Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ Marjaana Suorsa 1 , Ralph E. Regel 2 , Virpi Paakkarinen 1 , Natalia Battchikova 1 , Reinhold G. Herrmann 2 and Eva-Mari Aro 1 1 Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Finland; 2 Botanisches Institute der Ludwig-Maximilians Universita ¨ t, Mu ¨ nchen, Germany The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII–light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) pro- teins, PsbP and PsbQ, were completely lacking in DpsbL.In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence meas- urements and by the lack of PSII–LHCII supercomplexes. The DpsbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In con- trast, very young, vigorously expanding leaves of all psb- EFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were pre- sent and correctly assembled in all psbEFLJ operon mutants. Keywords: oxygen-evolving complex; photosystem II assembly; photosystem II small subunits; psbEFLJ operon; tobacco. Photosystem II (PSII) is a multisubunit pigment–protein complex that catalyses electron transfer from water to the plastoquinone pool with concomitant evolution of oxygen. The PSII reaction center core consists of the D1 and D2 proteins, cytochrome b 559 (Cyt b 559 ), the chloro- phyll a-binding antenna proteins CP43 and CP47, and a number of low molecular mass (LMM) proteins, the functions and locations of which in PSII are still largely unknown. They include both chloroplast-encoded (PsbH, I, J, K, L, M, N, T and Z) and nucleus-encoded (PsbR, W and X) proteins with generally only one membrane-spanning helix [1]. During the past few years, enormous progress has been made in determining the structure of PSII [2–4]. The functional form of PSII is apparently a dimer [5]. The oxygen-evolving complex (OEC) situated on the lumenal side of PSII is composed of the PsbO (33 kDa), PsbP (23 kDa) and PsbQ (17 kDa) proteins in higher plants. PSII dimers further associate with the light-harvesting complex II (LHCII) to form PSII–LHCII supercomplexes, the minor antenna proteins CP24, CP26 and CP29 probably serving as linker proteins [2,5,6]. It has been suggested that several LMM proteins participate in PSII dimerization [7,8]. However, despite the available structure of PSII at 3.8 and 3.7 A ˚ resolution [3,4], the exact locations and roles of most of the LMM proteins in the assembly and stability of PSII remain to be determined. Today it is a challenge to resolve the assembly steps of PSII. Various approaches have been fruitful in analysing the primary assembly steps of PSII [9]. The best-characterized LMM proteins of PSII, the a and b subunits of Cyt b 559 , probably function as an assembly core, which is required for the synthesis of the D2 protein [10]. Indeed, it has been shown that Cyt b 559 and the D2 protein exist as a complex in etiolated barley leaves [11]. The full-length D1 protein, however, is synthesized only in the light and is cotransla- tionally associated with the D2–Cyt b 559 complex [12]. Radiolabeling experiments have demonstrated that the subsequent assembly steps include association of CP47 followed by that of CP43 [13]. Labeling experiments, however, are unable to reveal all the different steps in the sequential and hierarchical assembly of multisubunit PSII. In particular, the assembly of the LMM subunits, except the Cyt b 559 subunits, has been difficult to address. This is because separation of the various subcomplexes after assembly of each of the LMM subunits is not possible Correspondence to E M. Aro, Department of Biology, Plant Physio- logy and Molecular Biology, FIN-20014 University of Turku, Finland. Fax: + 358 2333 5549, Tel.: + 358 2333 5931, E-mail: evaaro@utu.fi Abbreviations: BN, Blue-native; Cyt, cytochrome; LHCII, light- harvesting complex II; LMM, low molecular mass; OEC, oxygen- evolving complex; PSI, photosystem I; PSII, photosystem II. (Received 5 September 2003, revised 28 October 2003, accepted 4 November 2003) Eur. J. Biochem. 271, 96–107 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03906.x because of resolution problems and, furthermore, only some of the LMM subunits of PSII incorporate [ 35 S]methionine. Another approach to understanding the assembly of LMM subunits into PSII is to use specific PSII protein deletion mutants and to analysethe ability of such mutants to form various PSII subassemblies. This approach has only seldom been taken because of technical problems, and, when applied, the fractionation of PSII subcomplexes has been based in sucrose-density centrifugation with limited resolu- tion capacity [14]. Moreover, none of the numerous studies with Synechocystis 6803 mutants of the LMM subunits of PSII has addressed the PSII assembly process as such, but instead the focus has been on functional properties of PSII and the overall synthesis or composition of thylakoid polypeptides. Furthermore, despite remarkable similarities between cyanobacterial and chloroplast PSIIs [15], many of the PSII LMM subunits, which are completely dispensable for the assembly of PSII in Synechocystis, are necessary for the formation of functional PSII in the respective LMM mutants of Chlamydomonas reinhardtii.Representative examples of differential effectson the formation of functional PSII in Synechocystis and Chlamydomonas are the deletion mutants of psbH [16,17], psbI [18,19] and psbK [20,21]. However, it is not known at which assembly step these proteins are crucial for the formation