tris pH 7.0, 30% (w/v) sucrose, 500 mM -amino-n-caproic acid, 10 mM EDTA pH 7.0, 5% (w/v) Serva Blue G. The samples were loaded onto a 0.75 mm thick 5-12.5% acrylamide gradient gel (Hoefer SE250, GE Healthcare) and electrophoresis was performed at 4°C, on ice, by increasing voltage gradually from 50 V up to 200V during the run.
Lanes from the BN gel were excised and equilibrated in 2 ϫ SDS sample buffer containing 10% ␤-mercaptoethanol and 8 M Urea for 45 min at room temperature. The gel pieces were covered by a small volume of SDS sample buffer and fresh buffer was added during the incubation period to avoid gel drying. The lanes were rinsed in SDS-PAGE running buffer and laid onto in-house-cast 1 mm thick 14% SDS-PAGE gels with 6 M urea (Hoefer SE260, GE Healthcare) (33).
After electrophoresis proteins were visualized by Coomassie Brilliant Blue R250 staining solution (Bio-Rad).
Immunoblotting-WT and ⌬LepB1 samples containing 10 g protein in SDS-PAGE loading buffer with 4 M urea were applied on precast 12% acrylamide gels (Mini-Protean TGX gels; Bio-Rad, Hercules, CA) or in-house-cast 1 mm thick 12% SDS-PAGE gel (Hoefer SE250, GE Healthcare) with 6 M urea (33). For immunodetection the proteins were electrotransferred with a semidry blotter (Trans-Blot S.D., Bio-Rad) to PVDF membranes (Hybond-P, GE Healthcare). Membranes were blocked in 5% fat-free milk and incubated with primary antibodies as indicated in Fig. 3. Advanced ECL Prime (GE Healthcare) was used to develop the membrane after incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). All the blots were done at least three times with different preparations from independent cultures. The images were captured using an ImageQuant LAS 4000 system equipped with a Fujifilm Super CCD camera and were quantified using the MultiGauge software (v 3.0, Fuji). Antibodies against PsaA, CP43 and D1 were purchased from Agrisera AB, PsbO antibodies from AbCam. Cyt f and PetC3 antibodies were kind gifts from M. Rogner, PsaF antibodies a gift from J. D. Rochaix.
iTRAQ Proteome Analysis-The iTRAQ proteome analysis was performed as previously described (34) with some modifications. Three independent cultures were pooled together to make the total membrane fractions, as described above. Two hundred and fifty micrograms of total membrane proteins from the WT and LepB1 mutant strains were separated on 12% precast SDS-PAGE gels. The gel was run for one fourth of its total length for sample concentration and pigment removal. Lanes of each sample were sliced and diced into small pieces. Gel pieces were extensively washed with 50 mM triethylammonium bicarbonate (TEAB) buffer and then dehydrated by acetonitrile (ACN). Proteins were reduced by 5 mM Tris-(2-carboxyethyl)phosphine (TCEP) in TEAB buffer for 1 h at 60°C and alkylated by 10 mM methyl methanethiosulfate (MMTS) in TEAB for 30 min at RT in the dark. In-gel digestion was performed at 37°C overnight. The tryptic peptides were extracted by 5% acetic acid in 50% ACN and vacuum dried by SpeedVac.
