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Eur. J. Biochem. 269, 1877–1885 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02834.x Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides Francesco Francia1,2, Jun Wang1,*, Hans Zischka1,†, Giovanni Venturoli2 and Dieter Oesterhelt1 1 Department of Membrane Biochemistry Max-Planck-Institute for Biochemistry, Martinsried, Germany; 2Department of Biology, Laboratory of Biochemistry and Biophysics, University of Bologna, Italy The core complex of Rhodobacter sphaeroides is formed by the association of the light-harvesting antenna 1 (LH1) and the reaction center (RC). The PufX protein is essential for photosynthetic growth; it is located within the core in a 1 : 1 stoichiometry with the RC. PufX is required for a fast ubiquinol exchange between the QB site of the RC and the Qo site of the cytochrome bc1 complex. In vivo the LH1– PufX–RC complex is assembled in a dimeric form, where PufX is involved as a structural organizer. We have modified the PufX protein at the N and the C-terminus with progressive deletions. The nine mutants obtained have been characterized for their ability for photosynthetic growth, the insertion of PufX in the core LH1–RC complex, the stability of the dimers and the kinetics of flash-induced reduction of cytochrome b561 of the cytochrome bc1 complex. Deletion of 18 residues at the N-terminus destabilizes the dimer in vitro without preventing photosynthetic growth. The dimer (or a stable dimer) does not seem to be a necessary requisite for the photosynthetic phenotype. Partial C-terminal deletions impede the insertion of PufX, while the complete absence of the C-terminus leads to the insertion of a PufX protein composed of only its first 53 residues and does not affect the photosynthetic growth of the bacterium. Overall, the results point to a complex role of the N and C domains in the structural organization of the core complex; the N-terminus is suggested to be responsible mainly for dimerization, while the C-terminus is thought to be involved mainly in PufX assembly. The purple bacterium Rhodobacter (Rb.) sphaeroides can grow photosynthetically or heterotrophically via aerobic or anaerobic respiration. When growing photosynthetically, it uses light energy as a driving force to form ATP via a cyclic electron transfer. Photons are captured from the lightharvesting (LH) complex(es) and the excitation energy funnelled towards a bacteriochlorophyll (BChl) special pair (P), located in the reaction centre (RC). The excited P delivers an electron via an accessory BChl and a bacteriopheophytin molecule to a primary ubiquinone acceptor (QA). In a much slower reaction the electron is transferred to a second ubiquinone acceptor (QB). The full reduction of the quinone molecule at QB to quinol requires a second photoexcitation of the RC and is coupled to the uptake of two protons from the cytoplasmic space. The formed ubiquinol dissociates from the RC and is released into the membrane lipid phase [1]. Ubiquinol molecules are oxidized at the Qo site of the cytochrome bc1 complex (cyt bc1). Here the electron pathway branches into a high and a low potential chain. The first electron reduces in series an iron cluster centre and a cytochrome c1 in the high potential chain, while the second electron reduces the low potential chain, composed of cytochrome b566, cytochrome b561 and a ubiquinone molecule located at the Qi site. A second ubiquinol oxidized at the Qo site brings the electron to fully reduce the ubisemiquinone to ubiquinol on Qi. From the cytochrome c1, the electron is transferred to a soluble cytochrome c2 that is the physiological electron donor to the oxidized P. From the Qo site, protons are released into the periplasmic space of the cell. This cyclic mechanism of redox reactions acts as a proton pumping system, moving protons from the cytoplasmic to the periplasmic space. The formed H+ gradient is the driving force for synthesis of ATP that is used to power the metabolic reactions in the cell [2]. In Rb. sphaeroides the ability of the RC to capture light energy is largely increased by the presence of two LH complexes: LH1 and LH2. The LH1 complex is intimately associated with the RC in a fixed stoichiometry to form the core complexes (LH1–RC), while the LH2 is arranged peripherally with respect to the core. Both LHs are organized in circular supramolecular complexes, resulting from the repetition of a minimal building block commonly Correspondence to F. Francia, Department of Biology, Laboratory of Biochemistry and Biophysics, University of Bologna, Via Irnerio n.42, 40126 Bologna, Italy. Fax: + 39 051 242576, Tel.: + 39 051 2091300, E-mail: francia@alma.unibo.it Abbreviations: BChl, bacteriochlorophyll; cyt bc1, cytochrome bc1 complex; ICM, intracytoplasmic membranes; LH, light-harvesting complex; PMC, photosynthetic membrane complex; QA,QB, primary/ secondary electron acceptor; Qi, quinone reductase site of the cyt bc1 complex; Qo, quinol oxidase site of the cyt bc1 complex; RC, reaction center. *Present address: Department of Plant Biology, The Ohio State University, Columbus, OH, USA. Present address: GSF Forschungszentrum, Institut für Humangenetik, Oberschleißheim, Germany. (Received 6 November 2002, revised 12 February 2002, accepted 13 February 2002) Keywords: LH1-RC; photosynthesis; PufX; Rhodobacter sphaeroides. Ó FEBS 2002 1878 F. Francia et al. (Eur. J. Biochem. 