Oligomeric Characterization of the Photosynthetic Apparatus of

Nov 9, 2009 - Synopsis. Since membrane proteins tend to self-associate into noncovalent multimers due to their hydrophobic nature, the understanding o...
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Oligomeric Characterization of the Photosynthetic Apparatus of Rhodobacter sphaeroides R26.1 by Nondenaturing Electrophoresis Methods Gian Maria D’Amici,† Sara Rinalducci,† Leonardo Murgiano,† Francesca Italiano,‡ and Lello Zolla*,† Department of Environmental Sciences, Tuscia University, Viterbo, Italy, and Institute for Chemico-Physical Processes, National Research Council (CNR), Bari, Italy Received June 9, 2009

Blue and colorless native gel electrophoresis in combination with LC-ESI-MS/MS are powerful tools in the analysis of protein networks in biological membranes. We used these techniques in the present study to generate a comprehensive overview on a proteome-wide scale of intracytoplasmic membrane (ICM) associated proteins in order to investigate the native supramolecular organization of Rhodobacter sphaeroides R26.1 photosynthetic apparatus. The results obtained were compared with past proteomic data, as well as with models for the topology of photosynthetic membranes as derived from previously published atomic force microscopy studies. We identified 52 proteins organized in 10 different multiprotein complexes. We were able to demonstrate the existence of different oligomeric states for the integral membrane pigment-protein complexes dedicated to bacterial photosynthesis. Specifically, we found dimers and trimers, as well as supercomplexes of light-harvesting (LH) 2 at very high molecular weights (around 10 000 kDa). We recovered the monomeric form of the photochemical reaction center (RC), as well as the monomer and dimer of the reaction center-light harvesting 1-PufX (RC-LH1PufX) complex. Curiously, no type of LH1 complex was detected. Lastly, ATP synthase and cytochrome bc1 complexes were only recovered in their monomeric states. Purified ICM vesicles were shown to be rich in newly discovered gene products, including three proteins with unknown functions (RSP_2125, RSP_3238, RSP_6207), a possible alkane hydroxylase and a spheroidene monooxygenase. Other multiprotein complexes were found to be localized in the ICM, including succinate dehydrogenase in trimeric form and sarcosine oxidase in two different aggregation states. These findings contribute to the growing body of evidence that the bacterial ICM is a specialized bioenergetic membrane hosting, not only photosynthesis, but many other critical activities. Keywords: Rhodobacter sphaeroides bacteria proteomics • blue native PAGE • nondenaturing electrophoresis • membrane proteins

Introduction Rhodobacter sphaeroides is a Gram-negative, purple nonsulfur bacterium belonging to the R-3 subdivision of the Proteobacteria.1 It has a remarkable ability to grow under a variety of environmental conditions. It grows by aerobic respiration in the presence of oxygen; it also grows by anoxygenic photosynthesis in the presence of light or by anaerobic respiration in the presence of alternative electron acceptors, such as dimethyl sulfoxide and nitrate, under anaerobic-dark conditions.2-4 When grown anaerobically in the light or dark R. sphaeroides synthesizes an intracytoplasmic membrane (ICM) system, where the photosynthetic apparatus is located.2 R. sphaeroides is also known for its ability to survive high * Corresponding author: Prof. Dr. Lello Zolla, Tuscia University, Largo dell’ Universita` snc, 01100 Viterbo, Italy. Phone: 0039 0761 357100. Fax: 0039 0761 357179. E-mail: [email protected]. † Tuscia University. ‡ National Research Council (CNR).

192 Journal of Proteome Research 2010, 9, 192–203 Published on Web 11/09/2009

concentrations of heavy metal ions,5-7 to fix nitrogen and assimilate carbon dioxide.8 This organism has often been used as a model for the reaction center and more generally for the photosynthetic apparatus of photosynthetic species.9 R. sphaeroides is a facultative anoxygenic bacterium able to switch from aerobic to anaerobic growth depending on the partial pressure of oxygen. The strain we used in this study is R26.1, a partial revertant of R26. Both are deletion mutants that lack carotenoids,10-12 a group of photoprotective pigments whose absence makes these strains very sensitive to oxygen. Even at very low partial pressure, O2 inhibits photosynthesis, which proceeds only under strict anaerobiosis. Under these conditions, no aerobic growth is possible allowing the investigation to focus solely on photosynthetic metabolism. The photosynthetic unit (PSU) of R. sphaeroides is housed in ICM vesicles, specialized domains in the bacterial cytoplasmic membrane (CM) that contains a network of membrane10.1021/pr9005052

 2010 American Chemical Society

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Proteomic Analysis of R. sphaeroides R26.1 ICM Vesicles bound bacteriochlorophyll-protein complexes. Four membrane multiprotein complexes (MCPs) are involved in the conversion of solar energy into chemical energy. These are the lightharvesting complexes (LHCs), the reaction center (RC), the cytochrome bc1 complex (cyt bc1) and the ATP synthase (ATPase).2,13 Three-dimensional structures are available for all the PSU pigment-protein complexes.14-16 The RC complex contains three subunits: L and M subunits, that have very similar overall structures, each containing five transmembrane R-helices, and an H subunit anchored to the membrane by a single transmembrane R-helix.15 Like most purple bacteria, R. sphaeroides has two types of LHCs, termed LH1 and LH2. Both of these light-harvesting complexes are constructed on the same molecular design, based on an oligomeric organization of two small apoproteins (called R and β) noncovalently bound to bacteriochlorophyll a (Bchla) and carotenoids. LH1 surrounds the RC complex and consists of oligomeric repeats of a transmembrane heterodimeric R/β protein subunit. Scheuring et al.17 proposed that in R. sphaeroides LH1 assemblies contain 12 ( 1 LH1 R/β heterodimers organized to form an open ring of three-quarters of a full circle around the RC. Dimerization of the RC-LH1 has been linked to the presence of a small hydrophobic polypeptide of 82 amino acids named PufX which is located at the RC-LH1 dimer junction.17-19 It was demonstrated that the presence of PufX is essential for the formation of a long-range regular array of RC-LH1 complexes.20 The electron cryo-microscopy (cryo-EM) projection map showed a symmetric “S”-shaped [RC-LH1-PufX]2 ‘core’ complex.17 In this supramolecular organization, two molecules of the PufX polypeptide are at the LH1 “C”-shaped assembly open ring dimer junction. More recently, Qian and colleagues proposed a total of 14 LH1 R/β subunits,21,22 suggesting that the RC may be entirely circumscribed by LH1 and PufX. Cryo-EM of the LH2 complex obtained from R. sphaeroides shows a cylindric ring of nine R/β LH2 heterodimers.23 The inner wall of the cylindric structure is formed by a ring of nine R-apoprotein R-helices, whereas the outer wall is composed of nine β-apoprotein R-helices. A variety of species-dependent supramolecular arrangements was revealed for the LH2 complexes.24 In R. sphaeroides, it was shown that the ICM is packed with LH proteins and two types of organization were found.25 The purified bc1 complex contains four protein subunits with molecular masses of about 43, 31, 23, and 14 kDa.26 The complex contains three electron transfer subunits which bind four redox prosthetic groups. These are cytochrome b, which contains two b-type heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a high potential [2Fe-2S] Rieske protein.27,28 A decade ago, Jungas et al.18 proposed that the cyt bc1 was associated with the RC-LH1-PufX ‘core’ complex, but little structural evidence has emerged since then to support this hypothesis.17 It seems more likely that both bc1 and ATPase complexes are localized in the curved regions or at the poles of the isolated ICM vesicles.13 ATP synthase is a large mushroom-shaped asymmetric protein complex. The simplest bacterial version of this enzyme is composed of eight subunit types, of which five (R, β, γ, δ and ε) form the catalytic hydrophilic F1-portion. The proton translocating F0 portion is composed of 3 types of subunit named a, b and c. The catalytic portion of ATP synthase (F1) is formed by a R3β3 hexamer with a γ subunit inside it and an ε attached to the γ. Subunit δ is bound to the “top” of the hexamer and to subunit b. The hydrophobic transmembrane