iTRAQ Labeling and ERLIC Fractionation-The tryptic peptides were labeled with four-plex iTRAQ reagents (Applied Biosystems, Foster City, CA) according to the instructions from the manufacturer. Two different labeling schemes were applied in our experiments to minimize both systematic and experimental errors-Experiment 1: WT, 114; ⌬LepB1, 115/116/117 and Experiment 2: WT, 114/116, ⌬LepB1, 115/117. These labeled peptides were pooled and desalted using Sep-Pak C18 cartridges (Waters) and vacuum dried. The mixture of iTRAQ-labeled peptides was fractionated using a PolyWAX LP weak anion-exchange column (4.6 ϫ 200 mm; 5 m, 300 Å; PolyLC) on the same UFLC system, with UV detection monitored at a wavelength of 280 nm. Mobile Phase A (10 mM CH 3 COONH 4 in 85% ACN/1% formic acid [FA]) and mobile phase B (30% ACN/0.1% FA) were used to establish a 50 min gradient of 0 -28% buffer B for 40 min, 28 -100% buffer B for 5 min, and 100% buffer B for 5 min at a flow rate of 1 ml/min, with 30 fractions collected. The peptides were dissolved in 200 l mobile phase A for complete solution before being loaded to the column. Mobile phase A was prepared by dissolving CH 3 COONH 4 directly in 85% ACN/1% FA without any adjustment of its pH. The collected fractions were then dried by vacuum centrifuge at 45°C, combined into 20 fractions and reconstituted in 3% ACN/ 0.1% FA for LC-MS/MS analysis.
LC-MS/MS Analysis-The peptides were separated and analyzed on a home-packed nanobore C18 column (15 cm x 75 m; 5 m particles) with a Picofrit nanospray tip (New Objectives, Woburn, MA) on a Tempo TM nano-MDLC system coupled with a QSTAR® Elite Hybrid LC-MS/MS system (Applied Biosystems). Peptides from each fraction were analyzed in triplicate by LC-MS/MS over a gradient of 90 min. The flow rate of the LC system was set to a constant 300 nl/min. Data acquisition in QSTAR Elite was set to positive ion mode using Analyst® QS 2.0 software (Applied Biosystems). MS data was acquired in positive ion mode with a mass range of 300 -1600 m/z. Peptides with ϩ2 to ϩ4 charge states were selected for MS/MS. For each MS spectrum, the three most abundant peptides above a fivecount threshold were selected for MS/MS and dynamically excluded for 30 s with a mass tolerance of 0.03 Da. Smart information-dependent acquisition was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s.
Data Analysis-Protein identification and quantification were performed using ProteinPilot Software Version 2.01 (Applied Biosystems). The Paragon algorithm in the ProteinPilot software was used for the peptide identification, further processed by the Pro Group algorithm, where isoform-specific quantification was adopted to trace the differences between expressions of various isoforms. User defined parameters were as follows: (1) Sample Type, iTRAQ 4-plex (Peptide Labeled); (2) Cysteine alkylation, MMTS; (3) Digestion, Trypsin; (4) Instrument, QSTAR ESI; (5) Special factors, None; (6) Species, None; (7) Specify Processing, Quantitate; (8) ID Focus, biological modifications, amino acid substitutions; (9) Database, the Synechocystis PCC 6803 protein sequence database (from 2004) was downloaded from Cyanobase (http://genome.kazusa.or.jp/cyanobase/Synechocystis/genes.faa), which includes 3672 sequences and 1143081 residues; (10) Search effort, thorough; (11) MS and MS/MS mass tolerance, 0.2 Da. Protein confidence was set at 95% (equivalent to Unused ProtScore of 1.3). The peptide for iTRAQ quantification was automatically selected by the Pro Group algorithm to calculate the reporter peak area, error factor (EF) and p value. The resulting data was auto bias-corrected by the built-in ProteinPilot algorithm to eliminate any variations imparted because of the unequal mixing during combination of differentially labeled samples. During bias correction, the software identifies the median average protein ratio, corrects it to unity and then applies this factor to all results for quantification (35).
Respiration Measurements-Cells from three independent cultures for each strain were collected at OD 730 ϭ 1 by low speed centrifugation (1500 ϫ g) for 10 min at room temperature (RT). The cell pellet was gently washed once with BG-11 and resuspended to a final OD 730 ϭ 5 in fresh BG-11medium supplemented with 5 mM glucose. All cell culture concentration steps were performed under dim light. The concentrated cell suspensions were stirred with a magnetic stirrer to prevent cell sinking. Respiratory electron transport was measured at RT by using a Clark-type electrode (Hansatech), as described below. One milliliter of the cell suspension was added in the electrode chamber which was immediately covered with a thick black cloth. After 5 min of dark incubation, different electron transport inhibitors (KCN, PCP, or DBMIB) were added from freshly prepared stock solutions to the final concentrations, as indicated in the Results section. Following a further 5 min incubation, respiratory rates were read and calculated using the Oxygraph Plus software (Hansatech) using data up to 2 min of the start of the measurement. All individual experiments were repeated three times under the same conditions.