269) referred to as the a,b heterodimer. The a and b polypeptides span the membrane with a single hydrophobic a helix. This circular protein scaffold binds the pigments that are maintained in a spatial orientation that maximizes the efficiency of the energy transfer reactions. Structures of the LH2 [3,4] and RC [5], as well as of cyt bc1 [6] are known at atomic resolution but came from different organisms; on the contrary, high resolution structural data of the core complex are not yet available. In Rb. sphaeroides and Rb. capsulatus photosynthetic growth requires the presence of the PufX protein [7,8]. When an intact LH1–RC core complex is present, PufX is essential to promote an efficient ubiquinone/ubiquinol exchange between the RC and cyt bc1 [9], but is not necessary when the LH1 system is absent or reduced in size [10–12]. This evidence points to a complex structural relationship between the components of the photosynthetic system, in which PufX plays a central role [13]. Recently, several works have indicated that PufX is involved directly in the supramolecular organization of the photosystem: (a) the core complexes of Rb. sphaeroides are organized in a dimeric form [14], in which the presence of PufX induces a specific orientation of the RC inside the LH1 complex as well as the formation of a long range regular array of LH1– RC in the photosynthetic membrane [15]; (b) biochemical studies have shown that PufX is present in the LH1–RC complex in a 1 : 1 stoichiometry with the RC, and that the dimeric form of the core complex could only be isolated in the presence of PufX [16]; (c) the PufX protein has a strong tendency to interact with the LH1 a polypeptide, while no interaction was detected with the LH1 b polypeptide [17]; (d) the deletion of PufX increases the number of LH1associated Bchls per RC, suggesting an increased number of a,b heterodimers in the LH1 [18]. Moreover in the presence of PufX, electron density maps of the dimeric LH1–RC show unequivocally interruptions in the LH1 ring encircling the RC. The top view of the LH1–RC core complex presents two rings of LH1 in close contact forming a pattern which resembles the shape of the letter S; each interrupted ring contains an electron dense nucleus attributed to the RC [14]. All of these experimental results are consistent with the idea that PufX is responsible for these interruptions, allowing a faster lateral diffusion of ubiquinone/ubiquinol molecules toward/from the RC QB site. A previous work on Rb. sphaeroides demonstrated the role of the C-terminal amino-acid residues of the LH1 a polypeptide in the organization of the LH1–RC complex [12]. In the present work, we have investigated the possible involvement of the N-terminus and of the C-terminus of PufX in protein–protein interactions stabilizing the LH1–RC complex. To this aim two sets of mutant strains have been constructed. The N-terminal domain has been progressively shortened by deletions extending from the second residue of the primary sequence, while the C-terminal portion has been progressively shortened by introducing stop codons by sitedirected mutagenesis. We have obtained information on the involvement of the N- and C-terminal portions of PufX in its insertion in the membrane and dimerization of the core complexes. EXPERIMENTAL PROCEDURES Bacterial strains, plasmid, gene transfer, growth conditions, membrane preparations Bacterial strains and the plasmid used in this work were as described previously [16]. Growth conditions for Escherichia coli and Rb. sphaeroides have also been described [16]. All of the Rb. sphaeroides strains were grown semi-aerobically. Photoheterotrophic growth tests in liquid culture were monitored with a Klett–Summerson colorimeter as described by Farchaus et al. [7]; kanamycin and tetracycline were added at 25 lgÆmL)1 and 2 lgÆmL)1, respectively. Cultures were illuminated by two 120 W incandescent light bulbs; excessive warming was prevented by placing a 40-cm water bath between the lamps and the cultures. Intracytoplasmic membranes (ICM) were prepared as described previously [19]. PufX mutagenesis The PufX N-terminal deletion and C-terminal stop codon series were constructed using the pRKX plasmid [16] as DNA template and introducing the desired mutation/ deletion with the method given by Ausbel et al. [20]. The external primers used for the cited sequential PCR mutagenesis anneal, respectively, 330 bp upstream and 441 bp downstream the pufX gene on the plasmid. This fragment contains the HindIII and ClaI (8 bp upstream and 171 bp downstream the pufX gene, respectively) unique sites and allows, after