segment of subunit b is in contact with subunit a. Chloroplast ATP synthase and the enzyme from some photosynthetic bacteria have two different, although similar, b-type subunits in the proton translocating F0 portion, namely b and b′, one copy of each. Strong homology is found for most of the ATP synthase subunits from different bacteria and chloroplasts. Although three-dimensional structures are available for all the PSU pigment-protein complexes, their supramolecular organization is currently poorly understood. Recently, atomic force microscopy (AFM)25 and linear dichroism (LD)29 studies have contributed to a clarification of the supramolecular organization of the PSU of several purple bacteria. On the basis of this information, Sener et al.13 proposed a three-dimensional structural model for the spatial supramolecular organization of ICM vesicles. They proposed a high-density array of ordered pigment-protein complexes. This array is a linear assembly of the dimeric ‘core’ complex RC-LH1, separated by domains of LH2 complexes. However, it is difficult to effectively explore proteins interacting in an organism on a proteome-wide scale, due to the limited number of techniques available. One established method is the use of nondenaturing electrophoresis followed by SDS-PAGE. The blue native (BN) gel system30 allows hydrophobic membrane proteins and complexes to be separated in their native state, avoiding the well-known drawbacks associated with separation and quantification of membrane proteins by conventional two-dimensional gel electrophoresis (2DE). This study presents, for the first time, a complete overview of interacting and individual proteins in R. sphaeroides ICM vesicles using nondenaturing gel systems. The MPCs identified by these techniques were compared with previous proteomic and microscopy data,13,24,25,31 confirming that this approach may be used to bridge remaining gaps in knowledge in order to fully explain the functional and structural assembly of R. sphaeroides photosynthetic membranes.

Material and Methods Strains and Growth Conditions. The Feher/Okamura group (San Diego) kindly provided cells of R. sphaeroides strain R26.1. Bacterial cells were grown at pH 6.9 in DSMZ medium 27 (http://www.dsmz.de) in the light under anaerobic conditions, according to Giotta et al.5 Cell Fractionation and Purification of ICM Vesicles. Cells in their late exponential growing phase were harvested by centrifugation at 14 000g at 4 °C for 15 min from 1 L of culture medium. The resulting biomass (5 g) was washed in 100 mL of 20 mM Tris-HCl, pH 8.0, and 1 mM EDTA, kept on ice, centrifuged again and then resuspended in the same buffer to a final absorbance of 80 at 865 nm. Cell wall disruption was accomplished by a high pressure extrusion method, using a French pressure cell operating at 150 bar while the sample was kept at 4 °C in the presence of 100 µg/mL DNase I, 10 mg/mL RNase A and 1 mM PMSF. Unbroken cells and debris were removed by centrifugation at 18 000g for 20 min at 4 °C. The resulting crude cell extract was centrifuged at 180 000g for 2 h at 4 °C in order to separate the membranes and the soluble cell fraction. To minimize soluble protein contamination, the pellet was suspended and washed three times in 20 mL of 20 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The pellet (1 g) was loaded onto a 15/40/50% (w/w) sucrose gradient layered over a 60% sucrose cushion32,33 and centrifuged at 27 000 rpm for 10 h in a Beckman SW28 rotor. Pigmented bands were harvested and dialyzed for 4 h at 4 °C using a slide-A-Lyzer 3.5 Journal of Proteome Research • Vol. 9, No. 1, 2010 193

research articles K MWCO Dialysis Cassette (Pierce) against 100 vol of Tris-HCl, pH 7.5, and 100 mM NaCl. Dialyzed fractions were then centrifuged at 180 000g for 2 h at 4 °C and the pellet was used for proteomic analysis. Solubilization of the ICM Vesicles. Solubilization of protein complexes from ICM vesicles was performed according to Ku ¨gler et al.,34 and Suorsa et al.35 with the following modifications: photosynthetic membranes were washed with washing buffer (330 mM sorbitol, 50 mM BisTris-HCl, pH 7.0), and 250 mg/mL Pefabloc as a protease inhibitor (Roche, Indianapolis, IN), collected by centrifugation (3500g for 2 min at 4 °C), and resuspended in 25BTH20G (20% v/v glycerol, 25 mM BisTrisHCl, pH 7.0, and 250 mg/mL Pefabloc). An equal volume of resuspension buffer containing 1% (w/v) n-dodecyl-β-D-maltoside (DDM; Sigma-Aldrich, St. Louis, MO) was added under continuous mixing, and the solubilization of membrane protein complexes was allowed to occur for 3 min on ice. Insoluble material was removed by centrifugation at 18 000g for 15 min. After solubilization, protein concentrations were determined using a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. First Dimension. Native PAGE was performed at 4 °C using a Protean II xi Bio-Rad electrophoresis system (180 × 160 mm, 0.75 mm thick) and applying a constant voltage of 90 V overnight and gradually increasing this value up to 200 V the next day until the run was complete. For both BN- and CNPAGE, the same amount of protein (100 µg) was loaded onto a 0.75 mm thick 4-13% (w/v) acrylamide gradient gel. For BNPAGE, the solubilized ICM vesicles were mixed with 0.1 vol of Coomassie blue solution (5% w/v Serva blue G, 100 mM BisTrisHCl, pH 7.0, 30% w/v sucrose, and 500 mM ε-amino-n-caproic acid), whereas in the CN system, the solubilized ICM vesicles were mixed with 0.1 vol of the same buffer without Coomassie blue stain. The apparent molecular weight of bands was determined using a high molecular weight calibration kit for native electrophoresis (GE Healthcare, Buckinghamshire, U.K.). Reduction and Alkylation before Second Slabs. For the equilibration of gel strips after the first dimension, the lanes of the 1D BN-PAGE and 1D CN-PAGE gels were excised and incubated in 50 mM Tris-HCI buffer pH 8.8 containing 6 M urea, 30% (w/v) glycerol, 3% (w/v) SDS and 3% (w/v) DTT for 30 min followed by a second incubation with 12% (w/v) iodoacetamide in the equilibration solution buffer for 20 min. Second Dimension. Second-dimensional SDS-PAGE was done in a large format (180 × 160 mm, 1 mm thick) gel camera (Protean II xi, Bio-Rad). The lanes were then layered onto 14% (w/v) acrylamide and 6 M urea SDS gel.36 Proteins were separated overnight at 10 mA constant current per gel and 13 C°. The proteins were visualized using blue-silver stain method.37 Absorption Spectra of MPCs. Native protein complexes were extracted from CN gel pieces by adding water (10 times the gel volume) and letting the proteins diffuse out overnight. Absorption spectra of native MPCs were recorded between 650 and 950 nm at room temperature using a Jasco V630 UV-vis spectrophotometer (Jasco, Easton, MA). Determination of ATPase Stoichiometry. The ATPase complex was extracted from BN gel pieces by adding water and letting the complex diffuse out overnight. The supernatants were dialyzed overnight against distilled water, and concentrated by lyophilization. Protein concentration was estimated by the DC Protein Assay (Bio-Rad). ATPase subunits were separated by 12% SDS-PAGE (180 × 160 mm, 0.75 mm thick) 194