of functional PSII in Chlamydomonas. So far only a few studies have seriously searched for PSII assembly intermediates in the absence of any particular LMM subunit, in either Synechocystis or chloroplasts of Chlamydomonas and higher plants. The psbEFLJ operon of plant chloroplasts encodes four distinct LMM subunits of PSII, the a and b subunits of Cyt b 559 (encoded by the psbE and psbF genes) and two other small subunits, PsbL and PsbJ. Deletion of the psbE gene in Chlamydomonas [22] or the psbF gene in Synecho- cystis [23] resulted in loss of PSII activity. Similarly, the psbL deletion mutant of Synechocystis was not capable of PSII oxygen evolution [24]. The crucial role of PsbL has been suggested to be related to the function of the acceptor side of PSII at the level of Q A [25,26]. On the other hand, the psbJ deletion mutants of cyanobacteria were capable of slow photoautotrophic growth [27,28], whereas the growth of DpsbJ tobacco mutants was completely dependent on an external energy source [28,29], possibly because of an incorrectly assembled OEC [29]. Recently, very young leaves of tobacco psbEFLJ operon mutants were characterized in terms of functional, structural and biogenetic aspects [14]. To differentiate the mechanisms related to the rapid growth and division of chloroplasts in young leaves from mechanisms of the PSII assembly process as such, we mainly focused on mature, but not old, leaves of tobacco psbEFLJ operon mutants, where partial disassem- bly and assembly of PSII is constantly occurring because of turnover of the reaction center D1 protein [30]. In particular, the role of PsbL and PsbJ in the assembly and stability of PSII was addressed. To maximize separation of PSII subcomplexes, we applied 2D Blue-native (BN) gel electrophoresis followed by protein identification with immunoblotting and MS. In addition, comparative analysis of both very young and mature leaves was performed to examine the developmental aspects of PSII core and OEC protein accumulation in the psbEFLJ operon mutants with impaired PSII assembly. Materials and methods Transformation of tobacco chloroplasts Tobacco (Nicotiana tabacum cv. Petit Havanna) psbEFLJ operon mutants were constructed by replacing portions of the four individual genes of the operon with a terminator- less aadA gene cassette. A similar cassette with a terminator wasalsoinsertedintoanEcoRV site, located in the 3¢ UTR of the operon, to generate the RV control plants. The plasmid construct and the transformation, selection and culture of the transformants is described in detail elsewhere [14,28,31]. Mutants and controls (wild-type and RV plants) were aseptically grown in MS medium [32] supplemented with 3% (w/v) sucrose under low light conditions ( 10 lmol photonsÆm )2 Æs )1 )at25°C. Mature, fully expanded green leaves, but not senescing ones (hereafter referred to as mature leaves), were used for all experiments except the one in Fig. 1B where, for comparison, rapidly expanding small and very young leaves (hereafter referred to as young leaves) were used. Isolation of thylakoid membranes Leaves were briefly homogenized in 50 m M Hepes/KOH, pH 7.5, containing 330 m M sorbitol, 2 m M EDTA, 1 m M MgCl 2 ,5m M ascorbate, 0.05% BSA and 10 m M NaF, filtered through Miracloth and centrifuged at 2500 g for 4minat4°C. The pellet was resuspended in 50 m M Hepes/ KOH, pH 7.5, containing 5 m M sorbitol and 10 m M NaF and centrifuged at 2500 g for 4 min at 4 °C. The thylakoid pellet was resuspended in 50 m M Hepes/KOH, pH 7.5, containing 100 m M sorbitol, 10 m M MgCl 2 and 10 m M NaF, centrifuged at 2500 g for 3 min at 4 °C, and finally resuspended in the same buffer. Chlorophyll was extracted in 80% (v/v) buffered acetone (2.5 m M Hepes/NaOH, pH 7.5) and quantitated as described [33]. BN-PAGE, SDS/PAGE and protein identification Blue-native PAGE (BN-PAGE) was performed as des- cribed previously [34] with slight modifications. Thylakoid membrane suspensions containing 20 lg chlorophyll were used as starting material. Thylakoids were washed with 50 m M BisTris/HCl, pH 7.0, containing 330 m M sorbitol and 0.25 lgÆlL )1 Pefabloc (Roche), sedimented at 3500 g for 2 min at 4 °C, and resuspended in 25 m M BisTris/HCl, pH 7.0, containing 20% (w/v) glycerol and 0.25 lgÆlL )1 Pefabloc. Thylakoids were then solubilized with 1% (w/v) n-dodecyl b- D -maltoside (0.5 mg chlorophyllÆmL )1 )and incubated on ice for 2 min. After centrifugation at 18000 g for 15 min at 4 °C, the supernatant was supplemented with 0.1 vol sample buffer (100 m M BisTris/HCl, pH 7.0, 0.5 M e-amino-n-caproic acid, 30% (w/v) sucrose, 50 mgÆmL )1 Serva blue G) and subjected to BN-PAGE with a gradient of 5–12% acrylamide in the separation gel. The electro- phoresis was performed at 2 °C, 95 V overnight, followed by a progressive increase in voltage to 200 V for  4–5 h. After the run, a lane of BN-PAGE was cut out, solubilized with 5% (v/v) 2-mercaptoethanol in the sample buffer [35] for 40 min and run in the second dimension in SDS/PAGE with 15% acrylamide and 6 M urea. After electrophoresis, Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271)97 gels were silver-stained or electroblotted on to a poly(viny- lidene difluoride) membrane. Western blotting with chemi- luminescence detection was performed with standard techniques using protein-specific antibodies (D1, D2, PsbE, CP43, CP47, PsbO, PsbP, PsbQ, Cyt f, Lhcb1,2, CP26, CP29) or an antibody raised against the PSI complex. The AIS