digestion with HindIII and ClaI, the ligation of the final PCR product containing the mutated pufX gene with the pRKX vector digested with the same restriction enzymes. After transformations of E. coli S17-1 cells, single colonies of the putative transformants were grown overnight in 5 mL Luria–Bertani media with 10 lM tetracycline. The pRKX derived harbouring mutated pufX gene constructs from the E. coli S17-1 cells were introduced into Rb. sphaeroides DQ x/g cells by conjugation [16]. Mutations were confirmed by sequencing the plasmids isolated from transformed Rb. sphaeroides cells with the Qiagen Minikit. Isolation of core complexes and SDS/PAGE Core complexes were extracted from (ICM) according to the method described previously [16,21] except that the NaBr washing step was performed at 0.6 mgÆmL)1 total protein. The concentration of LH1–RC complex in the isolated bands was estimated on the basis of the total photooxidizable RC measured by flash kinetic spectrophotometry as described before [16]. Aliquots containing the same number of LH1–RC moles were treated with 10 vol. cold acetone/ methanol (7 : 2, v/v), vortexed for 2 min and centrifuged. The organic phase was discarded, and the protein pellet was dried at 40 °C for 30 min. The pellets were redissolved in SDS/PAGE loading buffer to final concentration of 2 lM LH1–RC in all of the samples. SDS/PAGE was carried out accordingly to Schägger & Von Jagow [22], with a separating gel of 19.5% (w/v) acrylamide, 0.5% (w/v) bis-acrylamide. Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1879 MS and sequencing of PufX54* protein Proteins were separated by SDS/PAGE as described above, and stained with Coomassie G250. Gels were washed extensively with H2O to remove residual acid from the destaining process. The band of interest was cut out with a razor blade and transferred to reaction tubes. Proteins were then subjected to a limited protease treatment overnight (0.5–1 lgÆband)1 endoproteinase LysC, Roche Molecular Biochemicals) [23]. Peptides were extracted from gel slices by altered incubation with 10% formic acid and acetonitrile. Pooled fractions were dried in a speed vac concentrator. Dried peptides were re-dissolved in 10 lL 10% acetonitrile/0.1% trifluoroacetic acid. Between 0.5 and 1 lL were used for MALDI-TOF analysis (adapted from [24]). The residual sample was applied to a reversed-phase HPLC system to separate peptides. Purified peptides were subjected to automated Edman degradation (with kind support of J. Kellermann, Max Planck Institute for Biochemistry, Martinsried). Time resolved spectroscopy on ICM The kinetics of cytochrome b561 reduction induced by a single actinic flash were measured under the following conditions: ICM were resuspended in a buffer composed of 50 mM Mops, 100 mM KCl, pH 7.0; valinomycin and nigericin were added at 10 lM to collapse the transmembrane proton gradient and to avoid spectral interference due to BChl and carotenoid electrochromic effects; 5 lM antimycin A was used to inhibit the Qi site of the cyt bc1. Measurements were performed in a nitrogen atmosphere under controlled redox conditions as described by Venturoli et al. [25]. One micromolar each of phenazine methosulfate and phenazine ethosulfate; 2 lM of 2,3,5,6-tetra-methyl-pphenylenediamine; 10 lM each of p-benzoquinone, duroquinone, 1,2-naphthoquinone, 1,4-naphthoquinone were used as redox mediators. The experimental apparatus is as described in Francia et al. [16]. Traces of cytochrome b561 reduction (Fig. 3) were analysed numerically in terms of pseudo first-order kinetics following an initial lag period, as described by Barz et al. [9]. In order to determine the best fitting parameters, the lag period following the time of the flash was varied stepwise: for each lag period the amplitude and rate constant of the exponential function were optimized using a nonlinear v2 minimization routine [26] and a plot of the minimized v2 vs. the lag period was constructed for each kinetic trace. For all traces this procedure yielded a minimum reduced v2 (v2min ) between 0.8 and 1.2. The confidence interval in the determined value of the lag was obtained by using an F-statistic to determine the probability p of a particular fractional increase in v2 according to: generally asymmetrical, due to the nonlinear nature of deconvolution. RESULTS Construction of the PufX N-terminally deleted series and C-terminally truncated series To obtain the strains with the mutated PufX protein reported in Table 1, two sets of plasmids were constructed. The first series consists of a progressive deletion at the N-terminus, extending from the second residue of the primary sequence; the second series consists of a progressive truncation of the C-terminal domain of PufX (Fig. 1) obtained by the introduction of stop codons in the gene sequence of pufX. In all the cases, the Rb. sphaeroides host strain was DQ x/g [10]. The pseudo wild-type strain used in this work was obtained reintroducing the complete puf operon via the plasmid pRKX (in