Deletion of LepB1 Specifically Decreases the Amount of PSI
and Cyt b 6 f Complexes but not PSII-To gain insight into the consequences of the LepB1 deletion for the organization of the photosynthetic apparatus, total membranes from WT and ⌬LepB1 strains were analyzed by BN-PAGE (Fig. 1), which has been shown to be a powerful tool in the analysis of LepB1 Deletion Affects Photosynthesis and Respiration membrane protein complexes in cyanobacteria (36). A remarkable decrease in the amount of PSI complexes, both in the trimer and the monomer form in ⌬LepB1 (Figs. 1 and 2) was observed on the gel. In contrast, the amount of PSII was approximately the same. BN gel lanes for the WT and ⌬LepB1 were further separated in the second dimension SDS-PAGE gel (Fig. 2), to resolve the protein composition of each complex. Spots from different complexes were identified by LC-MS/MS, confirming their respective identities (supplemental Data File S1). The 2D gel of ⌬LepB1 showed a general decrease of all observable PSI subunits in comparison to WT (Fig. 2). The Cyt b 6 f complex could not be identified in the first dimension BN-PAGE gel. However, after the second dimension SDS gel run three subunits of Cyt b 6 f could be identified on the WT gel, corresponding to Cyt f (spot 14a), Cyt b 6 (spot 15), as well as subunit IV (spot 16). In the ⌬LepB1 gel, three weak spots could be observed in the same positions but, of these, only Cyt f (spot 14b) could be identified by LC-MS/MS ( Fig. 2 and supplemetal Data File S1).
PSII is mainly detectable in its monomer form in our membrane samples, both for WT and ⌬LepB1, with similar intensity as shown in Fig. 1. In the second dimension SDS-PAGE, none of the four core subunits of PSII -CP47 (spot 8), CP43 (spot 10), D2 (spot 11), and D1 (spot 12), identified by LC-MS/MSappeared to change in abundance in the mutant, in comparison to the WT ( Fig. 2 and supplemental Data File S1).
To complement this analysis, several antibodies were used to compare the amount of specific proteins in total membrane samples of WT and ⌬LepB1. Blot signal intensities from three independent preparations were quantified (using MultiGauge - Fig. 3) and presented in the form of a histogram, with results expressed as abundance ratios between ⌬LepB1 and WT. In agreement with the results from 2D/BN-PAGE, the PSII core subunits CP43 and D1 remained largely unchanged, whereas the PSI core subunit PsaA and the Cyt b 6 f complex subunit Cyt f decreased to less than half the WT amounts in ⌬LepB1 (Fig. 3).
Influence of the LepB1 Deletion on Preprotein Maturation-Out of 300 -350 Synechocystis proteins with predicted (37) or verified signal sequences only limited numbers are part of the photosynthetic apparatus or important for its proper function and maintenance. PsaF in PSI and Cyt f in Cyt b 6 f complex, both integral membrane subunits, are synthesized with presequences and integrated into the respective complexes (38 -40). PSII contains at least three proteins with Sec-signal peptides, PsbO, PsbU, and PsbV, which constitute the membrane associated oxygen evolving complex (OEC), situated on the lumenal side of the PSII complex (41). As potential substrates PSI monomer bands from the WT and the ⌬LepB1 were cut out from the BN gel and separated on an SDS gel side by side (Fig. 4), more differences could be seen on the gel apart from the remarkable decrease of the amount in the mutant. Both cell types have four bands in the low molecular area, but it is clearly seen that the second band from the top migrates at a position 2-3 kDa higher in the mutant (marked with 3b in Fig.  4) than in the WT (marked with 3a in Fig. 4). The identity of these bands (3a and 3b) as PsaF was confirmed by LC-MS/MS as well as Western blot (Figs. 2, 3, and 4 and supplemental Data File S1). Synechocystis PsaF was previously shown to be an integral membrane protein with a cleavable signal peptide in the first 23 amino acids (38) and the 2-3 kDa increase in band 3b corresponds well with what is expected for pre-PsaF (38). All our attempts to identify PsaF presequence peptides by LC-MS/MS failed, most probably because of their highly hydrophobic nature. No processed PsaF was observed, either in 2D BN/SDS-PAGE gels (Fig. 4) or in Western blots (Fig. 3) of ⌬LepB1 total membranes. It can be concluded that the majority of PSI complexes in the mutant contain PsaF in the unprocessed form (Fig. 2).