Analytical Imaging Station (version 3.0 rev 1.7; Imaging Research Inc., Brock University, St Catharines, Ontario, Canada) was used for quantitation of the Western blots. For each quantitation, a minimum of three inde- pendent Western blots was used. Several protein components of PSII, OEC and LHCII complexesaswellasCytb 6 f and PSI were also identified by MS MALDI-TOF analysis. Protein in-gel digestion with modified trypsin (Promega) and sample preparation for MS analysis were performed manually [36]. Samples were loaded on to the target plate by the dried droplet method using a-cyano-4-hydroxycinnamic acid as a matrix. MALDI-TOF analysis was performed in reflector mode on a Voyager-DE PRO mass spectrometer (Applied Bio- systems, Foster City, CA, USA). Internal mass calibration of spectra was based on trypsin autodigestion products (842.5094 and 2211.1046 m/z). Proteins were identified as the highest ranking result by searching in the NCBI database using Mascot (http://www.matrixscience.com). The search parameters allowed for carbamidomethylation of cysteine, one miscleavage of trypsin, and 50 p.p.m. mass accuracy. For positive identification, the score of the result [)10 · log(P)] where P is the probability that the observed match is a random event had to be over the significance threshold level (P<0.05). Fluorescence measurement Fluorescence emission spectra at 77 K were measured on a diode array spectrophotometer (S200; Ocean Optics, Dun- edin, FL, USA) equipped with a reflectance probe [37]. Fluorescence was excited with visible light below 500 nm, which was defined by using LS500S and LS700S filters (Corion Corp., Holliston, MA, USA) in front of the slide projector. The emission between 600 and 780 nm was recorded. Thylakoid samples (100 lL) contained 10 lg chlorophyll per mL in 50 m M Hepes/KOH, pH 7.5, containing 100 m M sorbitol, 10 m M MgCl 2 ,and10m M NaF. Three independent measurements were made from each tobacco line. Results Polypeptide composition of thylakoid membranes The protein composition of thylakoids from mature leaves of psbEFLJ operon mutants and the controls, wild-type and the RV plants (see Materials and methods), was first determined using 1D SDS/PAGE and immunoblotting with protein-specific antibodies. DpsbE and DpsbF thylakoids were practically devoid of all PSII core proteins tested (including D1, D2, CP43, CP47, PsbE and PsbZ, Fig. 1A). Similarly, all three OEC proteins, PsbO, PsbP and PsbQ, were completely missing from thylakoids of these two mutants (Fig. 1A). PsbW, on the other hand, represented a PSII LMM protein that was present at reduced amounts in the thylakoids of both DpsbE and DpsbF (33 ± 11 of that in the control thylakoids). To investigate the apparent devel- opmental control of the accumulation of PSII proteins, we also isolated thylakoids from very young, rapidly expanding leaves of DpsbE and DpsbF and analysed their protein composition (Fig. 1B). In contrast with mature leaves, the young leaves of both DpsbE and DpsbF accumulated all Fig. 1. Immunoblots of thylakoid membrane proteins of the four tobacco psbEFLJ operon mutants and the controls (wild-type and RV). Thyla- koids were isolated from mature green leaves (A) and rapidly expanding young leaves (B). Proteins were separated by SDS/PAGE, electroblotted on to a poly(vinylidene difluoride) membrane and pro- bed with antisera against different thylakoid membrane proteins. Chlorophyll (1 lg) was loaded in each well, except for PsbW (0.3 lg) and PsbO and PsbP (0.5 lg). 98 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003 OEC proteins and also traces of D2 and the CP43 protein (Fig. 1B). Interestingly, traces of PsbE protein (the a subunit of Cyt b 559 ) could also be distinguished in young leaves of DpsbF (Fig. 1B). Other PSII core proteins (D1, CP47) were, however, similarly missing from both the young and mature leaves of the DpsbE and DpsbF mutants. As to the DpsbJ and DpsbL mutants, the thylakoids from mature leaves contained all major PSII core proteins (Fig. 1A), but in lower amounts than in the controls. The mean content of the major PSII core proteins (D1, D2, PsbE, CP43 and CP47) in DpsbL and DpsbJ was 14 ± 5% and 57 ± 18% of that in the control thylakoids, respect- ively. Interestingly, the recently identified small PSII protein PsbZ [38–40] was present in both DpsbL and DpsbJ,in quantities related to the amount of the D1 protein present in the thylakoid membrane (18% and 88% of that in the control thylakoids in DpsbL and DpsbJ, respectively). Also PsbW was present in both DpsbL and DpsbJ, amounting to 69 ± 4% of that in the control thylakoids. As to the OEC proteins, the thylakoids from mature leaves of the DpsbL and DpsbJ mutants clearly differed from both each other and the controls. Only scarce amounts of PsbO were found in thylakoids isolated from DpsbL (Fig. 1A; 11% of that in the control), while other OEC proteins were missing. Thylakoids of DpsbJ, on the other hand, contained consid- erable amounts of PsbO and also some PsbQ (up to 100% and 6%, respectively, compared with the control thyla- koids), whereas the PsbP protein was completely missing, in accordance with earlier observations [29]. It is noteworthy that, when the immunoblots were heavily overexposed showing traces of PsbP even in DpsbE, DpsbF and DpsbL, the PsbP protein could not be detected in DpsbJ thylakoids (data not shown). Young leaves of both DpsbL and DpsbJ, on the other hand, accumulated all OEC proteins in