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Plant Physiology 134:470-481 (2004) © 2004 American Society of Plant Biologists Towards Functional Proteomics of Membrane Protein Complexes in Synechocystis sp. PCC 68031Plant Physiology and Molecular Biology, Department of Biology, University of Turku, FIN-20014 Turku, Finland
The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43' protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.
Photosynthetic organisms experience continuous fluctuation in their growth environment, including changes in light conditions, temperature, and nutrient availability. Cyanobacteria, the probable progenitors of chloroplasts, possess a remarkable capacity to adapt to a wide range of environmental conditions. Cyanobacteria are primarily photoautotrophic, but some strains, such as Synechocystis sp. PCC 6803, can also grow mixotrophically or photoheterotrophically in the presence of external Glc. This, together with their flexibility to genetic manipulation and simple nutrient requirements for growth, has rendered cyanobacteria into a favored model in deciphering the structure-function relationships and acclimation strategies of the photosynthetic machinery.
Energy-transducing thylakoid membranes are the targets for modification by a number of environmental variables. Light, the most important environmental factor for photosynthesis, induces a well-defined acclimation response, including changes in the amount and ratio of the two PSs (Fujita, 1997
Increase in salinity of the growth medium likewise was reported to induce the expression of the isiAB operon (Vinnemeier et al., 1998
Limitation of inorganic carbon (Ci) in aquatic environments also poses a problem for photosynthesis. As a consequence, cyanobacteria have developed an extremely efficient carbon-concentrating mechanism, which operates to rise the CO2 concentration up to 1,000-fold around their relatively inefficient Rubisco enzyme (Badger and Price, 2003
During the past few years, genome-wide microarray studies of Synechocystis sp. PCC 6803 have provided a vast amount of information on global modification of gene expression in response to environmental variables (Hihara et al., 2001
Protein Complexes of Synechocystis sp. PCC 6803 Membranes: Photoautotrophic Growth Conditions Membrane protein complexes from wild-type (WT) Synechocystis sp. PCC 6803 cells grown photoautotrophically under air level of CO2 were first separated by blue native (BN)-PAGE. Approximately 20 distinct protein complexes could be observed in silver-stained BN gels (Fig. 1). To identify the protein complexes and to resolve their composition, the proteins from the BN gel lanes were separated in a second dimension by denaturing SDS-PAGE (Fig. 1), and the silver-stained protein spots were identified by mass spectrometric (matrix-assisted laser-desorption ionization time of flight [MALDI-TOF]) analysis or by immunoblotting (Fig. 2). Altogether, a total of 53 protein spots were identified as the products of 37 different Synechocystis sp. PCC 6803 genes (Table I).
Complexes Related to Linear Photosynthetic Electron Flow and ATP Synthesis PSI became distinguished as three major complexes, apparently representing a monomer, a trimer, and a high-molecular mass PSI supercomplex (Fig. 1). Moreover, a minor fraction of PSI resolved in BN gels apparently in a dimeric form. The reaction center subunits PsaA and PsaB, and PsaD, PsaF, and PsaL were identified by MALDI-TOF in the PSI complexes (Table I).
PSII complexes were resolved mainly as monomers but also as dimers or monomers lacking the CP43 subunit. The reaction center proteins D1 and D2, the Chl a-binding proteins CP47 and CP43, and the The cytochrome b6f (Cyt b6f) complex resolved mostly as a monomer, although a marginal amount of dimeric form was also observed in the gels. In both complexes cytochrome f, cytochrome b6, and subunit IV were identified.
The ATP synthase complex migrated in the BN gel slightly faster than the PSII dimer (Fig. 1). Subunits B and B' of the membrane integral CF0 complex, and the extrinsic CF1 subunits
Four complexes contained NDH-1 subunits. Based on their migration (and, correspondingly, the size), we denoted them as NDH-1L (large), NDH-1M (medium size), NDH-1S1 (small), and NDH-1S2 (Fig. 1). NDH-1L migrated in close proximity to ATP synthase, NDH-1M practically overlapped with the PSI monomer, NDH-1S1 migrated slightly faster than the CP43-less PSII monomer, and NDH-1S2 migrated between the NDH-1S1 and the Cyt b6f monomer. Both NDH-1L and NDH-1M contained the NdhH, NdhK, NdhI, and NdhJ subunits, which have been reported to belong to a NDH-1 subcomplex (Berger et al., 1993
A small protein complex migrating between the NDH-1S1 and NDH-1S2 complexes in the BN gel (Fig. 1) contained SbtA protein (Slr1512), which has been demonstrated as an essential component in a sodium-dependent bicarbonate transport in Synechocystis sp. PCC 6803 (Shibata et al., 2002a In addition, we discovered several minor, previously unknown protein complexes. One of them, containing the slr0151 gene product, was observed in the close proximity to the PSII monomers (Fig. 1). Two other protein complexes, containing gene products of sll1757 and slr0695, respectively, migrated between the NDH-1S2 and Cyt b6f monomer. The function of these novel protein complexes remains to be investigated.