trans) into the host Rb. sphaeroides DQ x/g, deprived of the chromosomal copy of the puf operon. Table 1. Bacterial strains and plasmids. The plasmid host strain in all the cases was Rb. sphaeroides DQ x/g. Strain Plasmid name Wild-type N-Terminus series PufXD2–4 PufXD2–7 PufXD2–19 PufXD2–26 C-Terminus series PufX54* PufX68* PufX72* PufX76* PufX81* PufDX pRKX pRKXD2–4 pRKXD2–7 pRKXD2–19 pRKXD2–26 pRKX54* pRKX68* pRKX72* pRKX76* pRKX81* pRKDX v2 =v2min ¼ 1 þ ½m=ðn ÿ mފ Fðm; n ÿ m; 1 ÿ pÞ where m is the number of parameters, n is the number of data points, and F is the upper (1–p) quantile for Fisher’s F distributions with m and (n–m) degrees of freedom [27]. Confidence intervals within 1 SD (P ¼ 0.68) calculated by this procedure are given in Table 2. These intervals are Fig. 1. Nature of the deletions and truncations on PufX. The helix transmembrane region of the PufX protein, predicted with the program PHDHTM [32], is indicated at the top of the figure and represented as an empty rectangle in the primary sequences of PufX showed below. The related Rb. sphaeroides strains are given on the left. Ó FEBS 2002 1880 F. Francia et al. (Eur. J. Biochem. 269) Photosynthetic growth curves Aliquots of bacteria corresponding to 1 absorbance unit at 700 nm from precultures grown semi-aerobically in darkness were transferred to 13 mL final volume of fresh media in 15 mL glass tubes. Air was eliminated from the tubes by using a vacuum water pump; tubes were then exposed to the light in a 30 °C chamber. The results of this photosynthetic assay are shown in Fig. 2. Mutants of the N-terminally deleted series (Fig. 2A) exhibit photosynthetic growth with the exception of the PufXD2)26 strain. The curves in Fig. 2A show a lag phase varying between 15 and 50 h. By comparing several independent growth curves for each mutant (data not shown), it appeared that a similar, large variability could be observed in any strain including wildtype (compare Fig. 2A,B). Therefore the observed lag phase did not show any correlation with the phenotype. Clearly photosynthetic-negative phenotypes are evidenced by the PufDX (as already reported previously [28]) and PufXD2)26 curves. Also the C-terminus mutants PufX76*, PufX72* and PufX68* exhibit a nonphotosynthetic phenotype, whereas the mutant with the shortest truncation (PufX81*) Fig. 2. Growth curves of the control and mutated PufX strains under photosynthetic conditions. The growth of the coltures was monitered by a Klett–Summerson colorimeter. (A) Wild-type (j), PufXD2–4 (s), PufXD2–7 (n), PufXD2–19 (,), PufDX (h); the growth curve of PufXD2–26 (not shown in the figure for visual clarity) coincides with that of PufDX. (B) Wild-type (j), PufX54*(s), PufX68* (n), PufX72* (,), PufX81* (e); the growth curve of PufX 76* (not shown) coincides with those of PufX68* and PufX72*, i.e. reveals inability of photosynthetic growth. is photosynthetically competent (Fig. 2B). Surprisingly, the most extended truncation (mutant PufX54*) does not affect the ability of photosynthetic growth. Kinetics of cytochrome b561 reduction induced by a single-turnover flash on ICM The rate of electron transfer through the Qo site of cyt bc1 can be measured in ICM by monitoring the reduction of the cytochrome b561 induced by a short actinic light flash in the presence of the inhibitor antimycin A [29]. Reduction of the cytochrome b561 typically shows a lag period prior to the onset of the reaction at its maximal rate. In wild-type ICM, the initial rate of this reaction, as well as the lag phase, depends on the redox state of the ubiquinone pool and on the ubiquinone/RC stoichiometry [2,25]. For a normal size of ubiquinone pool ( 25 ubiquinone molecules/RC)1), upon decreasing the ambient redox potential (Eh) from 250 to 100 mV at pH 7.0, the initial rate of cytochrome b561 reduction increases progressively, while the lag becomes shorter. This behaviour has been attributed to the increased availability of prereduced ubiquinone molecules in the pool reacting at the Qo site of cyt bc1. Keeping the Eh high enough, the only ubiquinol molecule which can react at Qo and reduce the cytochrome b561 is the one released by the RC following photoexcitation, as the ubiquinone pool is completely preoxidized [30]. Under this condition the lag period is maximal, typically 1 ms in wild-type ICM. A drastic increase of the lag phase, paralleled by a decrease in the initial reduction rate is observed in pufX-deleted strains as compared with wild-type [9]. Both of these effects are maximal at Eh > 180 mV (i.e. when the ubiquinone pool is fully oxidized) and reflect a dramatic impairment in the redox interaction between the QB site of the RC and the Qo site of the cyt bc1 in the pufX-deleted strain. We have measured the kinetics of cytochrome b561 reduction of all the N- and C-terminal PufX mutants (at Eh 180–220 mV) on ICM prepared from cultures grown semi-aerobically the dark. It must be pointed out that under these growth conditions there is no photosynthetic selective pressure that could