considerable amounts. However, the DpsbJ mutant was again the exception, accumulating only traces of PsbP compared with the other mutants (Fig. 1B). Otherwise the pattern of PSII proteins in young leaves of DpsbL and DpsbJ resembled that of the mature leaves (Fig. 1B). AswellasthePSIIcoreandOECproteins,we investigated the amounts of the LHCII, CP26, Cyt f,LHCI and PsaA/B proteins in mature leaves of the psbEFLJ operon mutants. All mutants were capable of accumulating these proteins and no clear differences were recorded compared with thylakoids isolated from control plants (Fig. 1A). Assembly of thylakoid membrane protein complexes in psbEFLJ operon mutants Simple detection of thylakoid proteins by immunoblotting does not reveal whether the proteins are assembled into complexes or whether they exist as free proteins in the membrane or lumen. The general assumption that good quality control in chloroplasts results in rapid degradation of unassembled proteins [41] does not always hold true. In rapidly expanding young leaves in particular, some of the PSII core proteins and all of the OEC proteins can accumulate in thylakoids in the absence of any assembly of PSII, as was evident for the DpsbE and DpsbF tobacco mutants (Fig. 1B). Thus, to understand the role of various LMM subunits in the stable assembly of PSII, it is necessary to isolate various PSII assembly intermediates. For these experiments we used only mature leaves to avoid accumu- lation of PSII proteins that do not become assembled. One-dimensional separation of thylakoid protein com- plexes in BN gels had already revealed major differences in the capacity for PSII assembly in the psbEFLJ operon mutants. Clear separation of intact PSII core monomers, PSII core dimers and PSII–LHCII supercomplexes was typical only for the control thylakoids (Fig. 2), whereas DpsbJ and DpsbL, and particularly DpsbE and DpsbF, showed clear deficiencies in their PSII assemblies. The PSII monomer was missing from DpsbE and DpsbF and was present only in minor amounts in DpsbL. In contrast, the two other thylakoid electron-transfer complexes, the PSI and Cyt b 6 f complexes, were present in similar amounts in all the mutants and control plants (Figs 2 and 3). More detailed information about various PSII (sub)assemblies and their polypeptide composition was obtained from 2D gel analysis (BN-PAGE followed by SDS/PAGE) combined with immunochemical detection (D1, D2, CP43, CP47, PsbE) and MS analysis (MALDI- TOF) of various PSII core and OEC proteins (Figs 3 and 4). In wild-type plants, the intact PSII core monomers, the PSII core dimers and PSII–LHCII supercomplexes (confirmed by immunoblotting to contain the D1, D2, PsbE, CP47 and CP43 proteins) were detected, and only a very minor amount of CP43-less PSII monomers was present (Fig. 3). The absence of free PSII core proteins after 2D electro- phoresis (see the immunoblots below the silver-stained gels) was an indication of the general stability of PSII core complexes on dodecyl maltoside solubilization and subse- quent electrophoretic separation of thylakoid protein com- plexes. Of the OEC proteins, the PsbO subunit was always detected in association with the PSII–LHCII supercom- plexes (Fig. 4A). The Cyt b 6 f complex was present in wild- type thylakoids mainly as a dimer, and the PSI complex Fig. 2. BN-PAGE of thylakoid protein complexes from mature leaves of the four tobacco psbEFLJ operon mutants and the wild-type and RV controls. Thylakoids (20 lg chlorophyll per well) were solubilized with 1% n-dodecyl maltoside before BN-PAGE. For identification of the complexes, see Fig. 3. Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271)99 Fig. 3. Two-dimensional gel analysis of the thylakoid protein complexes from mature leaves of wild-type and DpsbF, DpsbL and DpsbJ mutants of tobacco. Thylakoids were solubilized and subjected to BN-PAGE separation of the protein complexes as described in Fig. 2. After the run, a lane of BN-PAGE was cut out, solubilized with 5% (v/v) 2-mercaptoethanol, and placed horizontally on the top of the SDS/polyacrylamide gel. After electrophoresis, the gel was silver-stained. Similar gels were also electroblotted on to poly(vinylidene difluoride) membranes and probed with antisera against D1, D2, CP43, CP47 and PsbE (Cyt b 559 a subunit). Strips of such immunoblots are presented below the corresponding silver- stained gels. Some of the immunoblots are overexposed and thus cannot be compared quantitatively. The D1, D2, CP43 and CP47 proteins from the PSII complexes (PSII core monomers, CP43-less core monomers, PSII core dimers and PSII–LHCII supercomplexes) are circled. Positions of PSI, Cyt b 6 f dimers and various LHCII subassemblies are circled in the silver-stained gel of the DpsbF mutant lacking all PSII complexes and were identified by MALDI-TOF MS and immunoblotting (data not shown). 