Next, we tested the effect of the growth mode (photoauto-, mixo-, or photoheterotrophy) on the membrane proteome of Synechocystis sp. PCC 6803. To induce mixotrophic growth, the WT Synechocystis sp. PCC 6803 cells were cultured in the light in the presence of 5 mM Glc. Analysis of the membrane protein pattern, however, did not reveal any significant changes of whether the WT cells were grown in the absence (Fig. 1) or presence (data not shown) of Glc. To induce photoheterotrophic growth conditions, the cell cultures were supplied with the PSII electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in addition to Glc and light. These non-photosynthetic growth conditions induced notable changes in the pattern of membrane protein complexes (Fig. 3A). In particular, the NDH-1S1, NDH-1S2, and SbtA complexes were completely absent, and only traces of the NDH-1M complex were present. Conversely, two novel protein complexes in the range of 210 kD became strongly induced by these growth conditions and, therefore, were denoted as "Glc-induced" complexes. Mass spectroscopic analysis did not reveal the identity of the proteins belonging to these complexes, possibly because of strong hydrophobicity interfering the sample preparation.
To exclude possible DCMU-induced side effects (Schmitz et al., 1999
As a control to the mutant LC, we tested the autotrophic mutant strain AR cultured in the presence or absence of Glc. This strain contains the same four antibiotic resistance genes as LC but no mutations in the PSII reaction center protein D1 (Mäenpää et al., 1993
We also analyzed the protein complex profile of two other photoautotrophic mutant strains, an A2 mutant (Jansson et al., 1987
To study the effect of CO2 concentration on the accumulation of membrane protein complexes, we analyzed the membranes of WT cells grown under elevated CO2 concentration (1% [v/v] in air). The most pronounced changes induced by elevated CO2 were strong down-regulation of the NDH-1S1, NDH-1S2, and SbtA complexes and a marked reduction in the quantity of NDH-1M (Fig. 5). The content of the other membrane protein complexes showed no significant change in response to an elevated CO2 concentration (Fig. 5 versus Fig. 1).
The effects of iron starvation or increased salinity on the membrane protein complexes were studied with WT cells grown for 72 h in the absence of iron or in the presence of 0.4 M sodium chloride, respectively. Iron deficiency induced a pronounced accumulation of CP43' protein (Fig. 6A). This protein was found solely in association with the PSI trimers, forming a big PSI-CP43' supercomplex. This resulted in an increase in the amount of PSI-CP43' supercomplexes relative to PSI trimers. Otherwise, the membrane proteome strongly resembled that of the WT cells grown in iron-sufficient conditions (Fig. 1).
The increase in the salinity of the growth medium, on the other hand, did not induce any noticeable changes in the membrane protein complexes (Fig. 6B), as compared with the cells cultured in the absence of added NaCl (Fig. 1).
The expression patterns of membrane protein complexes described above represent a steady-state situation under each particular growth condition. To get insights into the synthesis and turnover of the membrane protein complexes, we performed [35S]Met labeling experiments with WT cells performing photoautotrophic, mixotrophic, or photoheterotrophic growth. The most distinct feature under all different growth conditions was the incorporation of radioactive Met into the PSII and its synthesis intermediates (Fig. 7). Labeling of subunit IV of the Cyt b6f complex was also observed under all growth conditions, suggesting a higher turnover rate for this protein as compared with the other Cyt b6f subunits. The NDH-1 subunits were synthesized in minor amounts. However, the synthesis of the NDH-1M and particularly the NDH-1S subunits NdhF3 and CupA was observed only during photoautotrophic and mixotrophic growth. Induction of photoheterotrophic growth by the addition of PSII inhibitor DCMU (15 min before labeling) completely abolished the incorporation of [35S]Met into these complexes. Labeling of the SbtA proteins followed closely that of the NDH-1 subunits under all growth conditions.