Cyt f and PsbO, were both affected by the LepB1 mutation, as shown in Fig. 3. Cyt f decreased in the mutant, similarly to PsaF. However, in contrast to PsaF, cyt f is only found in the processed form, which is in agreement with the prior observations of Zhbanko et al. (26). The signal peptide cleavage site of PsbO was previously verified and the mature protein has a MW that is 3 kDa lower than the preprotein (41). Using a PsbO specific antibody, we observed an accumulation of small amounts of pre-PsbO protein, despite the lack of decrease in the amount of mature PsbO. Consequently, 30% more of total PsbO (processed plus unprocessed) was found in the mutant (Fig. 3).
Protein Abundance as Revealed by iTRAQ-Our analysis using 2D BN/SDS-PAGE and specific antibodies revealed differences in the amount of some membrane complexes in the mutant. To obtain a more complete analysis of the changes induced by the mutation we subjected the membranes to iTRAQ. iTRAQ has previously revealed useful information in understanding the changes in the Synechocystis proteomic response to CO 2 limitation (42), increased salt concentration (43), and temperature shifts (44). The total membranes of WT and ⌬LepB1 were analyzed by combining iTRAQ and LC-MS/MS with SDS-PAGE gel-assisted digestion. Approximately 1350 proteins from Experiment 1 (Exp. 1) and 1200 proteins from Experiment 2 (Exp. 2) were identified with over 95% confidence (Unused ProtScore Ͼ 1.3), of which more than 90% of the proteins were overlapping in the two experiments. About 470 and 440 proteins were confidently

LepB1 Deletion Affects Photosynthesis and Respiration
quantified having p value Ͻ 0.05, respectively. All three data sets from the Exp. 1, together with the four data sets from the Exp. 2 were combined for statistical analysis. The geometric mean of the iTRAQ ratios and standard deviation (S.D. log ratios) of each identified protein from the seven sets of iTRAQ ratios of the two independent experiments were calculated, as shown in supplemental Data File S2. Only the proteins identified in both of the iTRAQ experiments with p value Ͻ 0.05 and ⌬LepB1/WT Ͻ0.7 and Ͼ1.5 fold difference in expression were selected as significantly down and up-regulated proteins (42,44). There were 46 down-regulated and 11 up-regulated proteins in the mutant (supplemental Data File S2), in comparison with the WT. Of the down-regulated eight proteins have a predicted/proven signal peptide and of the up-regulated three. Most of the quantified proteins were not significantly changed in the lepB1 mutant under the growth conditions used in this work. Table I shows the selected proteins present in the membrane fraction relevant to photosynthesis and respiration, which will be further described and discussed.
The decrease in expression in ⌬LepB1 of subunits of the PSI and Cyt b 6 f complexes, seen with 2D/BN-PAGE and specific antibodies, was confirmed by the iTRAQ analyses, with lower abundance ratios for all subunits quantified. Four integral and three peripheral proteins in the PSI complex decreased to 40 -52% of the WT levels and the Cyt b 6 f complex subunits to 59 -66% of WT amounts. Plastocyanin, which contains a Sec signal peptide, is a water soluble electron carrier found in the thylakoid lumen, carrying electrons from Cyt b 6 f to PSI. This protein was shown by our iTRAQ analyses to remain unchanged in the mutant, with a ratio of 1.37 fold, under the established limit for significance. Whether plastocyanin has an impaired processing like PsbO still needs to be confirmed.
