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Photosynth Res (2012) 111:125–138 DOI 10.1007/s11120-011-9707-4 REGULAR PAPER Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity Kamil Woronowicz • Oluwatobi B. Olubanjo • Hee Chang Sung • Joana L. Lamptey • Robert A. Niederman Received: 27 June 2011 / Accepted: 10 August 2011 Ó Springer Science+Business Media B.V. 2011 Abstract In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100 W/m2 and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-a and -b polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24 h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in This is a re-publication of the original article in the September 2011 issue of this journal. When referring to this article in future publications, please cite the original article, as follows: Woronowicz K, Olubanjo OB, Sung HC, Lamptey JL, Niederman RA (2011) Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity. Photosynth Res108(2–3):201–214. doi: 10.1007/s11120-011-9681-x K. Woronowicz O. B. Olubanjo H. C. Sung J. L. Lamptey R. A. Niederman (&) Department of Molecular Biology and Biochemistry, Rutgers University, Busch Campus, 604 Allison Road, Piscataway, NJ 08854-8082, USA e-mail: [email protected] Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a [2.5-fold enrichment in Puc1B levels between 3 and 24 h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48 h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24 h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band. Keywords Light-harvesting complexes Light regulation puc operon Proteomics Rhodobacter sphaeroides Abbreviations AFM Atomic force microscopy b-OG n-octyl b-D-glucopyranoside DOC Deoxycholate CM Cytoplasmic membrane COGs Clusters of orthologous groups ICM Intracytoplasmic membrane LH Light harvesting LH1 Core pigment-protein light-harvesting complex LH2 Peripheral pigment-protein light-harvesting complex RC Photochemical reaction center UPB Upper pigmented band 123 126 Introduction The photosynthetic units of the facultative photoheterotroph Rhodobacter sphaeroides, a member of the a-3 subclass of Proteobacteria, contain two integral membrane light-harvesting complexes, designated as LH1 and LH2, which function as core and peripheral antennae, respectively. Radiant energy harvested by LH2 is transferred to LH1, which directs these excitations within the photosynthetic unit cores to the photochemical reaction center (RC), where the excitation energy is trapped as a stable transmembrane charge separation (Hunter et al. 2009). The apoproteins of both LH complexes consist of heterodimers of a- and bpolypeptides containing 50–60 amino acid residues (Theiler et al. 1984, 1985), which in LH1, bind two molecules of BChl (B875 BChl with Qy absorption band at 876 nm and two carotenoids (Broglie et al. 1980), and in LH2, two B850 BChls, one B800 BChl (with Qy absorption bands at 850 and 800 nm, respectively) and one to two carotenoids (Hunter et al. 1988). The atomic-resolution structure of the LH2 complex from the related bacterium Rhodoblastus acidophilus has been determined by X-ray crystallography (Papiz et al. 2003) and consists of a circular arrangement of 18 B850 BChls sandwiched between two concentric cylinders of transmembrane helices, with the outer and inner nonameric cylinders made up of b- and a-polypeptides, respectively. The B850 BChls form a ring strucutre with overlapping bacteriochlorin moieties in which non-covalent associations alternate with the a- and b-polypeptides, while nine discrete B800 BChls form an outer ring. A nine-membered ring of ab-heterodimers has also been suggested for the Rba sphaeroides LH2 complex, based on a 6-Å projection structure obtained from negatively stained 2-dimensional crystals (Walz et al. 1998) and AFM topographs of isolated membranes (Bahatyrova et al. 2004). In Rba. sphaeroides, the LH2 polypeptides are encoded by the pucBA structural genes of the puc operon (Kiley and Kaplan 1987) which contains an additional gene (pucC), immediately downstream of pucA. The pucC gene, which encodes a member of the major facilitator superfamily, is cotranscribed with pucBA, acting posttranscriptionally in the membrane assembly of the LH2-a and b apoproteins (Lee et al. 1989; Gibson et al. 1992). Upon the availability of the complete genome sequence of Rba. sphaeroides (Mackenzie et al. 2001), it was found that in addition to this previously described puc operon (subsequently designated as puc1BAC), which encodes the respective LH2-b (51 amino acid residues) and LH2-a (54 residues) apoproteins subunits, a second puc operon exists (designated as puc2BA) which is located outside the photosynthesis gene cluster (Zeng et al. 2003). Expression studies suggested that while the Puc2B polypeptide (51 residues, 94% 123 Photosynth Res (2012) 111:125–138 identity to Puc1B) enters into LH2 complex formation, neither the full-length 263-residue Puc2A gene product, nor its N-terminal 48-amino acid Puc1A homolog (58% identity), were present in LH2 complexes. It thus appeared that puc1A was the sole source of the a–polypeptide for the LH2 complex, whereas *30% of the LH2 complexes were found to contain the puc2B-encoded b-polypeptide. Major differences in the regulation of the two operons were also revealed in which puc1BAC was shown to be under more stringent regulatory control than puc2BA. In addition, it was demonstrated that the presence of LH2 was dependent upon puc1BAC, while the ultimate cellular level of LH2 was also dependent upon puc2BA. In this report, we describe a proteomic analysis of the levels of the various puc1BAC and puc2BA operon-encoded polypeptides in the LH2 complexes assembled in Rba. sphaeroides exposed to a lowering of both incident light intensity and oxygen tension. For the light regulation study, cells grown photoheterotrophically at high incident light intensity were shifted to low light, circumstances under which an extensive remodeling of the intracytoplasmic membrane (ICM) occurs. This process is characterized by preferential synthesis and assembly of the LH2 complex, which results in marked expansion of the absorption crosssection largely by the addition of LH2 to the RC-LH1 core complex-enriched ICM formed under high illumination levels (Woronowicz et al. 2011). In the second approach, ICM development was induced gratuitously at low oxygen tension in concentrated suspension of pigment-depleted, chemoheterotrophically grown cells (Niederman et al. 1976). Under these conditions, photosynthetic units are sequentially assembled, with RC-LH1 core structures inserted initially into the cytoplasmic membrane (CM) in a form that is largely inactive in forward electron transfer (Koblı́zek et al. 2005). This is followed by the activation of functional electron flow, together with the addition of LH2, which results in further invagination and vesicularization of the membrane to form the ICM. During each of these ICM developmental processes, LH2 is thought to pack initially between linear arrays of dimeric core complexes observed by atomic force microscopy (AFM) (Bahatyrova et al. 2004). When these regions become fully occupied, the LH2 complex may ultimately form a light-responsive peripheral antenna complement by clustering into LH2-only domains (Hunter et al. 2005). Intracytoplasmic membrane vesicle (chromatophore) preparations and an upper pigmented band (UPB) containing ICM growth initiation sites were isolated from cells undergoing acclimation to low light intensity and gratuitous induction of ICM formation after the lowering of oxygen tension. The isolated membrane preparations were subjected to non-denaturing polyacrylamide gel electrophoresis (clear Photosynth Res (2012) 111:125–138 native electrophoresis, CNE) which gives rise to four wellresolved pigmented bands (Woronowicz and Niederman 2010). The slowest migrating (top) and fastest migrating (bottom) bands contained spectrally homogeneous preparations of the native RC-LH1 and LH2 complexes, respectively, while associations of these core and peripheral complexes were present in the two bands of intermediate migration. Surprisingly, a detailed proteomic analysis of the LH2 gel bands of chromatophores from cells acclimating to reduced light intensity revealed distinct alterations in the levels of the LH2-a and -b polypeptides encoded by the two separate puc operons (Zeng et al. 2003). These results demonstrate that two puc operons respond differently to changes in illumination levels during photoheterotrophic growth. (A preliminary report of this work was presented at the Photosynthetic Light Harvesting Satellite Meeting of the 15th International Congress of Photosynthesis, Tianjin, China, 2010). Materials and methods Cell growth and membrane isolation Rba. sphaeroides wild-type strain NCIB 8253 was grown photoheterotrophically at 30°C in l-l Roux bottles, in the medium of Cohen-Bazire et al. (1956). Cells were initially grown at high light intensity (1,100 W/m2) in a water-cooled plastic aquarium illuminated by two opposed 150 W tungsten lamps controlled by a rheostat. Exponentially growing high light cells were transferred to a refrigerated incubator and at a reduced light intensity (100 W/m2) provided by 40-W tungsten bulbs. Light intensities were monitored with a radiometer (YSI-Kettering). High light cells were also transferred to indirect weak illumination (30 W/m2) as described by Woronowicz et al. (2011). In addition, aerobically grown batch cells were concentrated and subjected to gratuitous induction of ICM formation at low-aeration as described by Koblı̀zek et al. (2005). For membrane isolation, which was performed in 1 mM Tris, pH 7.5, at 4°C, cells were centrifuged at 12,0009g, washed and resuspended. A few crystals of DNAseI (Roche) and protease inhibitor cocktail (Roche) were added and the washed cells were passed twice through a French pressure cell. Debris and unbroken cells were removed by centrifugation at 16,0009g. The supernatant was layered onto a 5–35% (w/w) sucrose gradient prepared over a 60% sucrose cushion and subjected to rate-zone ultracentrifugation for 3 h at 28,000 rpm in Beckman SW28 rotor. Two bands were collected: the UPB, containing membrane growth initiation sites and the main pigmented band, consisting of chromatophores. 127 Clear native electrophoresis For the isolation of pigment-protein complexes, chromatophore samples were solubilized with 15 mM each of n-octyl b-D-glucopyranoside (b-OG) and deoxycholate (DOC), or with 2 g digitonin/g total protein, and applied to a 5–10% polyacrylamide clear native gel as described by Wittig et al. (2007). For clear native electrophoresis (CNE) of the UPB fraction, solubilization was performed with digitonin. Protein concentration was determined using the BSA protein assay (Thermo) and 312 lg of total protein was loaded onto each gel lane. Electrophoresis was performed in a Vertical Slab Unit Model SE-400 (Hoeffer Scientific Instruments) at a constant current of 10 mA, until current was no longer measurable (*6 h). Unstained gels were scanned using a Typhoon imager (GE Healthcare) set in the fluorescence mode with a blue laser excitation source (488 nm) and a 670-nm emission filter; a plastic transparency (Tektronix) was placed on top of the gel to provide uniform background fluorescence. Gels were also scanned with a Canon visible light scanner before and after staining with Coomassie-blue (Gel Code blue safe, Thermo). Up to four pigmented bands were identified and their pigmentprotein complex composition was determined from absorption spectra of the excised bands obtained with a DU-640 spectrophotometer (Beckman), using excised gel containing no sample as a blank. Proteomic analysis Gel bands resolved in CNE were excised and fixed for at least 30 min in a 40% methanol, 10% acetic acid solution and subjected to in-gel digestion with trypsin, followed by LC-MS/MS using a Dionex U-3000 nanoflow liquid chromatography system in line with a Thermo LTQ linear ion trap mass spectrometer, operated in the nanoLC mode to obtain sub-femtomol sensitivity. Database analysis of peaks generated from LTQ data was performed as described by Sleat et al. (2009) in which tandem mass spectrometry data files were searched against the NCBI assembly of the annotated Rba. sphaeroides 2.4.1 genome (Mackenzie et al. 2001). The data were quantified by spectral counting, which measures protein abundance on the basis of the number of tandem mass spectral observations for all constituent peptides (Liu et al. 2004). Spectral count sampling statistics have been shown to have high technical reproducibility and can be successfully applied to relative protein abundance and differential protein expression studies in mass label-free LC-MS/MS (Zeng et al. 2007; D’Amici et al. 2010). 