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D’Amici et al. and the gels were stained and scanned. Assuming that the concentration of stain in a protein band is proportional to the mass concentration of proteins, we plotted the stain intensity (absorbance) versus loading amount of ATPase (sample load µL). The molecular ratios of the subunits were determined by using their theoretical molecular weight and the slope of the plot line according to the equation proposed by Abresch et al.38 In-Gel Digestion. Protein spots were carefully excised from blue-silver stained gels and subjected to in-gel trypsin digestion according to Shevchenko and colleagues39 with minor modifications. The gel pieces were swollen in a digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/µL of trypsin (modified porcine trypsin, sequencing grade, Promega, Madison, WI) in an ice bath. After 30 min, the supernatant was removed and discarded, 20 µL of 50 mM NH4HCO3 was added to the gel pieces and digestion allowed to proceed at 37 °C overnight. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Prior to mass spectrometric analysis, the peptide mixtures were redissolved in 10 µL of 5% FA (formic acid). Protein Identification by MS/MS. Peptide mixtures were separated using a nanoflow-HPLC system (Ultimate; Switchos; Famos; LC Packings, Amsterdam, The Netherlands). A sample volume of 10 µL was loaded by the autosampler onto a homemade 2 cm fused silica precolumn (75 µm i.d.; 375 µm o.d.; Reprosil C18-AQ, 3 µm, Dr. Maisch GmbH, AmmerbuchEntringen, Germany) at a flow rate of 2 µL/min. Sequential elution of peptides was accomplished using a flow rate of 200 nL/min and a linear gradient from Solution A (2% acetonitrile (ACN); 0.1% FA) to 50% of Solution B (98% ACN; 0.1% FA) in 40 min over the precolumn in-line with a homemade 10-15 cm resolving column (75 µm i.d.; 375 µm o.d.; Reprosil C18AQ, 3 µm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted directly into a High Capacity ion Trap (model HCTplus, Bruker-Daltonik, Germany). Capillary voltage was 1.5-2 kV and a dry gas flow rate of 10 L/min was used with a temperature of 230 °C. The scan range used was from 300 to 1800 m/z. Protein identification was performed by searching the National Center for Biotechnology Information nonredundant database (NCBInr, version 20081128, www.ncbi. nlm.nih.gov) using the Mascot program (in-house version 2.2, Matrix Science, London, U.K.). The following parameters were adopted for database searches: complete carbamidomethylation of cysteines and partial oxidation of methionines, peptide Mass Tolerance ( 1.2 Da, Fragment Mass Tolerance ( 0.9 Da, missed cleavages 2. For positive identification, the score of the result of (-10 × Log(P)) had to be over the significance threshold level (P < 0.05). Even though high MASCOT scores are obtained with values greater than 60, when proteins were identified with only one peptide, a combination of automated database search and manual interpretation of peptide fragmentation spectra was used to validate protein assignments. In this manual verification, the mass error, the presence of fragment ion series, and the expected prevalence of C-terminus containing ions (Y-type) in the high mass range were all taken into account. Moreover, replicate measurements have confirmed the identity of these protein hits. Prediction of Hypothetical Protein Function and Subcellular Localization. On the basis of MS analysis, we identified hypothetical proteins and checked their sequences against a number of databases, including SWISS-PROT and NCBI. To

Proteomic Analysis of R. sphaeroides R26.1 ICM Vesicles

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predict direct (physical) and indirect (functional) protein interactions, we applied STRING 8.0 database (http://string. embl.de/).40 Protein subcellular localization was predicted by PSORTb software (version 2.0.4, http://www.psort.org/psortb/).

Results To define the number and types of multiprotein complexes present within a photosynthetic membrane, a proteomic analysis was carried out on ICM vesicles from R. sphaeroides R26.1 using nondenaturing electrophoresis systems (BN- and CN-PAGE) followed by SDS-PAGE. ICM vesicles were prepared from R. sphaeroides cells grown under phototrophic conditions. The washing procedure and the sucrose gradient centrifugation (see Materials and Methods) were performed in order to reduce nonspecifically adsorbed soluble protein contamination and to obtain purified ICM vesicles. Separation of MPCs from ICM Vesicles by Monodimensional Nondenaturing Electrophoresis. Correct analysis of the oligomeric state of membrane proteins requires the use of detergent to prevent aberrant migration caused by the presence of lipids. We tested various detergents for their solubilization in ICM vesicles, including Triton X-100, digitonin, octyl glucoside, and DDM, in the range of 0.5-5% (w/v) (data not shown). Best results were obtained with 0.5% (w/v) DDM (see figure in Supporting Information) which was used in all subsequent BN and CN experiments. This concentration represented a good compromise allowing us to avoid either partial disintegration of native complexes caused by excess detergent, or the appearance of gel band smearing due to insufficient detergent content. Different first dimension gradients were also applied to achieve maximum resolution of MPCs, excluding any accidental comigration of proteins. The maximum resolution was achieved with a gradient gel running from 4 to 13% acrylamide. At last, the combined Coomassie blue-silver stain procedure ensured a high sensitivity and was completely compatible with mass spectrometry. Figure 1A shows a representative 1D BN-PAGE of MPC separation from ICM vesicles. Eight distinct bands with molecular masses from 74 kDa to approximately 10 MDa were revealed. Each band represents a protein complex, as demonstrated by electrophoresis on a second-dimension gel under denaturing conditions (see below). The protein composition of each complex was determined by digesting each entire BN gel band with trypsin and analyzing the peptide mixture obtained using RP-HPLC-ESI-MS/MS. Finally, proteins were identified by database search.41 On the basis of the identities ascertained for each complex through mass spectrometry, it was possible to claim that the three bands 1, 4 and 5, at about 10 000, 303, and 181 kDa, respectively, contained LH2 complexes (composed of R9β9 heterodimers) in different oligomeric states. To confirm that LH2 supercomplexes at ∼10 000 kDa were really stable in DDM, we increased the DDM/protein ratio from 1.5 to 4.0 (g/g). Interestingly, the aggregate was still not affected under these conditions (data not shown). The blue band 2 found at 580 kDa was identified as the ATP synthase monomer, whereas the blurred band 3 at 451 kDa contained some subunits of the photosynthetic RC plus LH1 and two subunits of the succinate dehydrogenase enzyme. Thus, we concluded that this band at 451 kDa represented the comigrating dimeric core complex [RC+PufX+LH1]2 and the succinate dehydrogenase enzyme complex. The largest band 4 at 303 kDa contained LH2 complexes (as mentioned before),