All the PSII integral membrane subunits identified and quantified in this work (D1, D2, CP43, CP47, PsbH, and Cyt b 559 ␣) had abundance ratios of roughly 1 between LepB1 mutant and WT (Table I). The lumenal subunits of the PSII complex, PsbO, PsbU, and PsbV, forming the OEC in a ratio of 1:1:1, contain Sec signal peptides, were also shown to be unchanged in the iTRAQ analyses. As described above, PsbO blots showed an increase in the mutant of roughly 30%. The average ratio from the iTRAQ data for PsbO is 1.21 (Ϯ0.03), a 21% increase in ⌬LepB1, below our established significance threshold. The abundance ratios for the other two proteins in the OEC, PsbU, and PsbV, are 1.46 and 1.21 respectively and they must all be defined as proteins with unchanged abundance. The expression of the other PSII associated proteins identified (Psb27, PsbQ, PsbP, and Psb28) also remains at same level in mutant and WT and the consistency in these results clearly indicates that all PSII subunits are expressed in similar amounts in WT and LepB1 depletion mutant.
Both the mutant and WT strains were grown in low light (Ͻ1E m Ϫ2 s Ϫ1 ) with glucose, and therefore both strains are totally dependent on respiration as a source for both ATP and reducing equivalents. In our iTRAQ data, we found no changes in abundance between WT and the mutant for the subunits belonging to NADH dehydrogenase type 1 or ATP synthase (supplemental Data File S2). Synechocystis contain three distinct respiratory terminal oxidases, CtaI, CtaII and CydAB (45,46). We were only able to identify and quantify CydA (Slr1379), which increased almost twofold (Table I) in the LepB1 mutant. PetC3, a Rieske protein with unknown function in respiration was also increased in the mutant (Table  I), as also seen in the Western blot (Fig. 3).
All 13 subunits of the phycobilisome complex were quantitatively identified and, of these, six had ratios Ͻ 0.7, indicating a significant decrease, whereas seven others had ratios between 0.7 and 0.9 ( Table I). The decrease of the phycobiliproteins was also reflected by the difference observed in the absorbance spectra of total membranes. The absorbance at 618 nm, which is the phycocyanin absorption peak (31), was only 70% of the WT intensity in the mutant (supplemental Data File S3). Three enzymes involved in the heme/chlorophyll biosynthesis pathway were found to decrease significantly in ⌬LepB1 (Table I), uroporphyrinogen decarboxylase (Slr0536), magnesium-protoporphyrin IX monomethyl ester cyclase (Sll1214) and geranylgeranyl reductase or ChlP (Sll1091). Synthesis of chlorophyll is correlated to the amount of chlorophyll binding proteins (47) and it was previously shown that deletion of PSI in Synechocystis leads to a reduced chlorophyll content to about 15% (31). In the LepB1 mutant the chlorophyll content is decreased, in comparison to WT (supplemental Data File S3), because of a lower amount of PSI. Thus it seems reasonable that the amount of chlorophyll synthesizing enzymes is decreased in the mutant.
We have also identified several other proteins with changed abundance ratios in the mutant (supplemental Data File S2), including some periplasmic and cytoplasmic proteins. These changes most likely reflect indirect effects of the mutation and work is currently ongoing in our laboratory to further investigate and clarify their importance for the overall adaptations of the LepB1 deficient strain.