123 128 Results Membrane isolation from cells acclimating to low-intensity illumination and separation of integral pigment–protein complexes by clear native electrophoresis Figure 1 shows the near-IR absorption spectra of cell extracts and membrane fractions isolated after 0, 3, and 24 h of acclimation from high (1,100 W/m2) to low intensity illumination (100 W/m2). Only low levels of LH2 were present (LH2/LH1 molar ratio = 0.39) in the initial exponential-phase high light cells (time = 0), with the predominance of the RC-LH1 core complex indicated by the LH1 absorption maximum at 875 nm and the presence of a small band appearing at *750 nm, especially notable in the isolated 0 h UPB membrane fraction (see also Fig. 2b), attributable to the RC bacteriopheophytin component. LH2/LH1 was initially 2.2-fold lower in the UPB relative to chromatophores and was maintained at 1.7- and 1.5-fold lower levels after 3 and 24 h, respectively, consistent with a role for these CM invagination sites as hotspots for initial assembly of LH1-RC cores and thereafter as LH2 assembly sites prior to ICM vesicularization. The gradual increase in molar ratios of LH2/LH1 resulted in a 4.3-fold higher level after 24 h in chromatophores, as compared to 6.1-fold in the UPB. Overall, these dramatic increases in LH2 levels demonstrated that the essential membrane fractions can be isolated during a light-intensity downshift to critically test the proposed mode for expansion of the functional absorption cross-section and remodeling of the LH apparatus during acclimation to lowered light intensities (Hunter et al. 2005). In order to assess the assembly of the LH2 and RC-LH1 complexes within the developing ICM, a non-denaturing electrophoresis procedure (CNE) was established for their isolation from the purified membranes (Fig. 2). This method was adapted from the high-resolution CNE procedure of Wittig et al. (2007) for direct in-gel functional and fluorescence assays of well-separated mitochondrial inner membrane protein complexes. Coomassie blue, which interferes with catalytic activity and fluorescence determinations in blue-native electrophoresis (Schägger and von Jagow 1991), was replaced with clear mixtures of anionic and neutral detergents in the cathode buffer. Membrane protein solubility during electrophoresis is enhanced in this manner, and like the Coomassie dye, a charge shift is imposed to augment their anodic migration. Protein aggregation and band broadening are prevented, resulting in superior resolution, while facilitating in-gel functional assays. In the current studies, migration of the pigment-protein complexes can be directly visualized, and their absorption spectra can be determined in the gel slices containing the resolved bands. 123 Photosynth Res (2012) 111:125–138 Figure 2a shows that after b-OG/DOC solubilization of chromatophores isolated from the adapting cells, four pigmented bands were resolved: a top RC-LH1 band of near spectral homogeneity (Fig. 2b); a bottom, spectrally homogeneous LH2 band (Fig. 2c); and two bands of intermediate migration containing mixtures of RC-LH1 and LH2. The upper intermediate complex is enriched in LH2 (designated LH2–LH1), while the lower one is enriched in RC-LH1 (designated LH1–LH2). These intermediate complexes may represent detergent-protein micelles arising from ICM regions seen in AFM where the LH2 and LH1 complexes are associated (Woronowicz and Niederman, 2010) (see below for further evidence of authentic LH2–LH1 associations). Relative increases in the intensity of the two LH2-enriched bands can be seen as the low-light acclimation process proceeds (Fig. 2a). It can be seen in Fig. 2d that the bottom LH2 band is free of contamination by RC and LH1 polypeptides, essentially confirming the absorption spectrum. Importantly, this clear native gel procedure has provided the basis for a critical proteomic analysis of the assembly of the pigment-protein complexes under these conditions. Protein composition of the LH2 CNE bands After the separation of intact pigment-protein bands by CNE, they were excised from the gel and subjected to direct in-gel trypsin digestion followed by LC-MS/MS as described in ‘‘Materials and methods’’ section. It should be emphasized that data obtained here by proteomic analysis in the absence of mass isotope labeling, only reflect the ability of trypsin to act at potential cleavage sites and are therefore semiquantitative in nature. This means that proteins that are not highly abundant with a large number of accessible cleavage sites give rise to higher spectral counts than more abundant proteins with far fewer sites; in extreme cases where no sites are available, no counts are found in the resulting pivot table, viz., pufB (LH1-b polypeptide) and Puc1A (LH2-a); however, the latter polypeptide could be detected after chymotrypsin cleavage (see below). Nevertheless, valid comparisons can be made of the same protein between different gel lanes, which were loaded with equal amounts of total protein, thereby providing valid data for protein expression studies of the clusters of orthologous groups as described here (Fig. 3). Differences in the number of trypsin cleavage