Figure 1. Resolution comparison of first-dimension blue native PAGE (A) and colorless native PAGE (B) using 0.5% (w/v) DDM solubilized ICM vesicles. One hundred micrograms of total proteins was applied on a gradient gel of 4-13% acrylamide. Gels were fixed and stained with blue-silver stain procedure. The prominent bands were identified both by trypsin digestion followed by RP-HPLC-ESI-MS/MS analysis and by their characteristic polypeptide patterns in second-dimension SDS-PAGE (Figure 3). The numbers and the lines indicate the MPCs listed in Table 1.

as well as all RC and LH1 subunits. Thus, this band contained the monomeric form of the core complex [RC+PufX+LH1], in agreement with its apparent molecular weight. In band 5, in addition to LH2 complexes (see above), two sarcosine oxidase enzyme subunits were found, whereas band 6 (130 kDa) contained the entire RC complex. The size of the latter suggest that the monomeric form of the RC (without PufX and LH1 polypeptides) migrated in this band. In band 7 (118 kDa) some subunits of the cyt bc1 complex were recognized suggesting the presence of the monomer. Interestingly, in the last band 8 at 74 kDa, a recently discussed31 alkane hydroxylase was identified together with other “free” proteins, so-called because we found no molecular mass increase due to interaction with other protein subunits. ICM membranes were also run by 1D colorless native (CN)PAGE, with the intended aim of corroborating the assignments above derived from BN gel analysis (Figure 1B). While BN-PAGE is a charge-shift system relying on the gentle binding of anionic Coomassie Brilliant Blue G-250 dye to the MPCs,42 in the CN electrophoresis gel migration of MPCs is driven solely by the intrinsic charge toward the anode and their electrophoretic mobility is determined by size and native pI.42 As a result, a considerably reduced electrophoresis migration was seen in the CN system, and the BN band 4 split into two different bands (bands 4′ and 5′). Most of the proteins identified by BN were Journal of Proteome Research • Vol. 9, No. 1, 2010 195

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Figure 2. The absorption spectra of MPCs extracted from CN gel bands were measured at room temperature. Bands 1′, 3′, 4′, 5′ and 6′ referred to CN gel bands (Figure 1B). Vertical dotted lines indicate the LH1, RC and LH2 absorption maxima.

confirmed using CN gels, with the exception of the cyt bc1 complex, missing in CN maps probably because its pI is higher than 5.4, the limiting pH in CN. Taking advantage of the fact that complexes are not stained in CN electrophoresis, we extracted the endogenous blue-green bands numbered 1′, 3′, 4′, 5′ and 6′ by simple diffusion from the native gel and their absorption spectra between 650 and 950 nm were recorded at room temperature (Figure 2). Similarly to the BN gel, we identified LH2 complexes in three blue-green bands, namely, band 1′ (∼10 000 kDa), band 4′ (385 kDa) and band 6′ (241 kDa) in different oligomeric states. Band 6′, associated with a dimeric form of LH2, showed an absorption spectrum with the most intense peak centered at 860 nm, 10 nm red-shifted with respect to the value found for monomeric LH2 in R. sphaeroides R26.1.43-45 Bands 1′ and 4′ showed a further red-shift of this peak to 863-865 nm. These results are in agreement with the finding that the absorption maximum depends on the extension of the exciton coupling in bacteriochlorophylls which tends to increase in multiple oligomeric states of LH2.46 The broad peak centered at 770 nm, visible in spectra from bands 1′, 4′ and 6′, can be attributed to free bacteriochlorophyll.47 In-gel digestion and MS/MS analysis showed the presence of RC components and LH1 polypeptides in the bands 3′ 196

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D’Amici et al. ([RC+PufX+LH1]2, 493 kDa) and 5′ ([RC+PufX+LH1], 318 kDa). Accordingly, the higher wavelength peak at approximately 870 nm in the spectra relevant to bands 3′ and 5′ (slightly redshifted in the dimeric form of the complex) is due to the light harvesting LH1 complex bacteriochlorophyll.38,47 A small shoulder centered at 800 nm due to the monomer bacteriochlorophyll of the reaction center,47,48 as well as a further peak at 760 nm due to bacteriopheophytin of the reaction center overlapping with the 770 nm peak, are also visible.47 The colorless bands at 594 kDa (band 2′) and 148 kDa (band 7′) contain, respectively, the ATPase complex and the sole three RC core polypeptides (subunit L, M and H), suggesting, in line with BN gel observations, that the RC core migrates in this band. Finally, in lower CN gel band (number 8′), there was comigration of some free proteins, for example, the possible alkane hydroxylase, and some not well characterized hypothetical proteins.31 Finally, Table 1 summarizes the protein identification results for both BN and CN bands. Second-Dimensional Denaturing Electrophoresis. For separation in the second dimension, both BN and CN gel strips carrying membrane proteins were transferred horizontally onto an SDS gel. Under these conditions, MPCs dissociated, and on the resulting two-dimensional gels, the subunits of distinct MPCs are ordered according to their molecular weight in vertical rows. Analysis (by tryptic digestion and MS/MS measurements) of the separated protein complexes on the second gel dimension allowed us to identify MPCs by their subunit composition. Figure 3A shows a representative 2D BN-SDSPAGE map of ICM membrane proteins. Fifty-one spots were recognizable on 2D BN-SDS-PAGE map (see Table 2). The highest molecular weight BN gel band 1 (∼10 000 kDa) can be resolved into 4 proteins (spots 1-4). Spot number 1 migrated with an apparent molecular mass of about 179.6 kDa and showed an endogenous stain on 2D denaturing gels. This observation suggested that some pigment molecules can bind covalently to that protein. MS analysis of spots 1 and 2 identified the R and the β subunits of the sarcosine oxidase enzyme. Spots 3 and 4, both with an apparent molecular mass 0.700). Interestingly, PSORTb software analysis placed these proteins in the cytoplasm as well. The blurred band on the BN gel at 451 kDa was resolved into 8 spots. Spots 17, 18, 19, 21, 22, and 23 were, respectively, identified as polypeptides M, L, H, PufX, LH1 R and LH1 β. All these proteins are components of the RC core complex. In this

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Proteomic Analysis of R. sphaeroides R26.1 ICM Vesicles a

Table 1. Multiprotein Complexes Identified on Native Gel Systems 1D BN-PAGE

1D CN-PAGE

MS/MS identification multiprotein complex

apparent apparent number of band no. of peptides molecular Protein no. of peptides molecular distinguishable identified identified massb (kDa) number protein name massb (kDa) band number name subunits on 2D gelsc

[LH2]n

∼10000

1

ATP synthase

580

2

[RC+PufX+LH1]2

451

3

[LH2]3

303

4

[RC+PufX+LH1]

303

4

[LH2]2

181

5

[RC]

130

6

M H

[Cyt bc1]

118

7

subunit IV

a

2D

MS/MS identification

LH2R LH2β AtpR Atpβ Atpγ Atpδ AtpB Atpε RSP_2125 M H PufX LH1β LH2R LH2β M H L LH1R LH2β