Respiratory Electron Pathway Mainly Proceeds Through CydAB-As pointed out earlier, the mutant cells can only grow under conditions of low light (Ͻ1E m Ϫ2 s Ϫ1 ) in the presence of glucose. Under these conditions, the cells rely on respiration for continuous growth and three terminal oxidases, CtaI, CtaII, and CydAB, have been identified which could participate in this respiration (45,46). Respiratory activity measured in the dark was quite similar in WT and ⌬LepB1, about 0.09 mmol O 2 h Ϫ1 (ml at OD 730 ϭ 1) Ϫ1 (Table II). Respiration in both strains was inhibited 90% by KCN (not shown), which inhibits all three terminal oxidases. PCP, a specific inhibitor for the quinol oxidase CydAB (46), caused 80 and 60% inhibition in WT and ⌬LepB1, respectively, indicating that this might be the major route for the electrons in dark grown cells. DBMIB is a potent inhibitor of

LepB1 Deletion Affects Photosynthesis and Respiration
the Cyt b 6 f complex (48), inhibiting the reduction of Cyt b 6 , with an inhibition constant of 80 nM (49). In our experiments, no inhibitory effect on the respiration in either strain was observed at any of the concentrations tested (from 40 nM to 40 M, not shown). Interestingly, DBMIB caused a concentration-dependent increase in the rate of O 2 -consumption. This was particularly noteworthy in the mutant, with an increase in respiratory activity of more than threefold at 40 M DBMIB (Table II). The addition of 5 mM KCN caused about 90% inhibition of oxygen consumption both in the absence and presence of DBMIB, for both strains, demonstrating that the DBMIB stimulated rate is dependent on a CN Ϫ sensitive oxidase (not shown).

DISCUSSION
In the previous study of the knockout mutants of the two leader peptidases in Synechocystis it was shown that LepB2 is essential for cell viability (26). The LepB1 mutant is highly prone to photoinhibition unless DCMU, an inhibitor of PSII is present indicating that the electron flow from PSII is the cause for the observed photodamage. In the present study we show a dramatic decrease in Cyt b 6 f and PSI, in combination with an unchanged amount of PSII in ⌬LepB1. This could produce an imbalance in electron flow upstream and downstream the PQ pool and be the direct reason for the light sensitivity. The LepB1 mutant could be propagated under heterotrophic conditions, although only at very low light intensity. It was previously shown that a PSI-less mutant, could only survive in low light, though keeping normal amounts of PSII (50), a direct parallel to the LepB1 mutant.
PsaF was exclusively observed in the unprocessed form in the mutant, strongly suggesting that when LepB1 is deleted, the remaining leader peptidase, LepB2, is not able to cleave the presequence of PsaF. The structure of Thermosynechococcus elongatus PSI has been determined at 2.5 Å resolution (39) and it was shown that PsaA and PsaB form the core reaction center located in the center of the monomer, surrounded by seven small integral membrane proteins. PsaF is situated in the periphery of the PSI complex according to the crystal structure. Studies on the PSI assembly process in Synechocystis concluded that PsaL is the last subunit to be added to the monomer, and that subsequent addition of PsaK  leads to trimerization of PSI monomers (51). PsaF should consequently be inserted in an earlier step of the monomer assembly at least before the PsaL subunit is inserted, although the dynamics leading to early PSI complex formation are still unknown (51,52). Furthermore, PsaF has been proposed to be important for the stabilization of both surfaces, lumenal and cytoplasmic, of PSI (53). The lower amounts of PSI that we observe could be explained by a slower assembly rate or a lower stability of the PSI monomer resulting from the presence of the signal peptide in PsaF. PsaF function has been well studied in plants and green algae, as well as in cyanobacteria. Unlike the eukaryotic PsaF, the cyanobacterial PsaF is not essential for docking of plastocyanin to PSI (54), because of absence of the basic N-terminal region present in plant and green algae, which is important for plastocyanin binding. The cyanobacterial PsaF-null mutant showed normal photoautotrophic growth when compared with the WT, suggesting that PsaF has dispensable accessory roles in the function and organization of PSI complex (38,55). A chimeric PsaF, containing the N-terminal domain of PsaF from C. reinhardtii and the C-terminal