sites and their accessibility explain the lack of congruence between the apparent absence of cross contamination of the LH2 gel band by RC-LH1 core complexes as observed in the nearIR absorption spectrum (Fig. 2c) and the presence of 7% of total spectral counts in this orthologous group cluster as demonstrated for the 3-h LH2 bands in Fig. 3. Accordingly, the total counts for all four CNE bands analyzed in Photosynth Res (2012) 111:125–138 A Crude Extract Upper Pigmented Band 0h Absorbance Fig. 1 a Near-IR absorption spectra of the crude cell extracts, UPB, and chromatophore fractions after isolation by rate-zone sedimentation on sucrose density gradients from cells undergoing acclimation to low light intensity. Crude extracts represent the supernatant material obtained from cell lysates after French-press treatment and centrifugation at 16,0009g. b LH2/LH1 molar ratios calculated after spectral deconvolution as described by Sturgis et al. (1988) 129 Chromatophores LH1 (B875) 800 875 850 800 875 850 3h LH2 (B800-850) 24 h 0.3 A 800 750 850 950 750 850 950 750 875 850 850 950 Wavelength, nm B 2 UPB LH2/LH1, mol/mol Chromatophores 1.5 Crude 1 0.5 0 0h this experiment were 3,224 for the RC-LH1-PufX core complex and 1,138 for the LH2 complex, giving at least a 2.4-fold bias for the cores, which are present at near equimolar levels with LH2 as judged from near-IR absorption of the 3-h chromatophores (Fig. 1). This would reduce the actual contamination by core complex protein to \3.0%, thereby accounting for the apparent lack of an obvious LH1 contribution to the absorption spectrum of the 3-h LH2 band (Fig. 2c). An analysis of the photosynthetic membrane proteome of Rba. sphaeroides 2.4.1 by Zeng et al. (2007) identified 98 3h 24 h proteins associated with ICM vesicles; among these, 70 were enriched relative to the outer membrane and soluble fractions. In a nondenaturing clear gel electrophoresis study of an n-dodecyl-b-D-maltoside solubilized ICM fraction purified from the carotenoidless Rba. sphaeroides strain 26.1 (D’Amici et al. 2010), proteomic analysis resulted in the identification of 52 proteins arising from 10 different bands containing multiprotein complexes. Among these were LH2 dimers, trimers, and high molecular mass supercomplexes, RC-LH1-PufX monomers and dimers, succinate dehydrogenase trimers and monomeric forms of ATP synthase, and 123 130 Photosynth Res (2012) 111:125–138 Fig. 2 Separation of intact pigment-protein complexes by CNE of c b-OG/DOC solubilized chromatophores isolated from cells undergoing acclimation to low-light intensity. a Left panel Typhoon fluorescence scanner (GE Healthcare) profile of unstained gel observed against a plastic film providing uniform background emission at 670-nm; pigments absorbing at the excitation wavelength (488 nm) appear as white images, defining the positions of the original pigmented gel bands. Note marked increases in the density of the bottom (LH2) band and upper intermediate (LH2–LH1 band) as a function of acclimation time. Right panel Scan of a typical unstained gel lane obtained with a visible light scanner (Canon). Approx. molecular masses of the pigmented bands are shown at right, obtained from plot of Rf versus Mr for native electrophoresis high molecular weight calibration kit (GE Healthcare) as described in Table 1. b, c Absorption spectra determined directly on indicated gel slices. In the top (RC-LH1) band, note the presence of the RC bacteriopheophytin absorption band at *750 nm and the RC monomeric BChl band at 803 nm. The blue shift of the LH1 B875 absorption band to 872 nm and the shoulder on the blue side indicate some contamination with LH2 (LH2:LH1 molar ratio = 0.21 determined as described by Sturgis et al. 1988); however, it has also been possible to isolate the RC-LH1 band in a spectrally homogeneous form (Woronowicz and Niederman 2010). Note the high level of spectral purity of the LH2 complex in the bottom band (maxima at 849 and 799 nm; LH2:LH1 molar ratio = [300:1). Comparison of this spectrum with that of the 24-h crude cell extract (Fig. 1a) indicates that during the isolation procedures, some diminution in the absorbance of the labile B800 band has occurred. Also noteworthy are the differences in carotenoid contents of the complexes, reflecting the BChl:carotenoid molar ratios near 1.0 reported for LH1 and *2.0 for LH2, as well as the blueshift in the position of the redmost vibrational splitting of LH1-bound carotenoid absorption (the S2(1Bu?) state of spheroidene) in comparison to that of LH2, with maxima at *507 and 510 nm, respectively (Hunter et al. 1988). From near-IR absorption spectra of the upper and lower intermediate bands, ratios LH2:LH1 ratios of 2.39–2.77 and 0.63–0.76, respectively, were obtained. d Portion of silver-stained gel of CNE bottom bands subjected directly to SDSpolyacrylamide gel electrophoresis, showing an absence of the RCLH1 apoproteins, essentially confirming the observed absorption spectra. The band above the LH2-a band apparently represents the 11.4-kDa hypothetical protein RSP6124; levels of RSP6124 were correlated with LH2 levels after purification of the chromatophore band by two-phase partitioning, accounting for 5% of clusters of orthologous groups (COGs) at 3 h and 12% at 24 h (Woronowicz and one of these (RSP1760) demonstrated that the RSP1760 gene Niederman 2010) the cytochrome bc1 complex. In both of these studies, a considerable number of proteins of unknown function were encountered, and an in-frame deletion of the gene encoding product is essential for both photosynthetic growth and ICM formation (Zeng et al. 2007). This has served to illustrate that these proteomic strategies show considerable promise for identification of new ICM proteins with unique functions in maintaining and optimizing photosynthesis. Table 1 Apparent aggregation states of pigment-protein complexes as estimated from electrophoretic migration in clear native electrophoresis Band Designation Apparent Mr (910-3)a