1 3 13 15 4 5 6 3 3 2 7 1 2 1 2 3 5 2 1 2

∼10000

1′

594

2′

493

3′

385

4′

318

LH2R LH2β AtpR Atpβ Atpγ Atpδ AtpB Atpc

1 3 9 2 2 2 3 1

M L H LH1R LH2R

3 2 8 2 1

6

5′

M L H

2 2 2

6

241

6′

148

7′

2

-

1 1 2 2 4 -

2

2 7

LH2R LH2β M L H -

-

2 10

2

3 3

b

The list of MCPs is limited to PSU complexes. The apparent molecular mass of protein complexes from ICM vesicles separated on BN-PAGE were estimated by comparing them with the molecular masses of standard protein complexes (GE Healthcare). c The number of subunits per complex is deduced from the two-dimensional gels reproduced in Figure 3A/B.

way, on the basis of both the apparent molecular mass of the first dimension band and the presence of the PufX polypeptide,17-19 we were able to conclude that the BN gel band at 451 kDa represented the dimeric form of the core complex [RC+PufX+LH1]2. The other two spots were identified as the flavoprotein subunit (SdhA, spot 16) and the iron-sulfur subunit (SdhB, spot 18) from the succinate dehydrogenase enzyme. All the [RC+PufX+LH1] subunits (spots 26, 27, 28, 29 and 31) were also identified from the abundant BN band at 303 kDa. Spots 30 and 32 were identified as LH2 R and β polypeptides indicating that in this band the monomeric form of the core complex ([RC+PufX+LH1]) and the trimeric form of LH2 heterodimers comigrated. In addition, a leucyl aminopeptidase (spot 24) and a probable HflK protein (spot 25) migrated in that band. The BN band at 181 kDa shows once again the LH2 R and β polypeptides (spots 38 and 39). The R and the β subunits of sarcosine oxidase enzyme migrated in the same band (spots 34, 35). Interestingly, spot 34 showed the same endogenous stain as spot 1. The RC core complex also showed a band around 130 kDa, and in this case, seconddimension electrophoresis showed that this complex consists of three subunits with molecular weights ranging between 45 and 24 kDa (spots 41, 42, 43) identified as the RC polypeptides M, L and H. Finally, there is a very thick, dark blue band just below 80 kDa on the BN gel containing “free” proteins. Cyt bc1 complex subunits were identified in spots 45, 46, and 47 (cytochrome c1 precursor, cytochrome C reductase iron-sulfur protein and cytochrome bc1 subunit IV, respectively). This result suggests that the cyt bc1 migrated in the first-dimension gel close to or together with the RC (around 100 kDa). The second-dimension gel from this band revealed different spots between 60 and 18 kDa. Spot 44 was found to contain the wellknown enzyme spheroidene monooxygenase, and in spot 48, the possible alkane hydroxylase enzyme was identified.31 The

large spot labeled 49 from the 2D gel was found to contain the hypothetical protein RSP_1760. MPCs separated first by CN-PAGE were also resolved into their individual subunits by denaturing SDS-PAGE (Figure 3B). Comparison between 2D BN-SDS-PAGE and 2D CN-SDS-PAGE maps was useful in assigning first-dimension bands. The 2D CN map was very similar to the 2D BN map with the exception that cyt bc1 complex subunits were not present. Also in the second dimension CN map, all the subunits derived from the [LH2]n (∼10 000 kDa), ATPase (594 kDa), [RC+PufX+LH1]2 (493 kDa), [RC+PufX+LH1] (318 kDa), [LH2]3 (385 kDa), [LH2]2 (241 kDa) and the RC (148 kDa) were easily recognized. To appreciate the best resolution of the CN system, Figure 3 compares 2D gel portions between 14 and 5 kDa from BN (panel C) and CN (panel D). It was possible to see how the CN system allows the unequivocal identification of the [LH2]3 which in the BN migrated close to [RC+PufX+LH1].

Discussion In this paper, we have shown that BN and CN gel systems, when combined with MS analysis, are reliable techniques for characterizing native multiprotein complexes and their protein composition. Data for MPCs of photosynthetically grown R. sphaeroides are presented here. The proteomic blueprint described agrees with previous biochemical or functional analyses as well as with proposed models for the topology of photosynthetic membranes based on AFM studies of the ICM.13,25,29 This means we can be confident in the assignments of the newly identified proteins to these vesicles as well as in formulating new hypotheses about photosynthetic membrane assembly or function. RC-LH1-PufX Core Complexes. About 20% of RC-LH1 complex in our gels, including PufX, is in a dimeric state Journal of Proteome Research • Vol. 9, No. 1, 2010 197

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Figure 3. (A) 2D BN-SDS-PAGE map of intracytoplasmic membrane (ICM) vesicles from R. sphaeroides R26.1. 1D BN strips were loaded and separated by 14% acrylamide 6 M urea SDS-PAGE. The BN strips were oriented with the top on the left and the bottom on the right and the gels were stained with blue-silver. The numbers represent spots that were excised and identified by MS/MS and correspond to the numbers shown in Table 2. (B) 2D CN-SDS-PAGE map of ICM vesicles from R. sphaeroides R26.1. Similarly to BN, CN strips were separated by 14% acrylamide 6 M urea SDS-PAGE and stained with blue-silver. (C and D) Portions of 2D gels showing the region containing proteins under 14.0 kDa. Crosses indicate the LH1 and LH2 polypeptides in the BN gel (A) and the CN gel (B), respectively.

([RC+PufX+LH1]2). Bands at 451 kDa in the BN gel and 493 kDa in the CN gel agree with the predicted molecular mass of about 487 kDa obtained by the crystallized RC-LH1 complex from R. sphaeroides.22 However, a large amount of core complex is present in the monomeric state ([RC+PufX+LH1]), in agreement with previous observations in sucrose gradient.17,19 Our native gel electrophoretic systems also showed that the RC complex was more abundant at higher DDM concentration, indicating that PufX+LH1 needed the detergent to be detached from RC. The semiquantitative analysis of the RC complex could be a potential tool to evaluate RC-LH1 core complex stability in the event of abiotic stresses. In contrast to previous studies,19,49 no type of LH1 complexes was identified here suggesting either that the detergent conditions are not adequate for solubilization of the LH1 alone or that LH1 polypeptides (R and β) can only interact in the presence of PufX and RC. This observation confirms that the interaction between LH1 and RC is unusually strong as evidenced by the fact that attempts to isolate pure LH1 complex under various conditions have not been successful.50 LH2 Complexes. LH2 complexes were very abundant on BN and CN gels after solubilization with DDM. We were able to identify three LH2 complex bands on native gels. The lower apparent molecular mass band (181 kDa on the BN gel and 198

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241 kDa on the CN gel) was recognized as the LH2 dimeric configuration ([LH2]2). In the past five years, native membranes from different LH2-containing photosynthetic bacteria, including R. sphaeroides, have been imaged by AFM,25,51,52 but in all these cases, there have been no reports of LH2 dimers, indicating that LH2 is a monomeric protein complex in vivo. However, our interpretation of the result we obtained is in agreement with Liu and co-workers,53 who described an AFM investigation on artificially created 2D-crystals of the LH2 complexes. The authors demonstrated the existence of LH2 dimeric complexes, and suggested that the presence of dimers in the crystals may reflect a more dense packing condition. More recently, Olsen et al.54 deduced that the formation of LH2 dimers through protein-to-protein contacts may also be driven by the requirement to minimize the exposure of the hydrophobic central belt of the complexes to the buffer solution. On native PAGE, LH2 formed other two aggregation states in addition to dimeric conformation: (i) a predominant complex of 303 kDa (385 kDa on CN gel), and (ii) a supercomplex with Mw ∼10 000 kDa. The complex at 303 kDa comigrated with the monomeric RC-LH1-PufX on the BN gel, whereas it was completely isolated on the CN gel. Since the apparent molecular weight measured by the CN gel is very close to the predicted value, it is reasonable to assume that this band