Cyanobacterial membranes are dynamic entities, changing their protein composition in response to a wide variety of environmental factors, including light quality and quantity (Fujita, 1997  ; Hihara, 1999), iron availability (Straus, 1994; Sandström et al., 2002), CO2 concentration (Murakami et al., 1997; Deng et al., 2003), and salinity (Murakami et al., 1997). These changes are necessary to balance the energy transduction processes and to minimize photooxidative damage, thus being crucial for the cyanobacterial growth in the ever-changing environment. Previous studies on acclimation of the thylakoid protein complexes have addressed mainly the dynamics of the two PSs. Compared with the vast amount of information available on the cyanobacterial photosynthetic complexes, far less is known about the composition, structure, and dynamics of other membrane-embedded protein complexes.
The most distinctive novel feature of Synechocystis sp. PCC 6803 membranes observed in this study was the multiplicity of complexes containing NDH-1 subunits. The NDH-1 subunits are encoded by at least 10 single copy genes (ndhAIGE, ndhB, ndhCJK, ndhH, and ndhL) and two multigene families (ndhF family with three members and ndhD family with six members; Badger and Price, 2003 The multisubunit NDH-1L and NDH-1M complexes showed a similarity in their protein composition. Both complexes contained the NdhH, -K, -I, and -J subunits (Fig. 1; Table I). In addition, they contained several other, still unidentified proteins. It is conceivable that these proteins represent more hydrophobic NDH-1 subunits because they were not effectively digested by trypsin to provide representative sets of peak masses. These subunits must therefore be identified by other methods than MALDI-TOF. Two smaller complexes, NDH-1S1 and NDH-1S2, contained a set of proteins different from that of NDH-1L or NDH-1M. NDH-1S1 consisted of four proteins, two of which comprised NDH-1S2. The first hints for the identity of these two common subunits came from MALDI-TOF analysis. In the spectrum of the protein with an apparent molecular mass of 32 kD, four peaks were observed that referred to NdhD3, and in the spectrum of the protein of 40 kD, three peaks were observed that referred to NdhF3. The reason for obtaining low-coverage data in the MALDI-TOF analysis is likely to be related to the high hydrophobicity of the NdhF3 and NdhD3 proteins, which apparently also induced an anomalous migration of these proteins in the SDS-PAGE. However, the protein specific antibodies confirmed the identity of the NdhF3 and NdhD3 proteins in the NDH-1S complexes (Fig. 2). It is noteworthy that the NDH-1L and NDH-1M complexes lacked the NdhF3 and NdhD3 subunits, whereas the NDH-1S complexes appeared to lack the subunits belonging to NDH-1L and NDH-1M (Fig. 2).
Reverse genetics approaches have revealed that NdhF3, NdhD3, NdhF4, and NdhD4 subunits contribute mainly to the uptake of CO2 (Klughammer et al., 1999
In addition to NdhF3 and NdhD3, we identified the CupA protein (Sll1734, also known as ChpY) in the NDH-1S1 complex. The genes for NdhF3, NdhD3, and CupA are clustered in the genomes of several unicellular cyanobacteria (Klughammer et al., 1999
Interestingly, the NDH-1S1 complex was found also to contain a fourth subunit, encoded by sll1735. The sll1735 gene is located immediately downstream of the ndhF3/ndhD3/cupA operon (Klughammer et al., 1999
We did not find the constitutively expressed, low-affinity CO2 uptake complex containing the NdhF4, NdhD4, and CupB proteins (Shibata et al., 2001
The NDH-1 complexes, particularly NDH-1S1 and NDH-1S2, were the most dynamic group of membrane protein complexes. The two NDH-1S complexes were strongly expressed in cells cultured photoautotrophically under air levels of CO2, whereas an increase in CO2 concentration drastically reduced the expression of these protein complexes (Fig. 1 versus Fig. 5). This observation is in accordance with the studies showing that the NdhF3, NdhD3, and CupA subunits play a role in high-affinity CO2 uptake, induced during acclimation of cells to low CO2 conditions (Klughammer et al., 1999