Aggregation stateb Aggregate identity Top Upper intermed. RC-LH1 LH2–LH1 748 600 2.31 1.02 [RC-LH1-PufX]2 [RC-LH1-PufX][LH2]2 Lower intermed. LH1–LH2 463 1.02 [RC-LH1-PufX][LH2] Bottom LH2 375 2.85 [LH2]3 a Obtained from an exponentially fitted plot of Rf versus Mr for native electrophoresis high molecular maker proteins run in a separate gel lane (molecular masses of 669, 440, 232, 140 and 67 kDa) b Calculated using monomer molecular weights based on polypeptide sequences and pigment contents. For RC-LH1-PufX, molecular mass of 324.0 kDa included RC Fe and two ubiquinone molecules, the RC-H, -M, and -L polypeptides (101.8 kDa total/monomeric RC), 14 LH1-a and -b subunits (molecular mass = 213.1 kDa) and PufX (9.1 kDa). For LH2, the molecular mass of 131.4 included nine LH2-a and -b subunits 123 Photosynth Res (2012) 111:125–138 131 Fig. 3 Proteomic analysis of CNE bottom (LH2) band of chromatophores isolated from cells undergoing acclimation to low light intensity. Distribution of spectral counts after in-gel trypsin digestion of chromatophore LH2 band isolated from cell samples taken 3 and 24 h after the light intensity downshift. The distributions shown are for COGs (Tatusov et al. 1997), in which the usual energy production and conversion category has been divided into subgroups to account for the distinct metabolic capabilities unique to photoheterotrophically grown Rba. sphaeroides In this study, the LH2 band of chromatophores isolated from the ICM undergoing acclimation from high to low intensity illumination (Fig. 3), showed a nearly threefold increase in the relative spectral counts attributable to LH2 between 3 and 24 h of acclimation, while RC-LH1 remained at 7% of the total counts obtained for the clusters of orthologous groups. In addition, a large array of comigrating proteins was revealed and in particular, the cytochrome bc1 complex and several F1FO-ATP synthase subunits gave unexpectedly high counts, given the inability to detect these components in AFM (Bahatyrova et al. 2004, Sturgis et al. 2009). Significant levels of general membrane assembly factors were also encountered, as well as numerous hypothetical proteins of unknown function, including high spectral counts for the RSP6124 that were maintained throughout the acclimation process. It is also noteworthy that a comparison of the 3- and 24-h LH2 bands showed a twofold reduction in spectral counts attributable to transport proteins, while those of the cytochrome bc1 complex increased by threefold. Thus, as expected, a higher level of these permease components, which serve as CM markers, are present in the vesicular ICM early during lowlight acclimation and are diluted out by LH and RC protein as membrane remodeling proceeds. On the other hand, cytochrome bc1 components catalyzing light-driven cyclic electron flow become preferentially localized in the remodeled vesicular ICM during the acclimation process, conditions under which the levels of the electron contributing RC-LH1 complex undergoes a [9-fold increase. Proteomic analysis reveals distinct light regulation of the puc1BAC and Puc2BA operons In addition to the puc1BAC operon, which encodes the respective LH2-b (51 amino acid residues) and LH2-a apoproteins (54 residues), as well as the PucC polypeptide involved in LH2 assembly, the Rba. sphaeroides genome contains a second puc operon, designated as puc2BA (Zeng 123 132 et al. 2003). These investigators also reported that while the LH2-b polypeptide encoded by the puc2B structural gene was assembled into LH2 complexes, neither the putative fulllength 263-residue Puc2A gene product, nor its N-terminal 48-amino acid Puc1A homolog, appeared in assembled LH2 complexes. In contrast, the present proteomics data demonstrate that the Puc2A polypeptide is actually a major component of LH2-containing complexes isolated in CNE (Fig. 4). Moreover, this LH2-a component was the second most abundant protein among the [170 proteins that gave rise to significant spectral counts after trypsin digestion of the CNE gel bands, and significant levels were also encountered in LH2 bands digested with chymotrypsin (Fig. 4a). Proteomic analysis of the LH2 CNE band showed a predominance of the Puc2A polypeptide in LH2 complexes formed at high light intensity, as reflected in chromatophores isolated at the outset (0 h) and 3 h into the shift from high to low light (Fig. 4a), which is followed by an enrichment in Puc1B levels after 24 h. As seen in the relative distribution of spectral counts (Fig. 4a, bottom panel), this amounted to [2.5-fold increase in Puc1B spectral count levels between 3 and 24 h that was accompanied by a nearly 1.8-fold decline in those of Puc2A. These data provide support for the suggestion by Zeng et al. (2003) that the puc1BAC operon is under more stringent light control than the puc2BA operon, thought to reflect differences in the puc2 upstream regulatory region. Unlike trypsin, chymotrypsin digestion resulted in cleavage of Puc1A and also revealed high levels of both Puc1A and Puc1B along with lower levels of Puc2A in the 24-h chromatophore LH2 band (Puc1AB/Puc2A = 5.6) (Fig. 4a). Note also that the presence of some Puc1B in the 3-h chromatophore preparation, which is apparently due to the reported dependence of puc2BA expression on the expression of all three puc1BAC genes (Zeng et al. 2003). Also noteworthy are the similarities in the ratios of the Puc1/Puc2 polypeptides for the 24-h UPB and chromatophore bands (1.6 and 1.1 for trypsin digestion and 5.0 and 5.6 for chymotrypsin, respectively), showing that LH2 assembly occurs in both of the distinct inner membrane domains that give rise to these membrane fractions. This is also supported by the similarities in the increases in molar ratios of LH2/LH1 after 24 h (6.1- and 4.3-fold in the UPB and chromatophores, respectively) (Fig. 1). Figure 4b demonstrates that during ICM induction under semiaerobic conditions, or during acclimation from high-intensity illumination to indirect diffuse light, no differential synthesis of Puc1A and Puc1B polypeptides has apparently occurred (Puc1B/Puc2AB ratios were 0.48 after 48 h of induction and 0.31 after 11 days of acclimation to diffuse light, respectively). This indicates that light regulation of the pucAB operons is confined to rapidly dividing cells acclimating to physiologically relevant downshifts in light intensity. 