4 2 2 7

51.6/113.0 41.9/49.8 25.4/28.8 107.5/185.4

20 24 25 33 34

13

9 7 18

2 2 8

5

34.5/39.0 31.6/34.3 28.1/25.8

41 42 43

1 3

30.3/33.4

5.6/13.8 5.4/12.8

38 39

1 3

18

5.6/14.1 5.4/12.9

30 32

3 2 6 2 2

65.6/113.6

34.3/39.2 31.6/34.7 28.1/25.9 9.0/16.4 6.8/13.7

26 27 28 29 31

2 2 7 2 2 2

16

34.5/38.9 31.4/33.4 28.1/25.7 9.0/16.4 6.8/13.3 5.6/12.7

17 18 19 21 22 23

31 16 8 14 14 10 5 3 1

107.5/179.6 45.1/52.4 46.9/92.2

55.2/65.0 50.3/61.7 31.2/43.7 19.3/25.3 18.7/23.2 19.9/22.4 13.7/21.4 11.8/18.3 7.6/12.9

6 7 8 9 10 11 12 13 14 15

1 3

no. of peptides identified by MS/MS

1 2 5

5.6/13.7 5.4/12.3

3 4

spot no.

Mr, kDa theor./exper.a

265 108 83 308

201

608

396 394 1064

185 76 419

60 150

64 181

194 122 313 147 98

185 112 423 98 81 94

1740 981 520 972 820 668 239 156 70

60 161

Mascot score

11 6 11 9

18

25

10 20 37

9 6 40

20 45

20 45

12 6 22 18 15

9 7 35 18 15 48

51 46 31 79 57 48 29 35 12

20 45

seq cov (%)

GRAVY

subcell localizationc

HMMTOP:1 TMHMM:0 HMMTOP:1 TMHMM:0 HMMTOP:3 TMHMM:3 HMMTOP:1 TMHMM:0

HMMTOP:0 TMHMM:0

0.68 -0.435 0275 -0.162

-0.289

Cytoplasmic Unknown ICM ICM

ICM

[LH2]n, Band 1 on BN Gel HMMTOP:1 TMHMM:1 1.009 ICM HMMTOP:1 TMHMM:1 0.439 ICM ATP Synthase, Band 2 on BN Gel HMMTOP:0 TMHMM:0 -0.144 ICM HMMTOP:1 TMHMM:0 0.480 ICM HMMTOP:0 TMHMM:0 -0.133 ICM HMMTOP:0 TMHMM:0 0.094 ICM HMMTOP:1 TMHMM:1 0.131 ICM HMMTOP:2 TMHMM:2 0.137 ICM HMMTOP:0 TMHMM:0 0.077 ICM HMMTOP:0 TMHMM:0 -0.367 Cytoplasmic HMMTOP:2 TMHMM:2 1.146 ICM [RC+PufX+LH1]2, Band 3 on BN Gel HMMTOP:5 TMHMM:5 0.43 ICM HMMTOP:5 TMHMM:5 0.581 ICM HMMTOP:1 TMHMM:1 -0.056 ICM HMMTOP:1 TMHMM:1 0.274 ICM HMMTOP:1 TMHMM:1 0.728 ICM HMMTOP:1 TMHMM:1 0.316 ICM [RC+PufX+LH1], Band 4 on BN Gel HMMTOP:6 TMHMM:5 0.432 ICM HMMTOP:5 TMHMM:5 0.571 ICM HMMTOP:1 TMHMM:1 -0.056 ICM HMMTOP:1 TMHMM:1 0.274 ICM HMMTOP:1 TMHMM:1 0.728 ICM [LH2]3, Band 4 on BN Gel HMMTOP:1 TMHMM:1 1.009 ICM HMMTOP:1 TMHMM:1 0.439 ICM [LH2]2, Band 5 on BN Gel HMMTOP:1 TMHMM:1 1.009 ICM HMMTOP:1 TMHMM:1 0.439 ICM [RC]1, Band 6 on BN Gel HMMTOP:5 TMHMM:5 0.430 ICM HMMTOP5 TMHMM:5 0.586 ICM HMMTOP:1 TMHMM:1 -0.056 ICM Other MPCs and Free Proteins HMMTOP:1 TMHMM:0 -0.162 ICM HMMTOP:1 TMHMM:0 -0.050 ICM HMMTOP:1 TMHMM:1 -0.324 Cytoplasmic ICM (may have multiple localization sites) HMMTOP:0 TMHMM:0 -0.253 ICM

TDb

YP_352960 YP_353432 YP_353747 YP_352745

YP_354063

YP_354060

YP_352745 YP_352742 YP_354752

YP_353329 YP_353330 YP_353365

YP_353391 YP_353390

YP_353391 YP_353390

YP_353329 YP_353330 YP_353365 YP_353328 YP_353331

YP_353329 YP_353330 YP_353365 YP_353328 YP_353331 YP_353332

YP_352352 YP_352354 YP_352353 YP_352351 YP_354121 YP_354120 YP_352355 YP_352180 YP_354122

YP_353391 YP_353390

NCBI accession number

succinate dehydrogenase flavoprotein subunit (SdhA) succinate dehydrogenase iron-sulfur subunit (SdhB) Not identified leucyl aminopeptidase Probable HflK protein biopolymer transport protein, TolQ putative sarcosine oxidase, alpha subunit

putative sarcosine oxidase, alpha subunit putative sarcosine oxidase beta subunit hypothetical protein RSP_3238

photosynthetic reaction center subunit M PufL, photosynthetic reaction center L subunit Reaction center H protein

LHII alpha, light-harvesting B800/850 protein LHII beta, light-harvesting B800/850 protein

LHII alpha, light-harvesting B800/850 protein LHII beta, light-harvesting B800/850 protein

photosynthetic reaction center subunit M PufL, photosynthetic reaction center L subunit Reaction center H protein Intrinsic membrane PufX protein LHI alpha, light-harvesting B875 protein

photosynthetic reaction center subunit M PufL, photosynthetic reaction center L subunit Reaction center H protein Intrinsic membrane PufX protein LHI alpha, light-harvesting B875 protein LHI beta, light-harvesting B875 subunit

F0F1 ATP synthase subunit alpha F0F1 ATP synthase subunit beta F0F1 ATP synthase subunit gamma Not identified ATPase, delta (OSCP) subunit F0F1 ATP synthase, subunit B’ F0F1 ATP synthase, subunit B ATP synthase, epsilon subunit hypothetical protein RSP_2125 F0F1 ATP synthase subunit C

LHII alpha, light-harvesting B800/850 protein LHII beta, light-harvesting B800/850 protein

protein ID [R. sphaeroides 2.4.1]

Table 2. Intracytoplasmic Membrane Proteins from R. sphaeroides R 26.1 Detected by MS/MS. ID Number of the Bands Relates to Numbers Shown in Figure 3

Proteomic Analysis of R. sphaeroides R26.1 ICM Vesicles

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When no annotation based on literature searches was available, a Experimental Mr values were determined by PDQuest software (Bio-Rad, version 8.0.1). b TD, predicted number of transmembrane domains. putative subcellular localization of the various targets was determined using PSORTb (http://www.psort.org/psortb/) program.