induce the suppression phenomenon reported in [10]. Kinetic traces recorded from the mutants PufXD2)26 and PufX54* are shown in Fig. 3. The continuous curves are best-fits to an exponential function; the lag duration was determined numerically as outlined in Experimental procedures. The properties of the complete N-terminally deleted and C-terminally truncated series are listed in Table 2. While the lag period of PufX54* is comparable to that measured in a typical wild-type, the PufX68*, PufX72*, PufX76* as well as the PufXD2)26 exhibit an increased lag period usually observed in the pufX-deleted strain. The PufXD2)4, PufXD2)7 and PufX81* show a lag period like that of wild-type, indicating that a short deletion at the N- and C-terminus does not affect this parameter. Measurements carried out on PufXD2)19 revealed an intermediate lag duration (see Table 2), making ambiguous the attribution of the PufXD2)19 strain to the wild-type or the PufDX cluster. Isolation of the photosynthetic complexes from the mutant strains As described previously [16], the photosynthetic complexes (PMCs) could be extracted by detergent solubilization from Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1881 the membranes and purified by centrifugation in continuous sucrose density gradients. Briefly, the final wild-type pattern consists of four bands, named PMC1, PMC2, PMC3 and PMC4 from top to bottom of the tubes (Fig. 4, tube 1). PMC1, PMC2, PMC3 and PMC4 represent LH2, LH1 Ôempty ringsÕ, LH1–RC monomers and LH1–RC dimers, respectively. A consequence of the deletion of the pufX gene is the lack of LH1–RC dimer bands in the gradients (see Fig. 4A, tube 6). Fig. 4 shows the patterns of the N-terminally deleted (Fig. 4A) and C-terminally truncated (Fig. 4B) series. PufXD2)4 and the PufX81* (the shortest deletion and truncation, respectively) mutants exhibit the wild-type like pattern, with all four bands present. In the mutant PufXD2)7, a very faint band in the correct position of PMC4 could be seen in the original photograph, whereas in the other N-terminally deleted strains, PufXD2)19 and PufXD2)26 PMC4 is undetectable. Also in the mutants PufX68*, PufX72* and PufX76* PMC4 is not seen; interestingly a fourth band not clearly separated from PMC3, with a position intermediate between those of PMC3 and PMC4 (between the LH1–RC monomer and the dimer), is present in the PufX54* gradient profile (Fig. 4B, tube 2). SDS/PAGE of the isolated LH1–RC complex Fig. 3. Cytochrome b561 reduction kinetics induced by single flash photoexcitation in ICM. The continuous vertical line indicates the instant when the actinic flash pulse was fired (time ¼ 0), the dotted vertical line marks the beginning of the cytochrome b561 reduction at its maximal rate. The time interval between the continuous and the dotted vertical lines corresponds to the lag phase of the reduction kinetics. Lag duration was evaluated by a numerical procedure as outlined under experimental procedures. The experimental trace is represented by a continuous line connecting the points sampled by the recording apparatus; the best-fitting mono-exponential function is indicated by a continuous curve. (A) ICM from strain PufXD2-26; (B) ICM from strain PufX54*. The RC : PufX stoichiometry in isolated LH1–RC complexes is 1 : 1 in the wild-type strain. The same unitary stoichiometry was determined in the monomeric (PMC3) and dimeric (PMC4) core complex bands [16]. The presence of the mutated PufX can therefore be assessed in PMC3 isolated from the mutants. Fig. 5A shows an SDS/PAGE of isolated PMC3 from the N-terminally deleted series. Only the small molecular weight region is shown. In Fig. 5A, lanes 2 and 3, corresponding to PMC3 from PufXD2)4 and PufXD2)7, respectively, above the dominant LH1 a and b bands, a band attributable to the mutated PufX is clearly visible. In lane 4 a faint band, attributable to PufXD2)19 is indicated by an arrow, while no band, except those of LH1 a and b, can be seen in the mutant PufXD2)26 and in the PufDX strain. Table 2. Summary of experimental data. Strain Light growth Cytochrome b561 reduction lag (ms)a Sucrose gradientisolated PMC Detection of PufX in PMC3b Wild-type PufXD2-4 PufXD2-7 PufXD2-19 PufXD2-26 PufX54* PufX68* PufX72* PufX76* PufX81* PufDX Yes Yes Yes Yes No Yes No No No Yes No 0.7 1.1 0.5 3.8 5.3 1.2 10.7 6.3 4.7 0.7 8.8 PMC3, PMC4 PMC3, PMC4 PMC3 (PMC4)c PMC3 PMC3 PMC3, PMC3/4 PMC3 PMC3 PMC3 PMC3, PMC4 PMC3 Yes Yes Yes Yes No Yes No No No Yes No a (0.2–0.8) (0.9–1.3) (0.0–1.0) (3.5–4.2) (4.3–5.8) (0.5–1.4) (9.5–11.1) (6.2–6.8) (4.0–5.8) (0.5–1.0) (8.8–9.1) The confidence interval within 1 SD is given in parentheses. b Detected by SDS/PAGE on sucrose gradient-isolated PMC 3. c Detectable as a weak band in the original photograph. Ó FEBS 2002 1882 F. Francia et al. (Eur. J. Biochem. 