123 Photosynth Res (2012) 111:125–138 Fig. 4 Proteomic analysis of LH2 polypeptides in CNE bands of c membrane fractions isolated from cells undergoing ICM development. Top panels in Figs. 4a–c show overall spectral count distributions, while bottom panels show % distribution of spectral counts. a Low-light acclimation profiles of LH2 polypeptides in bottom (LH2) band resolved from chromatophores and UPB. Because of the limited spectral counts in the 0-h chromatophores, it was not possible to calculate their % distribution. Note the 6.0-fold increase in spectral counts of Puc1B between 3 and 24 h as compared to the only 1.5-fold increase for Puc2A. In addition to revealing the presence of all four LH2 polypeptides encoded by the puc1BAC and pucBA operons through digestion with both trypsin and chymotrypsin, trypsin digestion of the 24-h UPB LH2 gel band yielded a single peptide sequence arising from the putative Puc1C gene product, involved in the assembly of LH2 polypeptides (not shown). b LH2 polypeptide profiles in bottom (LH2) band resolved from chromatophores isolated during ICM induction at low aeration and during acclimation from high-intensity illumination to indirect diffuse light as described by Woronowicz et al. (2011). c Lowlight acclimation profiles of LH2 polypeptides in CNE intermediate bands resolved from chromatophores Figure 4c shows the Puc1/Puc2 ratios arising from LC/MSMS of the CNE bands of intermediate migration, after digestion with trypsin. The Puc1B/Puc2AB for the LH2enriched LH2–LH1 upper intermediate band was virtually unchanged between 3 and 24 h following the light-intensity downshift (0.13 and 0.17, respectively), despite a [10-fold increase in LH2 cellular levels over this time period. These results have considerable bearing on the interpretation of the origins of the LH2-containing complexes isolated by CNE (Fig. 2), insofar as these essentially unchanged LH2 polypeptide ratios suggests that the LH2–LH1 band arises from regions of LH1–LH2 associations, thought to be laid down mainly during high light growth. These appear in AFM topographs as rows of aligned dimeric RC-LH1 core complexes interspersed with narrow rows of LH2 (Fig. 5a) (Bahatyrova et al. 2004; Hunter et al. 2005). On the other hand, the origin of the bottom LH2 band with its increased levels of new Puc1BA polypeptides at 24 h (Fig. 4a) can be attributed largely to the LH2-only domains that arise when LH2–LH1 associations are complete (Fig. 5b), which represent the light-responsive LH2 complement formed as the cells adapt to low intensity illumination. In contrast, for the LH1-enriched LH1-LH2 lower intermediate band (Fig. 4c), the Puc1B/Puc2AB ratio increased from 0.21 at 3 h to 0.58 at 24 h, suggesting that this CNE band arises in part from regions of RC-LH1 core-LH2 interactions at the outer RC-LH1 edges (Fig. 5a). Discussion Multigene families of puc operons The existence of a second operon encoding LH2 polypeptides in Rba. sphaeroides was not surprising in view of the presence of multiple puc operons in a variety of other purple bacteria, including Rhodopseudomonas palustris in which Photosynth Res (2012) 111:125–138 133 B Additional ICM induction protocols A Low light adaptation Bottom band – LH2 160 Puc1A – LH2- 150 Puc2A – LH2- 125 Puc1B – LH2Puc2B – LH2- 100 Chymotrypsin 75 50 25 0 80 Low O 2 induction Puc2A – LH2- + C-term. ext. Puc1B – LH2- 140 + C-term. extension Spectral counts Spectral counts 175 Puc2B – LH2- 120 100 Diffuse light 80 60 40 20 0h 3h 24 h Chromatophores 24 h 24 h UPB Chrom 0 24 h UPB Bottom band – LH2 60 24 h 48 h Chromatophores 11 d Bottom band – LH2 50 60 Spectral counts (%) Spectral counts (%) 70 50 40 30 20 10 0 0h 3h 24 h Chromatophores 24 h 24 h UPB Chrom 24 h UPB 40 30 20 10 0 24 h 48 h Chromatophores 11 d C Low light adaptation -- Chromatophores 150 LH2-LH1 Puc2A – LH2- Spectral counts 125 + C-terminal extension Puc1B – LH2Puc2B – LH2- 100 LH1-LH2 75 50 25 0 3h 24 h 0h 3h 24 h Low light adaptation -- Chromatophores Spectral counts (%) 100 LH2-LH1 LH1-LH2 75 50 25 0 3h 24 h 0h 3h 24 h 123 134 Photosynth Res (2012) 111:125–138 LH2 ring A LH2-LH1 band 50 nm B 25 nm C --RC-LH1 [RC-LH1-PufX] 2 --LH2-LH1 [RCLH1-PufX][LH2] 2 RC-LH1 band --LH1-LH2 [RCLH1-PufX][LH2] LH2-only domain LH1-LH2 band --LH2 [LH2] 3 LH2 band LH2 ring Fig. 5 Model for proposed origins of CNE gel bands. a Local arrangement of photosynthetic complexes in an ICM patch showing highly ordered linear arrays of the RC-LH1 core complex with LH2 rows interspersed between (Bahatyrova et al. 2004). Existing structural data were used to model AFM topograph of cytoplasmic surfaces of ICM vesicle patch. A dimeric RC-LH1 complex ([RCLH1-PufX]2) is outlined in red, which is proposed to be the source of the RC-LH1 band. The LH2–LH1 (LH2-enriched upper intermediate) band ([RC-LH1-PufX][LH2]2), outlined in orange, is shown arising from a monomeric RC-LH1 complex in association with two LH2 rings, while the LH1–LH2 (RC-LH1-enriched upper intermediate) band ([RC-LH1-PufX][LH2]), outlined in green, is shown arising from a monomeric RC-LH1 complex in association with a single LH2 ring. b Tapping mode AFM topograph showing cytoplasmic surface of ICM vesicle patch adsorbed onto mica in a liquid cell (Bahatyrova et al. 2004; Sturgis et al. 2009). The blue circle outlines an LH2-only domain from which the LH2 band is thought to arise in an [LH2]3 aggregation state. c Scan of 24-h unstained gel lane. The molecular masses for the gel bands shown in Fig. 2 support the aggregation states of each of the four bands as depicted here structural genes are present for encoding