14.3/19.5 46.4/45.5 24.9/23.1 19.9/22.2 13.5/19.3 15.6/19.3 47 48 49 50 51

3 3 3 2 4 3

124 137 160 88 252 188

22 3 16 11 39 31

HMMTOP:1 HMMTOP:6 HMMTOP:1 HMMTOP:1 HMMTOP:1 HMMTOP:0

TMHMM:1 TMHMM:4 TMHMM:2 TMHMM:1 TMHMM:1 TMHMM:0

c

ICM ICM ICM ICM ICM ICM -0.605 0.142 -0.212 -0.170 -0.076 -0.352

YP_352743 YP_351510 YP_351808 YP_354283 YP_353547 YP_353371

Spheroidene monooxygenase Cytochrome c1 precursor Ubiquinol-cytochrome C reductase, iron-sulfur protein cytochrome bc1 subunit IV alkane 1-monooxygenase hypothetical protein RSP_1760 hypothetical protein RSP_1200 Cytochrome c’ Cytochrome c2 YP_353346 YP_354474 YP_354476 -0.486 -0.201 -0.052 HMMTOP:1 TMHMM:0 HMMTOP:2 TMHMM:1 HMMTOP:0 TMHMM:1 42 20 6 688 202 79 35.6/55.9 30.8/46.0 20.1/20.8 44 45 46

14 4 1

36.9/36.4 27.0/25.5 43.7/50.0 36 37 40

2 1 4

109 85 247

7 5 9

HMMTOP:1 TMHMM:1 HMMTOP:4 TMHMM:4 HMMTOP:0 TMHMM:0

-0.229 0.367 -0.326

Unknown ICM Cytoplasmic ICM (may have multiple localization sites) ICM ICM ICM

YP_353431 YP_351810 YP_351551

putative sarcosine oxidase, beta subunit Probable HflC protein hypothetical protein RSP_1762 hypothetical protein RSP_6207 YP_352742 18 396 6 45.1/53.4

spot no.

D’Amici et al.

35

GRAVY TDb seq cov (%) Mascot score no. of peptides identified by MS/MS Mr, kDa theor./exper.a

Table 2. Continued 200

Other MPCs and Free Proteins HMMTOP:1 TMHMM:0 -0.050

ICM

subcell localizationc

NCBI accession number

protein ID [R. sphaeroides 2.4.1]

research articles

represents a trimeric conformation of LH2 complexes ([LH2]3). For unknown reasons, in the BN system, there was a discrepancy between the apparent molecular mass of the LH2 trimer and the predicted value. We supposed that both trimeric and supercomplex conformations were artificially formed during the solubilization steps, since this oligomeric LH2 organization has never been detected by AFM or other techniques. ATPase Complex. As the ATPase band was sharp and very reproducible on BN and CN gels, we were able to perform an accurate biochemical characterization of the R. sphaeroides ATP synthase. The extrinsic part of the enzyme (F1) is formed by five proteins present in the stoichiometry R3β3γδε; the intrinsic portion (F0) is formed by three proteins with a stoichiometry of ab2cn.55,56 Resolution of ATPase protein complex on the second gel dimension curiously revealed 10 subunits. Spots 6, 7, 8, 10, 11, 12, 13, and 15 were identified by MS analysis, respectively, as R, β, γ, δ, b′, b, ε and c subunits (see Table 2). A database search after MS/MS analysis of spot 9 did not yield any results, and on the basis of its second dimension apparent molecular weight, this spot was matched with a subunit of ATPase F0 portion. The lack of ESI-MS/MS identification could be due to the high hydrophobicity of the protein (GRAVY score: 1.013) that can reduce the access of trypsin enzyme to the cleavage sites on the amino acid sequence. Interestingly, spots 11 and 12 were identified as b′ and b subunits, respectively. This proved that R. sphaeroides codes the bb′ heterodimer and not the b2 homodimer, in line with other photosynthetic bacteria. In fact, when an additional gene is present in the ATPase operon, the b2 homodimer is substituted by a bb′ heterodimer.57 Crystallographic, mutagenetic and AFM data in different systems have indicated a different number of the c subunit forming the ring of transmembrane R-helix hairpins, whose exact number (cn) is still uncertain and might be species specific (e.g., 10 subunits in Saccharomyces cerevisiae58 and, possibly in Escherichia coli,59 11 copies in Ilyobacter tartaricus,60 and 14 in chloroplasts61). By means of the procedure described in Materials and Methods, we were able to estimate a γ/c subunit ratio of 1.00/11.0 ((0.56), supporting an ATP synthase subunit stoichiometry of R3, β3, γ, δ, ε, a, b, b′, c11. However, a high resolution structure is obviously required to accurately determine the exact ATP synthase subunit stoichiometry for R. sphaeroides. On the basis of this stoichiometry, the predicted ATPase molecular weight could be about 530 kDa despite the fact that on the BN gel the complex migrated at 580 kDa (594 kDa on the CN gel). Interestingly, this value differed from that of chloroplast ATPase that migrated on BN-PAGE in a band around 560 kDa.62 This discrepancy between predicted and experimental molecular weight values is in agreement with the identification of a hypothetical protein associated with the ATPase complex. In fact, spot 14 on 2D BN-SDS-PAGE (Figure 3A) was identified by MS analysis with the hypothetical protein RSP_2125. Analysis of the primary sequence of the RSP_2125 protein with bioinformatic softwares predicted a GRAVY score of -0.367 and no transmembrane helices (HMMTOP, see Table 2). On the basis of protein subcellular localization prediction (see above), we can conclude that the RSP_2125 protein was a cytoplasmic protein that interacts with the CF0 portion of ATPase. Cyt bc1 Complex. It is well-known that the R. sphaeroides cyt bc1 complex exists in a dimeric form both in crystal formation and in solution.63 The apparent molecular mass of cyt bc1 complex on our BN gels (118 kDa, Figure 1) appeared to be too low to accommodate two bc1 complexes, but it was