sequence of the cyanobacterial PsaF was found to assemble into PSI complexes in Synechococcus with plastocyanin docking as well as PSI activity (56). Similarly to what we observe in the Synechocystis LepB1 mutant, the incorporation of this chimeric PsaF into PSI decreased the amount of PSI to one third of WT. The presence of the signal peptide introduces an extra N-terminal domain on the lumenal side of PSI which could result in a slower assembly rate or a lower stability of PSI in the mutant. In contrast to PsaF, Cyt f in ⌬lepB1 was only found in its processed form, albeit in significantly lower amounts. The Cyt b 6 f complex is shared by both the photosynthetic and the respiratory chains, catalyzing electron transfer between the plastoquinone pool and either plastocyanin or Cyt c 6 , The crystal structures of the Cyt b 6 f complexes from both the thermophilic cyanobacterium Mastigocladus laminosus (40) and the filamentous cyanobacterium Nostoc sp. PCC 7120 (57) contain four large subunits (Cyt f, Cyt b 6 , PetC1, and subunit IV) and four small subunits (PetG, -L, -M, and -N). Of all these, Cyt f is the largest subunit, containing a prosthetic c-type heme group. Mutants of CcsB (Sll1513), a protein responsible for heme insertion in c-type cytochromes, showed large accumulation of free pre-apo Cyt f in the thylakoid membrane lacking the covalently bound heme group. This indicates that heme binding and processing of Cyt f are closely coordinated in Synechocystis (58). In the iTRAQ analysis CcsB amounts did not change in the mutant (supplemental Data File S2). The normal level of CcsB and the decreased level of Cyt f in the LepB1 mutant may indicate that the processing by the remaining LepB2 is too slow and that the newly synthesized but unprocessed pre-Cyt f is degraded because of the presence of a degradation system that is able to remove nonfunctional proteins (58,59).
A third scenario is observed for the OEC subunit PsbO, which is found both in processed and unprocessed forms in the mutant. The presence of pre-PsbO in the mutant despite the WT levels of the mature form indicates a LepB1 substrate preference and low activity of LepB2 on the processing of pre-PsbO. In contrast to PsaF and Cyt f, PsbO is a soluble lumenal protein. As such it could be grouped together with the PsbU, PsbV and plastocyanin, three other soluble lumen located proteins containing a sec signal peptide. Neither of these decreased in the mutant, even if, because of the lack of proper antibodies and an inability to detect their respective presequence peptides in our iTRAQ data, we were unable to confirm whether or not these proteins have deficient processing as well.
The different behavior observed for individual LepB1 substrate proteins leads to the conclusion that the deletion has a more profound effect on integral membrane proteins than on lumenal or periplasmic proteins. This could, in the case of integral membrane proteins, reflect a need for correlation between processing and integration into the complex and also a lower stability of nonassembled proteins.
As previously mentioned, LepB1 and LepB2 were identified in TM and PM, respectively (24,25). However, the results in this work seem to implicate substrate overlapping and a dual location for LepB2. In E. coli the single leader peptidase always works together with the Sec translocon. When LepB1 is mutated, LepB2 becomes the only leader peptidase in Synechocystis and, even though the mutation caused a severe decrease of the PSI and Cyt b 6 f and concomitant light sensitivity, the cells can still grow in dim light. Most of the proteins we identified and quantified in the iTRAQ analysis were kept at the same expression level as in WT cells, including the PSII, NADH dehydrogenase 1 and ATP synthase complexes. The limited effect of the LepB1 mutation indicates that LepB2 is the major leader peptidase and probably has a dual membrane location in Synechocystis. Should LepB2 only exist in the plasma membrane of Synechocystis, this would implicate a very complicated movement of proteins between the periplasm and the lumen, as well as between PM and TM. YidC, which is required together with the Sec translocon for insertion of integral membrane proteins was previously shown to be present exclusively in TM (7). Therefore LepB1 might also exist only in TM to process photosynthetic signal peptide containing integral membrane proteins, inserted by the Sec translocon-YidC complex, which