Intriguingly, accumulation of the NDH-1S complexes was also affected by the growth mode of the cells. Pulse labeling experiments of photoautotrophically grown WT cells revealed ongoing synthesis of NDH-1S complexes (Fig. 7A), which resulted in high steady-state amounts of these complexes in the membrane (Fig. 1). Similar results were obtained using mixotrophically cultured WT cells (Fig. 7B), indicating that the cells relied on active CO2 uptake even in the presence of external organic carbon source. Analysis of metabolic fluxes using the carbon isotope labeling technique has demonstrated that during mixotrophic growth of WT Synechocystis sp. PCC 6803, carbon is assimilated mainly via the Calvin cycle, whereas the catabolism of Glc has only a minor role in carbon metabolism (Yang et al., 2002
Similar to photoheterotrophic WT cells, the nonphotosynthetic PSII mutant strains were devoid of NDH-1S complexes (Fig. 3B; data not shown). This confirms the importance of PSII function for NDH-1S accumulation and excludes the possibility that the lack of NDH-1S in photoheterotrophic WT cells was a nonspecific side effect caused by DCMU. Somewhat surprisingly, the down-regulation of NDH-1S content was also observed when the photosynthetic mutant cells AR, A2, and
It is noteworthy that the accumulation of the NDH-1S and the NDH-1M complexes was closely paralleled under all growth conditions. This further supports the idea obtained by mutant analysis that these two complexes probably function in cooperation. The observed down-regulation of the NDH-1M content under elevated CO2 (Fig. 5) is consistent with the recent results of Deng et al. (2003
Previous studies of thylakoid adaptation mainly have addressed the dynamics of the two PSs and their light-harvesting antenna (Fujita, 1997
Higher expression levels of the isiAB operon also have been observed in cells grown under salt stress (Vinnemeier et al., 1998
Despite the relatively static image of PS complexes obtained from the steady-state data, the pulse labeling experiments revealed active synthesis of these complexes in cells grown photoauto-, mixo-, or photoheterotrophically (Fig. 7). The most notable feature was the active synthesis of PSII, and particularly of the PSII reaction center protein D1, under all conditions tested. The D1 protein is prone to continuous light damage, degradation, and de novo synthesis to maintain the photosynthetic activity (Aro et al., 1993
A protein complex containing the SbtA protein(s) was among the most dynamic complexes in the cyanobacterial membranes. Inactivation of the sbtA gene severely depresses the Na+-dependent HCO3- uptake in Synechocystis sp. PCC 6803 (Shibata et al., 2002a
Expression of two novel protein complexes, denoted as the "Glc-induced" complexes, was strongly induced under photoheterotrophic growth conditions (Fig. 3) and in mixotrophic mutant cultures (Fig. 4B). Although the exact role of these protein complexes awaits the identification of their constituent subunits, their induction under conditions where the cells were likely to rely on external Glc for their growth strongly suggests that they are involved either in the uptake of Glc or in the oxidation of NADPH formed by Glc catabolism. The scarcity of these complexes in the mixotrophic WT cultures is in line with the observation that the Glc catabolism plays only a minor role in the mixotrophically grown WT cell cultures (Yang et al., 2002 The rest of the membrane protein complexes showed no drastic modulation under the various growth conditions. However, we observed three novel protein complexes, including sll1757, slr0151, and slr0695 gene products, respectively. Although the physiological role of these proteins remains to be solved, we show here that these proteins are components of membrane-embedded complexes. We conclude that the BN/SDS-PAGE is a powerful method for analysis of the composition and dynamics of membrane protein complexes of cyanobacterial cells. Of particular interest was the observation of four distinct NDH-1 complexes, whose accumulation was regulated by environmental cues. Furthermore, we demonstrate that the gene products of the ndhF3/ndhD3/cupA operon associate together as a protein complex and introduce a new subunit for this CO2 uptake complex. In addition to the already known cyanobacterial membrane components, several new protein complexes, formed by proteins with unknown functions, are reported. The function of these novel protein complexes remains to be elucidated.