100 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Fig. 4. Presence of the 33-kDa PsbO protein of OEC in different PSII assemblies of the control and DpsbJ and Dpsb L mutant thylakoids isolated from mature leaves. (A) Protein components of PSII (sub)complexes from wild-type and DpsbJ and DpsbL mutants of tobacco. The gels for the wild-type and DpsbJ mutant are enlargements from Fig. 3 (the 27–50-kDa region). The corresponding region from the DpsbL mutant was obtained after only partial solubilization of thylakoid complexes with n-dodecyl b- D -maltoside and separation of the complexes with a mini-gel system, which allowed disclosure of the PSII core monomer complex with attached PsbO. Arrows indicate the location of PsbO protein in PSII–LHCII supercomplexes of the wild-type control thylakoids and in the PSII core dimer or in a distinct PSII core monomer complex of the DpsbJ and DpsbL mutants, respectively. Cyt f of the Cyt b 6 f dimer complex (identified by both immunoblotting and MS; not shown) is indicated in the silver-stained gels with an asterisk. (B) Representative mass spectrum and the peptide masses of the PsbO protein (straight arrows with closed square) and the overlapping D2 protein (tilted arrows with open circles) from the PSII–LHCII supercomplex of control thylakoids. Tilted arrows with a cross show the trypsin self-digest products used for MS calibration. Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271) 101 migrated in a BN gel in close proximity to the PSII dimer (Fig. 3). LHCII proteins, despite forming the PSII–LHCII supercomplexes, were present in various subcomplexes detached from PSII. In the absence of either PsbF (Fig. 3) or PsbE (not shown) the 2D BN-PAGE profiles of the main thylakoid protein complexes were very similar. No PSII core proteins were found assembled into any kind of complexes, neither did they accumulate as free proteins. Other thylakoid protein complexes, such as Cyt b 6 f dimer, PSI and various LHCII subassemblies, were present in DpsbE and DpsbF in comparable amounts to that in the wild-type. Complete (DpsbE and DpsbF) or partial (DpsbL and DpsbJ;Fig.4) depletion of the PSII complexes thus had no effect on the assembly and accumulation of other multiprotein photo- synthetic complexes in the thylakoid membrane. This differs from a recent study in which the amounts of some PSI proteins were reduced in tobacco DpsbJ mutant [29]. Analysis of DpsbJ by 2D BN-PAGE revealed that both PSII core monomers and dimers were correctly assembled (Fig. 3). Considerable amounts of PSII monomers lacking CP43 were, however, also present, although the relative amount of free CP43 was much less than in DpsbL (see below). It is noteworthy that not even traces of PSII–LHCII supercomplexes were present in DpsbJ thylakoids. In the absence of PSII–LHCII supercomplexes, the PsbO protein of the OEC was found to be associated with the PSII core dimers (Fig. 4A) in the thylakoid membranes of DpsbJ. The DpsbL mutant was capable of partial assembly of the PSII core monomers, whereas PSII core dimers and supercomplexes were completely missing (Fig. 3). Small amounts of both types of PSII core monomers, an intact PSII monomer and a CP43-less monomer, were observed (Fig. 3). It is noteworthy that, in DpsbL, the portion of free CP43 compared with that assembled into the PSII core monomer was extremely high (91 ± 5%). In wild-type thylakoids, only a minor amount (2 ± 1%) of CP43 was found free and unassembled into the PSII complexes under similar experimental conditions. This indicates that, in the absence of PsbL, the assembly of CP43 and thus the formation of stable intact PSII core monomers is severely impaired. None of the other PSII proteins were found free after 2D BN-PAGE of DpsbL thylakoids (except for a tiny amount of PsbE; Fig. 3), indicating no general disassembly of PSII core complexes during electrophoretic separation. Further, the presence of a small amount of PsbO detected by immunoblotting of DpsbL thylakoid proteins (Fig. 1A) prompted us to search for a PSII subcomplex with attached PsbO protein. Only after using a mini-gel system and partial solubilization of the thylakoid complexes for fast and gentle separation of the PSII subcomplexes did we succeed in isolating a novel PSII core monomer with attached PsbO (Fig. 4). This complex migrated slightly more slowly in the BN-gel than the normal intact PSII core monomer. The gentle separation system did not reveal the presence of this novel PSII core monomer–PsbO protein complex in the control or DpsbJ thylakoids (data not shown). It did confirm the association of PsbO with PSII–LHCII super- complexes in the wild-type and with the PSII core dimers in DpsbJ, as well as the absence of PSII–LHCII supercom- plexes from DpsbJ, and both the supercomplexes and PSII core dimers from DpsbL (Fig. 4A). However, although useful in detecting the PSII core monomer–PsbO protein complex in DpsbL, the gentle mini gel system could not be used for PSII assembly studies in general because of a background smear and tailing of protein bands. 77 K fluorescence emission spectra All mutant thylakoids harbored considerable amounts of LHCII complexes, which, however, could not be isolated in supercomplexes with PSII cores. To investigate whether there was energy transfer from LHCII to the PSII core, the fluorescence emission spectra at 77 K were recorded from thylakoids of the four psbEFLJ operon mutants after excitation with visible light below 500 nm. The wild-type and RV thylakoids showed well-defined PSII emission peaks at 685 nm (CP43) and 695 nm (CP47) as well as the PSI emission peak at 735 nm (Fig. 5) [42]. DpsbE, DpsbF and DpsbL lacked the emission peaks at 685 and 695 nm and instead had a prominent peak at 680 nm, characteristic of free LHCII. The 730-nm PSI peak was shifted to a lower wavelength. Interestingly, in DpsbJ, the 680-nm (LHCII), 685-nm (CP43) and 695-nm (CP47) 77 K fluorescence emission peaks were all present, in addition to the promi- nent PSI emission peak. Fig. 5. 