269) Fig. 4. Isolation of the PMCs on a sucrose gradient. The final detergent extracts from the ICM were loaded on the top of a 10–40% sucrose gradient and centrifuged for 19 h at 230 · 103 g. The gradient was buffered with 50 mM Na-glycylglycine to pH 7.8, the detergents octylglucoside and Na-cholate were added to the gradient at a final concentration of 0.6% and 0.2% (w/v), respectively. (A) Tube 1, wild-type; tube 2, PufXD2–4; tube 3, PufXD2–7; tube 4, PufXD2–19; tube 5, PufXD2–26; tube 6, PufDX. (B) Tube 1, wild-type; tube 2, PufX54*; tube 3, PufX68*; tube 4, PufX72*; tube 5, PufX76*; tube 6, PufX81*. In Fig. 5B, data for the C-terminally truncated series are shown. The presence of PufX81* is evident in lane 2, while no PufX band was observed in the mutants PufX76*, PufX72* and PufX68* (lanes 3, 4, 5, respectively). A thin band very close to the LH1 a band, indicated by the arrow, is apparent in the PufX54* mutant (lane 6). This band was excised from the gel after SDS/PAGE and the protein was identified by using the method described in the Experimental procedures. Briefly, the band was digested with 0.5 lg of the proteolytic enzyme endoproteinase Lys-C and parts of the resulting peptide mixture were analysed by MS (MALDI-TOF). The peptide mass fingerprint obtained corresponded to fragments 17–29 (TNLRLWVAFQMMK) and 5–16 (TIFNDHLNTNPK) of the PufX protein. The identity of PufX in the band isolated was examined further by subjecting part of the Fig. 5. SDS/PAGE on sucrose gradient-isolated core complexes. The proteins of the PMC3 bands isolated from the sucrose gradients (see Fig. 4) were subjected to SDS/PAGE according to Schägger & Von Jagow [22]. The concentrations of acrylamide and bis-acrylamide were 19.5% and 0.5% (w/v), respectively, in the separating gel and 3.9% and 0.1% in the stacking gel. For each lane, 24 pmol PMC3, corresponding to the monomeric form of the core complex, was loaded. Only the region of low molecular mass proteins is shown in the figure. (A) Lane 1, wild-type; lane 2, PufXD2–4; lane 3, PufXD2–7; lane 4, PufXD2–19; lane 5, PufXD2–26; lane 6, PufDX. (B) Lane 1, wild-type; lane 2, PufX81*; lane 3, PufX76*; lane 4, PufX72*; lane 5, PufX68*; lane 6, PufX54*; lane 7, PufDX. The position of the faint band attributed to the PufXD2–19 protein is indicated by an arrow in lane 4 A, the position of the detected PufX54* is indicated by an arrow in lane 6 B. peptide mixture to separation by reversed-phase HPLC. Purified peptides were subjected to sequence analysis by Edman degradation. The sequence KTIFNDHLNTN, corresponding to the 4–14 fragment of PufX, was identified. DISCUSSION Effects of N-terminal and C-terminal PufX deletion on LH1–RC dimerization In the LH1–PufX–RC core complex of Rh. sphaeroides, the RC : PufX stoichiometry is 1 : 1 [16]. The role of PufX as a structural organizer of the core complex has been discussed recently in several works (rewiewed in [13]). Data on the sequence of assembly of the LH1–PufX–RC complex in vivo [31] and on protein–protein interactions between the single polypeptides of the complex [17] are consistent with the hypothesis that PufX interrupts the continuity of the LH1 ring and switches the structure of the complex from a ÔclosedÕ monomeric form to an ÔopenÕ dimeric form. Moreover, linear dicroism studies have demonstrated the role of PufX in the orientation of the RC inside the LH1 Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1883 [15]. These results indicate that the PufX protein is in contact with the LH1 and the RC subunits inside the core complexes. When secondary structure prediction was performed on PufX [32] the final output revealed a strong tendency to build a helices at both the N- and C-termini [33] and a transmembrane a helix in the central region (Fig. 1). On this basis, and in view of the finding that the C-terminal part of the LH1 a polypeptide plays an important role in the structure of the core complex [12], we decided to investigate the possible structural role of the N-terminus and the C-terminus of PufX. To this aim, nine strains of Rb. sphaeroides with mutated PufX were constructed. The dimeric form of the core complex purified from ICM [16] has been confirmed by electron microscopy [14]. We consider, therefore, the presence of the dimeric form (PMC4) upon isolation as an indication for dimerization in vivo. The shortest deletion in the PufXD2)4 and the shortest truncation in PufX81* do not impair the ability of PufX to facilitate dimerization, as a clear PMC4 band can be detected in the gradient (Fig. 4). Interestingly in the gradient of the N-terminus mutant PufXD2)7 a very faint PMC4 band is visible in the original gradient photograph (undetectable in Fig. 4). Apparently this deletion strongly destabilizes the dimer to the extent that it cannot withstand fully the membrane detergent extraction. The presence of the dimer in vivo in the mutant PufXD2)7 and presumably in PufXD2)19 is therefore not excluded. We have shown previously that in vitro an irreversible dissociation of the dimeric to the monomeric form of the complex from the wild-type exists: the dimer dissociates gradually into the monomer when the octyl-glucoside concentration is increased from 0.6 to 1.2% [16]. This