Strains and Growth Conditions
A Glc-tolerant strain of Synechocystis sp. PCC 6803 (Williams, 1988
All strains were grown in 200-mL batch cultures of BG-11 medium (Stanier et al., 1971
To induce mixotrophic or photoheterotrophic growth of WT or photoautotrophic mutant cultures (AR, A2, and
Membranes were isolated essentially as described by Gombos et al. (1994
The Chl a content of the isolated membranes was determined according to Arnon (1949
BN-PAGE was performed according to Kügler et al. (1997 ), with the modifications of Cline and Mori (2001). Cyanobacterial membranes were washed with washing buffer (330 mM sorbitol, 50 mM BisTris [pH 7.0], and 250 µg mL-1 Pefabloc [Roche Diagnostics, Indianapolis]), collected by centrifugation (18,000g for 10 min), and resuspended in resuspension buffer (20% [w/v] glycerol, 25 mM BisTris [pH 7.0], 10 mM MgCl2, 0.1 units µL-1 RQ1 RNase-Free DNase [Promega, Southampton, UK], and 250 µg mL-1 Pefabloc) at Chl a concentration of 1 mg mL-1. An equal volume of resuspension buffer containing 4% (w/v) n-dodecyl  -D-maltoside (Sigma) was added under continuous mixing, and the solubilization of membrane protein complexes was allowed to occur for 50 min on ice, followed by 10 min at room temperature. Insoluble material was removed by centrifugation at 18,000g for 20 min. The supernatant was mixed with 0.1 volumes of Coomassie Blue solution (5% [w/v] Serva blue G, 100 mM BisTris [pH 7.0], 30% [w/v] Suc, 500 mM  -amino-n-caproic acid, and 10 mM EDTA) and loaded onto 0.75-mm-thick 5% to 12.5% (w/v) acrylamide gradient gel (Hoefer Mighty Small mini-vertical unit, Amersham Pharmacia Biotech, Uppsala), containing 500 mM -amino-n-caproic acid. Electrophoresis was performed at 4°C by increasing voltage gradually from 50 up to 200 V during the 4.5-h run. For separation of proteins in the second dimension, the lanes of the BN gel were excised and incubated in SDS sample buffer containing 10% (v/v) -mercaptoethanol and 6 M urea for 45 min at room temperature, followed by 15 min at 40°C. The lanes were then layered onto 1-mm-thick SDS-PAGE gels (Laemmli, 1970), with 14% (w/v) acrylamide and 6 M urea in the separating gel. The proteins were visualized by silver staining (Blum et al., 1987, except that the stop buffer was replaced by 0.4 M Tris and 2.5% [v/v] acetic acid), by autoradiography ([35S]Met labeled proteins), or by electroblotting the proteins onto a polyvinylidene difluoride membrane (Immobilon P, Millipore, Bedford, MA) and immunodetection by protein-specific antibodies using the CDP-Star Chemiluminescent Detection Kit (New England Biolabs Ltd., Hertfordshire, UK). The NdhD3 antibody was raised against the amino acids 185 to 196 and 346 to 359, and NdhF3 antibody was raised against the amino acids 28 to 41 and 439 to 453 of the respective proteins of Synechocystis sp. PCC 6803 (Eurogentec, Seraing, Belgium).
Synechocystis sp. PCC 6803 WT cells were grown photoautotrophically or mixotrophically under standard growth conditions until the logarithmic growth phase and adjusted to Chl a concentration of 10 µg mL-1. Thereafter, the cell suspensions were pulse labeled with 3 µCi mL-1 [35S]Met (>1,000 Ci mmol-1, Amersham Biosciences UK Ltd.) for 30 min under conditions supporting photoautotrophic, mixotrophic, or photoheterotrophic growth. The photoheterotrophic growth conditions were induced by supplying the photoautotrophically grown cell culture with 15 µM DCMU 15 min before labeling. Labeling was terminated by adding 1 mM nonradioactive Met and 0.5 mg mL-1 chloramphenicol, and the cells were harvested by centrifugation at 4°C. Membranes were isolated and subjected to BN/SDS-PAGE as described. After BN/SDS-PAGE, the gels were dried, and the proteins were visualized by autoradiography.
In-gel trypsin digestion and sample preparation for MALDI-TOF analysis was done manually according to Shevchenko et al. (1996
We thank Dr. Teruo Ogawa for the  ndhD3 mutant, Dr. Bruce Diner for the psbA1 mutant, and Dr. Wim Vermaas for the PSI-less mutant. The mass spectrometer group in the Turku Centre for Biotechnology is thanked for advice in protein analysis. Received August 26, 2003; returned for revision September 17, 2003; accepted October 14, 2003.
1 This work was supported by the Academy of Finland.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032326. * Corresponding author; e-mail evaaro{at}utu.fi; fax 358-2-333-8075.
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Zhang L, Paakkarinen V, van Wijk KJ, Aro E-M (2000) Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II. Plant Cell 12: 1769-1781 This article has been cited by other articles:
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-subunit of cytochrome b559 were identified in this study. Most small subunits of PSII, however, failed the identification by MALDI-TOF, whereas subunits of the oxygen-evolving complex apparently released from PSII upon membrane solubilization. 
, 
, and 
-fragment (streptomycin and spectinomycin resistance cassette) after the psbA2 gene (Mäenpää et al., 1993