five ab-apoprotein pairs (Tadros and Waterkamp 1989; Tadros et al. 1993; Gall and Robert 1999), Rbl. acidophilus, capable of encoding at least four different ab-apoproteins (Gardiner et al. 1996) and Phaeospirillum molischianum in which evidence for multiple puc operons has also been reported (Mascle-Allemand et al. 2010). In these organisms, the various puc structural genes are expressed differentially in response to changes in light intensity, which in Rps. palustris is manifested in the formation of a nonameric B800-850 LH2 complex at high light intensity and an octameric 800-nm absorbing LH4 complex under low light (Evans et al. 1990; Scheuring et al. 2006). The peripheral LH4 complex (Hartigan et al. 2002) which is regulated by a specific phytochrome (Evans et al. 2005; Giraud et al. 2005), is present under high-intensity illumination at approximately the same level as the nonameric LH2 complex, while a 10-fold increase in the LH4 level relative to LH2 is found under low light. Recently, evidence obtained by single molecule spectroscopy by Brotosudarmo et al. (2009) has suggested that a B820 band is also present in the purified low-light peripheral antenna protein of Rps. palustris. These individual low-light antenna complexes arise as a result of a heterogeneous polypeptide composition provided by the multigene family of puc operons, resulting from the assembly of LH2 rings from polypeptides encoded from the distinct genes of the different operons (Tharia et al. 1999). Moulisová et al. (2009) explained the splitting of long-wavelength exciton bands by a heterogeneous composition of LH2 apoproteins that provides some of the BChls in the B850 ring with B820-like site energies, accounting for the disorder in the LH2 structure, as reflected in a strong inhomogeneity of B850 excitons in the low-light samples. In Rbl. acidophilus, besides the structurally characterized LH2 complex (Papiz et al. 2003), an additional peripheral antenna with absorption bands at 800 and 820 nm (LH3) is formed when grown under low-light conditions (Bissig et al. 1988; Gardiner et al. 1993, McLuskey et al. 2001). Using Psh. molischianum, Mascle-Allemand et al. (2010) showed that octameric LH2 rings (bands at 802 and 849 nm) (Koepke et al. 1996) form the predominant pigment-protein complex in the normal-light membranes, while in cells adapting to low-light, an LH3 complex clearly predominated, ultimately replacing LH2. Mascle-Allemand et al. (2010) also demonstrated antenna mixing in Psh. molischianum, in which this modulation can result in a complete shift between the production of octameric LH2 complexes absorbing maximally near 850 nm to an LH3-type complex with a long-wavelength absorption band near 820 nm. The ab-apoproteins making up the individual LH2 rings contained polypeptides that were derived from different operons, demonstrating a lack of strict coupling of polypeptide synthesis, cofactor addition and insertion into the membrane. Since the LH3-type complex serves to increase the spectral range of solar energy capture, polypeptide chain mixing could be important in maintaining photosynthetic efficiency during acclimation to low light intensity. 123 Possible distinct roles for Puc1 and Puc2 LH2 complexes It is tempting to speculate on a physiological role for the low-light intensity responsive differential synthesis of Puc Photosynth Res (2012) 111:125–138 polypeptides in Rba. sphaeroides, in which a predominantly Puc2 LH2 complex would function at high light intensity and a predominantly Puc1 LH2 complex would be required for optimal photon collection and excitation energy transfer under low intensity illumination. However, this awaits proteomic analysis of a functional, highly purified LH2 protein preparation isolated from high light cells, since the Puc2A peptide detected here arose from the 215 amino acid residue C-terminal extension. This large protein fragment does not contain any apparent membrane spanning regions, and it is possible that it is not part of the functional complex and instead, has arisen from in vivo enzymatic cleavage, representing an adventitious attachment to the CNE LH2 band. The recent study of Wang et al. (2009), however, has demonstrated that when the puc2BA genes were amplified and cloned into an appropriate plasmid vector under control of the puc promoter, both the Puc2A and Puc2B polypeptides are expressed normally. The puc2BA-encoded polypeptides were assembled into a membrane-associated Puc2BA LH2 complex in which a large puc2A-encoded polypeptide was present. The puc2BA genes were also expressed when integrated into the Rba. sphaeroides genome and Puc1C was necessary for synthesis of LH2 complexes from puc2BA. The resulting LH2 was spectroscopically distinct from the Puc1BA LH2 with a blue shifted B850 absorption band at 846 nm but the functional activity of the Puc2BA LH2 complex, especially under high light intensity, remains to be determined. Nevertheless, these results make it likely that in the present study, a Puc2BA complex is indeed formed in our wild-type strain and that it is this authentic complex that predominates under high light intensity. It is noteworthy that in the Puc2A-encoded polypeptide, the essential conserved Tyr residues (Tyr44 and Tyr45) are present in the conserved 48-residue N-terminal region. These residues have been shown to be involved in binding the 2-acetyl carbonyl groups of the B850 BChls (Fowler et al. 1992), suggesting that the Puc2BA complex can be assembled in a fully functional state. In contrast to this study and the report of Wang et al. (2009), Zeng et al. (2003) reported that the Puc2A-encoded polypeptide was not found in assembled LH2 complexes. For these studies, the PucA and PucB polypeptides were tagged by constructing PhoA fusion proteins and expressed in a puc1BAC deletion strain, which did not produce LH2 complexes. Fusion proteins containing full-length PucB and PucA polypeptides were formed, indicating that Puc2A had not undergone