77 K fluorescence emission spectra of thylakoid membranes of tobacco psbEFLJ operon mutants and controls (wild-type and RV). Thylakoids were excited with visible light below 500 nm. 102 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Discussion PSII contains several chloroplast-encoded and nuclear- encoded LMM subunits, the role of which in the assembly and stability of the complex has remained poorly under- stood. We have used a reverse genetics approach to elucidate the role of proteins encoded by the psbEFLJ operon, with special attention to PsbL and PsbJ, in the stable assembly process of the PSII core subunits, the LHCII antenna polypeptides, and the proteins of the OEC. PsbJ is essential for correct association of LHCII Although stable PSII core dimers were assembled in DpsbJ, the PSII–LHCII supercomplexes were completely missing. This indicates the importance of PsbJ in the steady-state higher organization of the PSII complexes. This conclusion, deduced from the 2D gel analysis (Fig. 3), was further supported by the 77 K fluorescence emission spectrum of DpsbJ revealing a distinct emission peak directly from LHCII at 680 nm, in addition to the two emission peaks from the PSII core (685 nm and 695 nm referring to CP43 and CP47, respectively; Fig. 5). This strongly suggests that the light energy absorbed by LHCII is not properly transferredtothePSIIreactioncenter.Invariancewitha recent study with tobacco DpsbJ mutant [29], we did not find any reduction in the contents of CP26 (Fig. 1A), the minor LHCII antenna protein thought to mediate the transfer of excitation energy from LHCII antennae to the PSII reaction center [6]. PsbJ is therefore probably essential in providing the PSII core dimer with a conformation that allows correct association with the LHCII complex and thereby efficient capture of excitation energy for PSII. Whether PsbJ exerts its effect on LHCII association directly or via its effects on the assembly of OEC remains to be resolved. PsbL is required for stable assembly of CP43 Comparison of the assembly of PSII in DpsbL and DpsbJ clearly demonstrates that PsbL is essential at earlier assembly steps than PsbJ, and therefore probably also represents a more intrinsic core protein than PsbJ in the structural hierarchy of PSII. Stable PSII core dimers were formed despite the absence of PsbJ, whereas in the absence of PsbL the PSII core proteins accumulated in minor amounts and successfully assembled only into PSII core monomers with unstable association of CP43 (Fig. 4). As shown with wild-type thylakoids, the correctly assembled PSII core monomers preserve their intactness during electrophoretic separation, whereas there are large amounts of free CP43 with the DpsbL thylakoids. It is thus conceivable that PsbL is an essential protein component of PSII for ensuring the stable assembly of CP43, and therefore, in DpsbL, the CP43 protein readily becomes detached from the PSII core monomer during the elec- trophoretic run. On the basis of the crystal structure of PSII [3], it was suggested that a transmembrane a-helix in the vicinity of CP47 possibly represents PsbL. We are inclined, however, to suggest that, rather than being located in the vicinity of CP47, PsbL is one of the unassigned transmem- brane a-helices in the vicinity of CP43 and D1 [3]. This suggestion is also supported by the fact that CP47 stably assembles with PSII core monomers even in the absence of PsbL (Fig. 3). Recently there has been a growing consensus in favour of PSII dimers being the functional forms of PSII [2,5,6]. Whether PsbL has a direct role in PSII dimerization, as was suggested by Barber and coworkers [7], is difficult to assess. It is probable that problems in stable assembly of CP43 exert secondary effects on PSII dimerization, and thus the role of PsbL in the dimerization process itself may be indirect. The exact mechanism of PSII dimerization is not known but it is conceivable that several small PSII subunits collectively control the successful dimerization of PSII [7,8]. The presence of CP43 in PSII is a prerequisite for association of PsbO whereas PsbL and PsbJ are needed for correct association of PsbP and PsbQ Three-dimensional OEC structures from spinach [2], Chlamydomonas and Synechococcus elongatus [43] were recently published. In all of these evolutionarily divergent species, the PsbO protein was suggested to be located towards the CP47/D2 side of the PSII reaction center core whereas the PsbQ and PsbP proteins (in cyanobacteria PsbV and PsbU, respectively) were located towards the N-terminal lumenal loop of the D1 protein. Such structures are in accordance with our results on the assembly of PsbO with the PSII core monomer in the mature leaves of DpsbL mutant. A lack of PsbL still allows a stable assembly and orientation of the CP47 side of the PSII core, which probably is required for stable association of PsbO. It should be noted, however, that the novel PSII core monomer–PsbO complex could be demonstrated only when CP43 was also present in the complex (Fig. 4A). Indeed, PsbO was found to be absent (as assessed by MALDI-TOF analysis and silver staining) from the CP43-less PSII core monomer. It is thus conceivable that the extended lumenal loops of CP43 are also involved in stabilization of the attachment of PsbO to the PSII core. In fact, the close proximity of PsbO and CP43 has been predicted previously from various in vitro studies with PSII membranes [44–47]. On the other hand, the lability and possibly incorrect conformation of