is indicated from the lack of PsaF processing. Fig. 5 shows the proposed scheme for respiratory and photosynthetic electron transport in TM of Synechocystis (46,48). In dark respiration, different dehydrogenases reduce PQ, which is then oxidized by CtaI (via Cyt b 6 f) or CydAB. It was previously concluded that in WT cells grown at normal light intensities (50 E m Ϫ2 s Ϫ1 ) in the presence of glucose the Cyt b 6 f complex provides the major route for PQH 2 oxidation in the respiration electron flow (48,60). On the other hand, in cells grown in low light (Ͻ1 E m Ϫ2 s Ϫ1 light and glucose) the CydAB oxidase seems to be the major route for oxidation of PQH 2 , because addition of PCP caused 80 and 60% inhibition in WT and mutant, respectively (Table II). WT and ⌬LepB1 grown at low light intensities showed similar respiratory rates despite the large decrease in Cyt b 6 f content in the mutant cells. This could be explained by a compensation by the almost twofold increase in CydAB in the mutant (Table I) DBMIB was shown to inhibit Cyt b 6 f with high affinity in cultures grown at normal light intensities (48,49). Surprisingly, in our case there was no inhibition of oxygen consumption but rather a dose-dependent stimulation. DBMIB is also a redox active molecule and is able to act as an electron shuttle (48,61). Therefore, the stimulatory effect of DBMIB could caused by electron shuttling from the PQ pool, or dehydrogenases, to the quinol oxidase CydAB. In the absence of DBMIB the rate-limiting step in respiration is before CydAB, because the same rate of respiration is obtained for WT and mutant. On the other hand in the presence of DBMIB CydAB becomes rate-limiting and the more than doubled respiration rate observed in ⌬LepB1 (Table II) corresponds well with the increase in content of CydAB, as shown by the iTRAQ analysis (Table  I). Increased CydAB activity has earlier been associated with impairments in Cyt b 6 f (62) and a ⌬PetC1 mutant was shown to have increased levels of CydA (63).
The lower extent of inhibiton by PCP (60% versus 80% in the WT) could indicate additional unidentified changes in the respiratory electron transport in the mutant. Though relatively little is still known about respiration in PM (64), CtaII was found only in this subfraction (7,24,46,48,65) and CydAB in both TM and PM (45,48). The Rieske protein PetC3, which was found to increase in the mutant as seen in Western blot and iTRAQ analyses, was shown to be present only in PM (64) and, similarly, a GFP-PetC3 construct was targeted to PM (66). Because of its unusually low redox potential (ϩ135 mV), PetC3 cannot be a component of the "classical" Cyt b 6 f complex and oxidize plastoquinone (60). There is little information regarding the electron transport chain in very low light conditions and the discrepancy seen between protein expression levels and oxygen consumption could perhaps be caused by the presence of uncharacterized respiratory chain components in the plasma membrane (64).
In conclusion, deletion of LepB1 strongly affects the assembly and integration of both PSI and Cyt b 6 f in the thylakoid membrane, resulting in considerably reduced amounts of these complexes. This leads to cells that are prone to photoinhibiton and unable to grow photoautotrophically because of an imbalance in photosynthetic electron transport. The obvious targets of the mutation are PsaF and Cyt f, integral membrane proteins with a signal peptide. The assembly of PSII is not affected by the mutation, even though PsbO processing is slower than in the WT. The different effects on PSI and Cyt b 6 f relative to PSII most likely reflects the fact that PsaF and Cyt f are integral membrane proteins inserted into the core complexes in early steps of assembly, whereas PsbO is a soluble extrinsic PSII subunit and assembled late. Our results also indicate a difference in specificity for the two leader peptidases, as well as an overlapping function for LepB2. LepB1 seems to be the natural peptidase for processing of PsaF, Cyt f, and PsbO. LepB2 is able to partially replace LepB1 in the processing of Cyt f and PsbO-although at lower rates-but not of PsaF. When cells were grown in the dim light with glucose added a number of adaptive changes for the LepB1 mutant to overcome the loss of Cyt b 6 f, such as twofold increase of CydAB oxidase, as well as decreased expression of chlorophyll biosynthesis pathway enzymes and phycobiliproteins were also established.