result suggested that hydrophobic interactions are involved in maintaining the dimeric form. The data obtained on the PufXD2)7 and PufXD2)19 strains indicate that important protein–protein hydrophobic interactions are made by the PufX N-terminus. In the case of the longest N-terminal deletion (strain PufXD2)26), the PufXD2)26 protein is not detectable in the core complex (see below and Fig. 5A, lane 5). Correspondingly only the monomeric form of the complex can be seen in the gradient (Fig. 4A, tube 5). Two main points of interest arise from the results obtained from the C-terminal truncation series. First, three mutants (characterized by a nonphotosynthetic phenotype), PufX76*, PufX72* and PufX68* show no dimers of the isolated core complex, whereas from the PufX54* strain a fourth band, with different sedimentation characteristics on sucrose gradients, has been isolated. In the following we refer to this band, located in an intermediate position between the monomer (PMC3) and the dimer (PMC4), as PMC3/4. We propose three alternative interpretations: (a) PMC3/4 represents a dimeric form in which the LH1 rings assume a different curvature, leading to a different sedimentation coefficient; (b) PMC3/4 is formed by two LH1 rings that lost one or two reaction centers; (c) when the C-terminal part of PufX is deleted the equilibrium between the monomer and the dimer is not attained during sedimentation. The second interesting point is that PufX76*, PufX72* and PufX68* mutants are photosynthetically incompetent, whereas the PufX54* mutant grows photosynthetically, demonstrating that a complete removal of the C-terminus is tolerated by the cell, while a partial truncation is photosynthetically lethal. The absence of PufX in PufX76*, PufX72* and PufX68* (Fig. 5B) could in principle either reflect an impairment in the insertion into the membrane of the shortened protein and/or in the assembly of PufX in the LH1–RC, or resides at transcriptional/post-translational level. The PufX54* protein possesses only the N-terminus and the hydrophobic transmembrane helix, whereas the other mutants have in addition part of the C-terminus. We suggest that the presence of a partial C-terminus leads to a misfolding that impedes the insertion/assembly of PufX in the membrane complex. Parkes-Loach et al. [34] have recently reported that mature forms of PufX extracted from cells of Rb. sphaeroides and Rb. capsulatus contains 12 and nine fewer amino acids, respectively, at the C-terminal end of the protein than are encoded by their pufX genes. These data are inconsistent with our previous report [16], where a PufX with a C-terminal six-histidine tail has been used to determine the RC : PufX stoichiometry by Western blot analysis with anti-His6 antibodies. However the genetic background of the strains used is different: in our studies (present paper and [16]) both the LH2 and the LH1 antenna systems are present, while in the work of Parkes-Loach et al. an LH2–, LH1– strain and an LH2– strain from Rb. sphaeroides and from Rb. capsulatus, respectively, have been used to extract PufX. We can suppose that the discrepancy is related to the presence of the LH2 which could influence the shortening processes of the assembled PufX protein. The exchange of ubiquinone between the RC and the cyt bc1 in the presence of mutated PufX protein The role of the PufX protein in facilitating the ubiquinone/ ubiquinol exchange between the QB site of the RC and the ubiquinone pool has been demonstrated in Rb. sphaeroides wild-type strains [7,8]. It has been proposed that PufX facilitates ubiquinone exchange by determining the structural supramolecular organization of the LH1–PufX–RC complex [12]. In this work, PufX has been detected by SDS/PAGE in core complexes (Fig. 5) isolated from the N-terminus mutants PufXD2)4, PufXD2)7, PufXD2)19 and from the C-terminus mutants PufX54*, PufX81*. The evidence that these are the only mutants which are photosynthetically competent (see Table 2) is in accordance with previous results on the requirement of the PufX protein for photosynthetic growth and suggests that the assembly of the wild-type or mutated PufX protein in the core complex is necessary for efficient light energy transduction. In the other mutants examined, PufXD2)26, PufX68*, PufX72* and PufX76* no PufX protein could be detected on SDS/ PAGE after isolation of the complex. Assaying on ICM the reduction kinetics of the cytochrome b561 induced by a single actinic flash in the mutants PufXD2)4, PufXD2)7, PufX54* and PufX81*, we found a lag time between the flash excitation and the onset of cytochrome b561 reduction close to that observed in wildtype ICM. This is indicative of a fast ubiquinone exchange between the reaction center QB site and the cyt bc1 Qo site. In the case of the shortest N-terminal deletion and C-terminal truncation (PufXD2-4 and PufX81*, respectively) Ó FEBS 2002 1884 F. Francia et al. (Eur. J. Biochem. 