the D1/CP43 side seems to prevent the assembly of PsbP and PsbQ with the PSII core monomer, despite the presence of PsbO, as evidenced by the complete absence of these OEC proteins from DpsbL.Themature leaves of DpsbJ showed a more stable association of CP43 than DpsbL, and indeed traces of PsbQ were also present, in addition to PsbO, whereas PsbP was completely missing. There seems to be no tight mutual control in the assembly of the three OEC proteins. Although the binding of PsbO apparently occurs first [48] and may be a prerequisite for the assembly of the other OEC proteins, it does not seem to provide any direct binding site for either PsbP or PsbQ, which does not support the previous suggestion [49]. It is evident that the presence of both PsbL and PsbJ is critical in providing proper docking sites, either directly or indirectly, by modifying the conformation of PSII on the lumenal side, making efficient binding of PsbQ and PsbP of the OEC possible. A fundamental difference in the DpsbJ mutants between cyanobacteria and the chloroplasts of higher plants was Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271) 103 recently described: only the cyanobacterial mutant is capable of slow photoautotrophic growth [28]. This is reflected in the capacity of the mutants to oxidize Q A – .Itis, however, likely that the water splitting and donation of electrons to P680 + also play a role in the better performance of the cyanobacterial than the tobacco DpsbJ mutant. Requirements for OEC proteins in cyanobacteria seem to be less stringent than in eukaryotes. In cyanobacteria, the presence of either PsbO or Cyt c 550 (PsbV) confers photo- autotrophic growth [50,51], whereas three distinct proteins form the OEC in eukaryotes [51]. Both PsbO and to a lesser extent PsbQ were present in mature leaves of tobacco DpsbJ mutant (Figs 1A and 4A), but, owing to the lack of PsbP, the oxygen-evolving capacity of this mutant is severely hampered [29]. In line with this notion, a Chlamydomonas mutant lacking PsbP was defective in oxygen evolution, which, however, could be restored by the addition of chloride ions [52]. The absence of psbEFLJ operon encoded proteins affects the accumulation of PSII core and OEC proteins in a development-dependent manner Studies with Chlamydomonas have demonstrated a com- plete lack of PSII assembly in the absence of Cyt b 559 [22]. An absolute requirement for PSII assembly of both the a and the b subunit of Cyt b 559 , encoded by the plastome psbE and psbF genes, was corroborated by this study using mature tobacco leaves. In fact, no PSII core or OEC proteins accumulated in thylakoids of mature leaves of the psbE and psbF inactivation mutants (Fig. 1A). This is completely opposite to the situation in young leaves, which, despite the lack of PSII assembly, kept accumulating all three proteins of the OEC and traces of the D2 and CP43 core proteins as well (Fig. 1B). The presence of minor amounts of D2 in both DpsbE and DpsbF supports the suggestion that the D2 protein is a component of the primary ÔreceptorÕ for the synthesis and cotranslational assembly of D1 [53,54]. In addition, Cyt b 559 has been found in barley etioplasts as a complex with D2 [11], emphasizing the role of these two subunits as primary assembly partners for construction of the PSII complexes. Indeed, the PsbE protein was also present in tiny amounts in the thylakoid membranes of young, developing leaves of DpsbF.Ofthe internal antenna proteins of PSII, the assembly of CP47 possibly also occurs cotranslationally because no free protein was found in the membrane, whereas the assembly process of CP43 seems to be less stringent [9,54,55] and some free CP43 was found in the thylakoid membrane of young developing leaves (Fig. 1B). Apparently a change in the developmental program upon leaf maturation and cessation of chloroplast division leads to down-regulation of both the chloroplast-encoded and nucleus-encoded PSII proteins (Fig. 1A), avoiding the wasteful synthesis of proteins when their assembly into functional complexes is prohibited. The possible signaling mechanisms leading to complete down-regulation of PSII core and OEC proteins in the absence of PSII assembly, manifested in DpsbE and DpsbF upon leaf maturation (Fig. 1A,B), are not known. However, the notion of the strict regulation of OEC protein synthesis in mature leaves also is supported by the identification of PSII subcomplexes that bind the PsbO protein in DpsbL and DpsbJ thylakoids (Fig. 4). Demonstration of the association of PsbO with PSII subcomplexes implies that free OEC proteins do not accumulate in the thylakoid lumen of mature leaves, in contrast with rapidly expanding young leaves (Fig. 1B). Fig. 6. Scheme demonstrating the ability of mature leaves of the DpsbE , DpsbF, DpsbL and DpsbJ mutants to form PSII–LHCII assemblies. In wild-type thylakoid membranes, PSII core dimers together with associated LHCII and OEC proteins form PSII–LHCII supercom- plexes. In the absence of PsbJ, the PSII core dimers can harbor the oxygen-evolving PsbO protein and also some PsbQ, but the LHCII complexes remain completely detached. Lack of PsbL results in more severe problems for the assembly of PSII: only PSII core monomers can be assembled with labile association of CP43, and, of the oxygen- evolving proteins, only PsbO is attached to the core monomer, pro- vided that CP43 is also present. Mature leaves of the DpsbE and DpsbF mutants do not accumulate any PSII core or OEC proteins but the LHCII complexes remain free in the thylakoid membrane. Oxygen- evolving proteins are shown as O (PsbO), P (PsbP) and Q (PsbQ). For clarity, only the major subunits are included. 