research articles

Proteomic Analysis of R. sphaeroides R26.1 ICM Vesicles in accordance with the predicted molecular weight (115 kDa) for a monomeric conformation. For this reason, it is our opinion that the monomeric cyt bc1 complex was formed by degradation of the native dimeric form during solubilization. By means of a second-dimension gel, we were only able to identify three subunits of the cyt bc1 complex (the cytochrome c1, [2Fe-2S] Rieske protein and subunit IV), missing the cytochrome b (fbcB). Surprisingly, we detected subunit IV (spot 47) in our 2D map. This protein is normally lost upon crystal formation;26 therefore, our finding confirmed the presence of subunit IV in solution. Several results have suggested that this subunit is essential for photosynthetic electron transfer activity.27 Identification of Hypothetical Proteins. Herein we newly identified three hypothetical proteins showing that these proteins exist and are indeed expressed in ICM vesicles from R. sphaeroides (see Table 2). A hypothetical protein is a protein whose existence has been predicted, but for which there is no experimental evidence that it is expressed in vivo. During genome analysis, when the bioinformatic tool used for gene identification finds a large open reading frame without an analogue in the protein database, it returns an answer of hypothetical protein as an annotation remark. Three of the identified hypothetical proteins (RSP_1200, RSP_3238, RSP_6207) were not clearly attached to a MPC and migrated as “free proteins”, whereas the proteins RSP_1762 and RSP_2125 migrated as a portion of the sarcosine oxidase enzyme and ATP synthase, respectively. Analysis of the primary sequence with GRAVY predicting software showed that only the RSP_1762 protein has a high grand average of hydropathicity (GRAVY) score (0.367). On the other hand, transmembrane predicting software (HMMTOP) found four membrane-spanning regions for RSP_1762 and one membrane-spanning region for proteins RSP_3238, RSP_1200, RSP_6207. In contrast, no membranespanning regions were found for the RSP_2125 protein. Accordingly, it is possible that RSP_1762 is an integral ICM membrane protein, whereas the other ones (RSP_3238, RSP_1200, RSP_6207, RSP_2125) may be peripheral ICM membrane proteins. On chromosome 1, the RSP_1762 gene was located near the RSP_1760 gene (spot 49, Figure 3) at about 1.6 Mb counterclockwise from a 70-kb region of the genome that encodes many proteins which are required for photosynthetic growth.2 Obviously, no clue can be obtained as to their function by analyzing the nearby DNA sequences in chromosome 1; however, it is interesting that Callister et al.64 showed that both RSP_1760 and RSP_1762 are only expressed in photosynthetic cells. Moreover, the same group demonstrated recently that a mutation in the RSP_1760 gene results in the failure of cell growth under photosynthetic conditions, but not under aerobic or dark-anaerobic conditions.31 With respect to any generalizations which may be made from these observations, on the basis of our BN gel data, it could be hypothesized that the RSP_1762 protein may be involved in a ICM vesicle complex of about 180 KDa together with the probable HflC protein (spot 36) or with the sarcosine oxidase enzyme. In fact, the real advantage of native techniques, such as blue native electrophoresis, is to create a bridge between interactomics and structural biology by providing new insights into the possible functional interactions of proteins in a system. Thus, the interesting aspect of a study such as this one is not so much the function of a single protein, but how the knowledge of native interactions may help the understanding of activities in which an unknown protein is involved. In line with this, we could not advance any

hypothesis about the possible role of the identified hypothetical gene products which migrated as “free” proteins (RSP_1200, RSP_3238, RSP_6207), whereas the RSP_2125 protein has been discussed in the ATPase section (see above). Other Complexes. In addition to the four MCPs involved in the photosynthetic process, our experimental approach successfully identified another two important MPCs. In accordance with their predicted molecular weights on the BN gel, the succinate dehydrogenase and the sarcosine oxidase (TSOX) comigrated with [RC+PufX+LH1]2 and with [LH2]2, respectively. Succinate dehydrogenase (respiratory complex II) is an enzyme complex which is bound to the inner membrane of many bacterial cells and participates in the bacterial electron transport chain.65 This complex with a predicted molecular weight of 369 kDa is a trimer of a heterotetramer composed of the four subunits SdhA/B/C/D. Here we were only able to identify two subunits: SdhA and B (spots numbered 16 and 18). Interestingly, these two subunits can exhibit succinate dehydrogenase activity in the absence of SdhC/D, which are the membrane components. On the basis of second-dimension apparent molecular weights, we can hypothesize that the subunits SdhC/D comigrated in the unidentified spot 20. As with spot number 9 of the ATPase F0 portion, we can ascribe the missed MS identification in the present case to the high hydrophobicity and to the low molecular weight of proteins contained in it. Detection of succinate dehydrogenase under our experimental conditions showed that this enzyme is also expressed when R. sphaeroides is grown anaerobically in the light (photosynthetically) and the complex is located in the ICM. This result agrees with previous reports which showed that Sdh is located in the chromatophores of phototrophically grown R. sphaeroides.66 Recently, Zeng et al. have found that two subunits of the succinate dehydrogenase are also found in the ICM.31 This finding corroborates previous studies which showed that some respiratory electron carriers were present in the ICM, either due to their role in the bioenergetic light reactions of photosynthesis or in the control of expression of the photosynthetic apparatus.67 Concerning sarcosine oxidase, we can say that it is a heterotetrameric sarcosine-inducible bacterial enzyme playing an important role in the catabolism of sarcosine (N-methylglycine), which is a common soil metabolite that can act as the sole source of carbon and energy for many microorganisms.68 TSOX contains four different subunits (R, β, γ, δ) that range in size from 100 to 10 kDa. The isolated enzyme contains three coenzymes: the noncovalently bound FAD and NAD+ and the covalently bound FMN. Here we identified two components of the TSOX enzyme, the R- (in spots 1 and 34) and β (in spots 2 and 35) subunits, respectively. Spots 1 and 2 were originated from BN gel band 1 (∼10 000 kDa), whereas spots 34 and 35 were from band 5 at 181 kDa. This confirmed that the TSOX complex is also an ICM-localized enzyme31 and that, in BN native gel, the enzyme migrated both as a complex (band 5, 181 kDa) and a supercomplex (band 1, ∼10 MDa).

Conclusions The use of simple and reproducible techniques, such as BN and CN gels, has allowed us to build the photosynthetic membrane assembly of our chosen model R. sphaeroides R26.1. Through this proteomic approach, our investigation has also revealed the existence in these complexes of new gene products allowing us to know their location and hypothesize about their functional and structural relationships to the process of solar Journal of Proteome Research • Vol. 9, No. 1, 2010 201

research articles energy utilization. It is worth pointing out that these results have been obtained using small amounts of sample, with respect to other alternative techniques and by means of simple instrumentation. Moreover, a semiquantitative estimation of the photosynthetic complexes can be obtained through the gel image analysis, so that the effects of several environmental stresses can be further elucidated in terms of changes in the composition of photosynthetic membranes. Finally, the absolute absence of any staining in the CN system allows a direct measurement of absorption spectra in pigment-binding complexes. This aspect is not to be underestimated because of its potential in the study of adaptive responses to stress. Abbreviations: ACN, acetonitrile; ATPase, ATP synthase; BN, blue native; CN, colorless native; Cyt bc1, cytochrome bc1 complex; GRAVY, grand average of hydropathicity; DDM, n-dodecyl-β-D-maltoside; ESI-MS/MS, electrospray ionizationtandem mass spectrometry; ICM, intracytoplasmic membrane; FA, formic acid; LC, liquid chromatography; LHC, light-harvesting complex; PAGE, polyacrylamide gel electrophoresis; PSU, photosynthetic unit; RC, reaction center; RP-HPLC, reversedphase-high performance liquid chromatography; SDS, sodium dodecyl sulfate; MPCs, multiprotein complexes; TSOX, sarcosine oxidase.

Acknowledgment. The authors are grateful to Dr. Massimo Trotta for his critical reading of the manuscript and helpful suggestions. This work was financially supported by the Italian National Blood Centre (CNS-ISS) and the “GENZOOT” research program funded by the Italian Ministry of Agriculture. Dr. G. M. D’Amici was the beneficiary of a fellowship granted by the Italian Proteomic Association (ItPA). Supporting Information Available: Table including sequences, m/z values, charge, start-end positions of peptides in protein; figure displaying the effects of the solubilization conditions on complexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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