269) this result was expected; in these two mutants a dimeric form of the core complex could be isolated. On the contrary, we obtained evidence of a less stable dimer in mutant PufXD2)7 and observed a band intermediate between that of the monomeric and the dimeric form (see above) in mutant PufX54*. As in these last two mutants a short lag was observed (see Table 2), apparently the presence of a stable dimer is not a necessary requisite for a fast RC/bc1 redox interaction, which is associated with a photosynthetic phenotype. As an alternative explanation the monomeric and the dimeric form of the LH1–PufX–RC could both be present in vivo; in the presence of an intact PufX the dimeric form would prevail, while altered equilibria arising from mutations on PufX could affect the stationary concentration of the dimer in the membranes. In the PufXD2)19 strain the dimeric form is even more destabilized, as no PMC4 can be isolated. Measurements of the lag in cytochrome b561 reduction in ICM from this mutant yielded values intermediate between those usually obtained in the wild-type and in the PufDX strain, with some variability between preparations from different cultures. Considering that the same amount of LH1–RC has been loaded in all lanes of the SDS/PAGE gel in Fig. 5A, the weaker intensity of the PufXD2)19 band (lane4) suggests that the amount of PufX per LH1–RC complex is lower in this mutant. Therefore it is likely that a mixture of monomeric LH1–RC with and without PufXD2)19 is isolated on the sucrose gradient. The occurrence of a mixed population of LH1–RC core complex in the ICM would explain the variability in the duration of the lag of cytochrome b561 reduction kinetics. The presence of the PufXD2)26 within the isolated core complex cannot be excluded in the SDS/PAGE shown in Fig. 5A, due to a possible overlapping with the a subunit of the LH1 complex. However a significant efficiency of PufXD2)26 insertion in the core complex seems unlikely, due to the nonphotosynthetic phenotype of this strain and to the pronunced lag in the cytochrome b561 reduction kinetics (see Table 2), systematically found in chromatophores from the pufX-deleted strain. Organization of the Q-cycle complexes The dimeric organization of the LH1–PufX–RC has been demonstrated directly in the membranes of Rb. sphaeroides by electron microscopy [14]. In this paper, the authors tentatively attribute a positive electrondense region in the two-dimensional projection of the dimer to cyt bc1 and interpret the S-shaped structure of the projection map as a supercomplex formed by the LH1–RC and cyt bc1 in a 2 : 1 stoichiometry. Some considerations on this point can be made in the light of our results. The presence in vitro of a less stable dimer in the mutant PufXD2)7 neither affects the photosynthetic capability of the bacteria nor the efficiency of exchange of the ubiquinol molecules between the RC and the bc1, as judged from the reduction kinetics of cytochrome b561 measured in ICM. Also the mutant PufD2)19, in which the dimeric form cannot be detected in the isolated core complex, exhibits a photosynthetic phenotype. In these two mutants, the photosynthetic phenotype suggests the presence in vivo of an open monomeric complex (or a prevalence of it with respect to the wild-type situation), consisting of an incomplete single LH1 ring containing one RC. The photosynthetic ability in PufX54* is consistent with the fast RC/bc1 ubiquinol exchange observed in ICM; on the other hand, the structural organization of the core complexes and/or the possible monomer–dimer equilibrium seem to be appreciably perturbed also in this mutant as judged from the different position of the PMC3/4 band (Fig. 4B) after isolation of the photosynthetic complexes on linear sucrose gradient. It is possible that the dimer form is not required as long as a reorganized core complex can efficiently shuttle quinones between the RC and the bc1 complex. The PMC3/4 isolated complex stimulates our interest, and further studies are in progress to understand the nature of the PufX54* mutant. In conclusion, our data indicate that both the N- and C-terminal portions of the PufX protein play a complex role in organizing the structure of the LH1–RC complex; the N-terminal region would be responsible mainly for the formation of a stable dimer, whereas the C-terminal portion would be involved mainly in PufX insertion/assembly. The transmembrane helix region of PufX appears to be sufficient to allow a fast quinone exchange between the core and the cytochrome b561 complex. Interestingly this conclusion fits well with the recent work of Parkes-Loach et al. [34], showing that the interaction between the hydrophobic PufX region and the LH1-a polypeptide has an inhibitory effect on the formation of the LH1 complex. This result suggests that the central core of the PufX protein is responsible of the break in the continuity of the LH1 ring in vivo [14], allowing a faster diffusion of the quinone molecules from/toward the RC QB site. ACKNOWLEDGEMENTS We thank B. A. Melandri and P. 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