processing. While expression of puc2BA did not require the puc1BAC operon, and Puc2B and Puc2A passed through the membrane in an apparently normal manner, immunoprecipitation with an anti-PhoA antibody showed that in a puc1 operon background, LH2 spectral complex were formed with the chimeric 135 polypeptides containing Puc2B and the N-terminal 48 amino acid puc1A orthologue encoded by puc2A, but not in the case of the chimera with the full length Puc2A polypeptide. Moreover, the LH2 complex was not assembled when the C-terminal Puc2A extension was added to Puc1A, suggesting that it must be removed for LH2-a assembly. The differences between these results and those of Wang et al. (2009) in which Puc2B and the full length puc2A-encoded polypeptides were assembled into a membrane-associated LH2 complex are presently not understood. Regulation of Puc polypeptide expression in Rba. sphaeroides In the initial puc operon expression studies (Kiley et al. 1988: Lee et al. 1989), 0.5- and 2.3-kb puc-specific transcripts were detected with respective abundance levels of up to 25:1. Both of these transcripts started at the same 50 end, 117 nucleotides upstream of the pucB start codon and their expression was regulated by oxygen tension and light intensity. After deletion of the puc1BA genes (Lee et al. 1989), a highly homologous 1.1- to 1.3-kb transcript appeared. In this pucBA- strain, no LH2 complexes were assembled from the puc2B and puc2A transcripts that were formed, which can now be explained by the requirement for Puc1C in the assembly of the Puc2BA polypeptides (Zeng et al. 2003). The mRNA from the puc2B start codon to the termination puc2A codon has a length of *963 bp, substantially longer than the corresponding length of the puc1BA mRNA (337 bp), accounting for the size of the 1.1- to 1.3-kb transcript detected by using a homologous puc1BA probe. Putative regulatory regions upstream of the puc2B gene include two PpsR binding motifs located at positions similar to the same sequence motifs upstream of puc1B. In addition, an FnrL binding sequence was located upstream in a unique region closer to the puc2B start codon than for puc1B (Zeng et al. 2003). This has suggested that like the puc1 operon, expression of the puc2 operon is under regulatory control by both PpsR and FnrL. PpsR forms part of the AppAPspR antirepressor-repressor system in which AppA integrates both redox and light signals and serves as a blue-light photoreceptor, in addition to functioning as a redox sensor of the quinone pool redox state (Masuda and Bauer 2002; Braatsch et al. 2002). FnrL is a positive, global anaerobic regulator required for enhanced expression of puc and selective structural genes encoding oxidoreductases and enzymes of pigment biosynthesis (Zeng and Kaplan 2001). It was suggested by Zeng et al. (2003) that the difference in location of the FnrL sequence upstream of puc1BAC may be responsible for the more 123 136 stringent light control of this operon than that seen for the puc2BA operon. Puc polypeptide composition of LH2-containing CNE gel bands supports structural model for their origin in native ICM Prompted by the elucidation of the supramolecular surface arrangement of ICM vesicles by AFM (Bahatyrova et al. 2004) together with membrane fractionation and radiolabeling studies of BChl insertion into developing pigmentprotein complexes, Hunter et al. (2005) have proposed that during acclimation to lowered light intensity, new LH2 rings initially cluster between expanding linear arrays of dimeric LH1-RC core complexes at membrane invagination sites (Fig. 5a). As the membrane matures further and these regions become fully occupied with rows of LH2 rings, ICM domains consisting exclusively of LH2 are formed by the packing of newly synthesized complexes into membrane regions that represent the light-responsive peripheral antenna complement (Fig. 5b). For the isolation of the developing pigment-protein complexes by Hunter et al. (2005), the isolated UPB and chromatophore membranes were fractionated into their constituent LH2, LH1, and RC components by solubilization in lithium dodecyl sulfate at 4°C in the dark, followed by polyacrylamide gel electrophoresis at this temperature (Broglie et al. 1980). This procedure was sufficiently gentle to preserve regions of contact between LH2 and LH1, giving rise to series complexes of intermediate migration, containing variable LH2:LH1 stoichiometries. In contrast, clear native electrophoresis as described here affords a less complex native electrophoresis procedure to assess membrane regions of putative LH2/LH1 associations, insofar as only two intermediate complexes appear, an upper one enriched in LH2 and lower LH1-enriched one. Accordingly, from the relative distribution of Puc1- and Puc2-encoded LH2-a and -b polypeptides during acclimation to reduced light intensity, it is possible to make a more explicit interpretation of the membrane origins of the CNE gel bands. Because the Puc2AB/Puc1B polypeptide ratio was largely unchanged in the LH2-enriched LH2– LH1 upper intermediate band during the light intensity downshift (Fig. 4c), it is proposed that this band arises from early LH2–LH1 association represented by the narrow rows of LH2 rings between the linear arrays of dimeric LH1-RC core complexes (Fig. 5a). Since these are thought to represent the first regions to be assembled, they do not subsequently exchange with the new LH2 complexes formed during the remainder of the membrane remodeling process. On the other hand, for the LH1-enriched LH1– LH2 lower intermediate band, the nearly three-fold increase in the Puc1B/Puc2AB ratio (Fig. 4c) suggests that 123 Photosynth Res (2012) 111:125–138 this band arises from isolated regions of dimeric RC-LH1 core-LH2 associations at the outer edges of the rows of dimeric RC-LH1 core complexes (Fig. 5a). Finally, suggested origins for the RC-LH1 top and the bottom LH2 bands are also presented in Fig. 5b. 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