104 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003 When assembly partners are not available, the chloro- plast-encoded major PSII core proteins (D1, D2, CP47) are typically down-regulated at the level of translation (for reviews, see [9,10,54]). The regulation of the synthesis of nuclear-encoded OEC proteins is still not understood, but may occur at the level of transcription. According to our results, it is likely that the regulation mechanisms for chloroplast-encoded PSII core and nuclear-encoded OEC proteins are different and independent of each other, as suggested previously [54]. Another explanation for the observed differences between young and mature leaves may be the enhanced proteolytic activity in mature leaves suffering from photo-oxidative stress because they either lacked PSII (DpsbE and DpsbF) or had a defectively assembled PSII (DpsbL and DpsbJ), as was discussed in the recent report on the DpsbJ tobacco mutant with dramatically reduced photosynthetic performance [29]. Lessons from psbEFLJ operon mutants on the role of PsbW and PsbZ subunits PSII assembly studies on psbEFLJ operon mutants also provided some information on the two other small PSII proteins, PsbW and PsbZ. Nuclear-encoded PsbW has been found to accumulate in the thylakoid membranes of both mature (Fig. 1A) and young [14] leaves of psbEFLJ operon mutants, even in the complete absence of PSII complexes (DpsbE and DpsbF). Similarly, PsbW was present, but at reduced amounts, in tobacco DpsbA mutant with no PSII assembly and activity [56]. Less stringent mutual regulation of the accumulation of PsbW and the other PSII core proteins was also evident in psbW antisense mutants of Arabidopsis [8]. All this suggests that PsbW is not under the same strict regulation and/or quality control as the other PSII core proteins and the OEC proteins in mature leaves. Recently characterized chloroplast-encoded PsbZ [38– 40], on the other hand, accumulated in mature leaves of DpsbL and DpsbJ, in comparable amounts to assembled PSII complexes, while being absent from DpsbE and DpsbF (Fig. 1A). The presence of PsbZ even in DpsbL may suggest the location of PsbZ in a very central core of PSII. Such a central location in PSII, however, seems to contradict the fact that PsbZ is not required for correct assembly of the oxygen-evolving PSII complexes and photoautotrophic growth of mutant plants [38–40]. Concluding remarks The capacity of mature leaves of the psbEFLJ operon mutants to assemble PSII (sub)complexes is schematically presented in Fig. 6. When either PsbE or PsbF is missing, the synthesis and accumulation of other PSII core proteins and the OEC proteins are strictly prevented. Thus, in contrast with young leaves [14], the absence of PSII assembly partners in mature leaves either evokes a signal to prevent the synthesis of other PSII proteins, of either chloroplast or nuclear origin, or enhances the proteolytic activity. Such control is, however, not exerted on the synthesis and assembly of the nuclear-encoded LHCII polypeptides or PSII LMM protein PsbW. Association of PsbL with PSII subcomplexes, in particular, promotes the stable and correct assembly of CP43 and thereby probably also facilitates the dimerization of PSII. Assembly of PsbJ is a subsequent step to the association of PsbL, and probably occurs only after the assembly of the PSII core monomer, or even the dimer, is accomplished. Of the OEC proteins, the binding of PsbO is clearly dependent on the presence of CP43 in the PSII core complex, whereas the correct association of PsbP and PsbQ additionally requires the presence of both the PsbL and PsbJ subunits. It remains to be investigated whether the PsbL and/or PsbJ proteins offer a direct docking site for PsbP and PsbQ or whether PsbL and PsbJ only modulate the structure and mutual orienta- tion of the PSII proteins on the lumenal side, making the association of PsbP and PsbQ feasible. Finally, PsbJ is clearly required for stable formation of PSII–LHCII supercomplexes, thereby allowing greater organization of PSII complexes in the thylakoid membrane. Acknowledgements Elena Baena-Gonzalez and Mika Kera ¨ nen are thanked for help with the 77 K fluorescence measurements, and Drs Roberto Barbato, Toril Hundal, Stefan Jansson, Wolfgang Schro ¨ der and Francis-Andre Wollman for the gifts of antibodies. This work was supported by the Academy of Finland, the Finnish Ministry of Agriculture and Forestry (NKJ project), the German Research Foundation (SFB-TR1) and Fonds der Chemischen Industrie. References 1. Hankamer, B., Morris, E., Nield, J., Carne, A. & Barber, J. (2001) Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II. FEBS Lett. 504, 142–152. 2. Nield, J., Orlova, E.V., Morris, E.P., Gowen, B., van Heel, M. & Barber, J. 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It remains to be investigated whether the PsbL and/ or PsbJ proteins offer a direct docking site for PsbP and PsbQ or whether PsbL and PsbJ. hold true. In rapidly expanding young leaves in particular, some of the PSII core proteins and all of the OEC proteins can accumulate in thylakoids in the absence of any assembly of PSII, as was. Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ Marjaana Suorsa 1 , Ralph E. Regel 2 , Virpi Paakkarinen 1 ,