Proteomics of Synechocystis sp. Strain PCC 6803
2002; Elsevier BV; Volume: 1; Issue: 12 Linguagem: Inglês
10.1074/mcp.m200043-mcp200
ISSN1535-9484
AutoresFang Huang, Ingela Parmryd, Fredrik Nilsson, Annika Persson, Himadri B. Pakrasi, Bertil Andersson, Birgitta Norling,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoCyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F0 membrane part of the ATP synthase. Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F0 membrane part of the ATP synthase. Cyanobacteria are unique prokaryotes due to the presence of a differentiated membrane system. Similar to other Gram-negative bacteria, cyanobacteria have an envelope consisting of an outer membrane, a peptidoglycan layer, and a plasma membrane (1.Stanier R.Y. Cohen-Bazire G. Phototrophic prokaryotes: the cyanobacteria.Annu. Rev. Microbiol. 1977; 31: 225-274Google Scholar, 2.Gantt E. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 119-138Google Scholar). In addition, these organisms have a distinct intracellular membrane system, the thylakoids, which are energy-transducing membranes and the site for both photosynthesis and respiration (2.Gantt E. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 119-138Google Scholar, 3.Cooley J.W. Vermaas W.F. Succinate dehydrogenase and other respiratory pathways in thylakoid membranes of Synechocystis sp. strain PCC 6803: capacity comparisons and physiological function.J. Bacteriol. 2001; 183: 4251-4258Google Scholar). The role of the cyanobacterial plasma membrane as an energy-transducing membrane is also crucial since a large number of biological processes are coupled to transmembrane ion potentials or dependent on membrane-bound ATPases as well as the respiratory chain (4.Peschek G.A. Wastyn M. Trnka M. Molitor V. Fry I.V. Packer L. Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans.Biochemistry. 1989; 28: 3057-3063Google Scholar, 5.Neisser A. Fromwald S. Schmatzberger A. Peschek G.A. Immunological and functional localization of both F-type and P-type ATPases in cyanobacterial plasma membranes.Biochem. Biophys. Res. Commun. 1994; 200: 884-892Google Scholar, 6.Norling B. Zarka A. Boussiba S. Isolation and characterization of plasma membranes from cyanobacteria.Physiol. Plant. 1997; 99: 495-504Google Scholar). The motility of the cell, nutrient uptake, and efflux pumps are also energy-dependent activities associated with the plasma membrane. The unicellular, naturally transformable cyanobacterium Synechocystis sp. PCC 6803 (henceforth referred to as Synechocystis) has been widely used for genetic and biochemical studies of photosynthesis and other metabolic processes (7.Pakrasi H.B. Genetic analysis of the form and function of photosystem I and photosystem II.Annu. Rev. Genet. 1995; 29: 755-776Google Scholar). It is the first photosynthetic organism for which the complete genome sequence is known (8.Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.DNA Res. 1996; 3: 109-136Google Scholar). The genome contains 3168 open reading frames (ORFs). 1The abbreviations used are: ORF, open reading frame; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; ASB-14, tetradecanoylamidopropyl dimethylammoniopropanesulfonate; PSI, photosystem I; PSII, photosystem II; Vipp1, vesicle-inducing protein in plastids; TAT, twin arginine translocation; EF-Tu, elongation factor Tu; Som, Synechococcus outer membrane; ABC, ATP-binding cassette. However, for about 40% of these ORFs it is not known whether they are transcriptionally active, and the potential gene products are known only as hypothetical proteins. Furthermore the cellular localization of these hypothetical proteins is not established, and this is also the case for many of the other proteins with a suggested function deduced from homology comparisons. Proteomic studies require, apart from the total genome knowledge, that each cellular compartment can be isolated without cross-contamination. As a crucial first step toward the analysis of the composition and function of the cyanobacterial plasma membrane, a procedure was developed for its complete biochemical purification (9.Norling B. Mirzakhanian V. Nilsson F.N. Morré D.J. Andersson B. Subfractional analysis of cyanobacterial membranes and isolation of plasma membranes by aqueous polymer two-phase partitioning.Anal. Biochem. 1994; 218: 103-111Google Scholar, 10.Norling B. Zak E. Andersson B. Pakrasi H. 2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis PCC680.FEBS Lett. 1998; 436: 189-192Google Scholar, 11.Norling B. Hatti-Kaul R. Methods in Biotechnology, Aqueous Two-phase Systems; Methods and Protocols. 11. Humana Press, Totowa, NJ1999: 185-192Google Scholar). In this procedure, two separation techniques, one based on surface properties (aqueous polymer two-phase partitioning) and the other on densities (gradient centrifugation) of the membrane vesicles, are combined to yield completely pure plasma and thylakoid membranes from Synechocystis cells. In the present work we have used the purified plasma membrane in proteome studies based on two-dimensional gel electrophoresis, trypsin treatment of excised spots, and MALDI-TOF analysis with powerful database identification. The identified plasma membrane proteins (a total of 57) show the following predicted membrane topology: 17 integral membrane proteins and 40 peripheral proteins of which 20 are located on the periplasmic side and 20 are on the cytoplasmic side of the membrane. Cells of Synechocystis 6803 were grown at 30 °C under 50 μmol of photons m−2 s−1 of white light in BG-11 medium (12.Allen M.M. Simple conditions for growth of unicellular blue-green algae on plates.J. Phycol. 1968; 4: 1-4Google Scholar). Liquid cultures were grown with vigorous bubbling with air. The cells were harvested at a density of 1.6–2.0 × 108 cells ml−1. Plasma membranes from Synechocystis were purified according to Norling et al. (10.Norling B. Zak E. Andersson B. Pakrasi H. 2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis PCC680.FEBS Lett. 1998; 436: 189-192Google Scholar) with the modification that sucrose density centrifugation was the first step and aqueous two-phase partitioning was the second step. Cells were broken with glass beads, and the total membranes were collected from the cell homogenate by centrifugation at 103,000 × g for 30 min. The pellet was homogenized in 20 mm potassium phosphate (pH 7.8), and solid sucrose was added to a concentration of 42% (w/w). A discontinuous sucrose gradient containing 20 mm potassium phosphate (pH 7.8) was made consisting of 5 ml of 60% (w/w), 5 ml of 50% (w/w), 7 ml of the sample in 42% (w/w), 6 ml of 38% (w/w), 6 ml of 35% (w/w), 6 ml of 30% (w/w), and 1 ml of 10% (w/w) sucrose. The gradient was centrifuged at 131,500 × g for 15 h at 4 °C. The membrane fraction from 38 to 42% sucrose density was collected and diluted 3-fold with 20 mm potassium phosphate (pH 7.8) followed by a centrifugation at 187,000 × g for 45 min at 4 °C. The pelleted membranes were resuspended in a small volume of 0.25 m sucrose and 5 mm potassium phosphate (pH 7.8), and the plasma membrane was isolated by aqueous two-phase partitioning (10.Norling B. Zak E. Andersson B. Pakrasi H. 2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis PCC680.FEBS Lett. 1998; 436: 189-192Google Scholar). Pure plasma membranes were recovered in the fifth top phase after three initial partitionings in a two-phase system consisting of 5.8% dextran T-500 and 5.8% polyethylene glycol 3350 and two more partitionings in a system containing 6.2% of both polymers (10.Norling B. Zak E. Andersson B. Pakrasi H. 2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis PCC680.FEBS Lett. 1998; 436: 189-192Google Scholar). The plasma membranes were washed with 0.1 m sodium carbonate (13.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Google Scholar). Plasma membranes containing 8 mg of protein were resuspended in 0.25 mm sucrose and 5 mm potassium phosphate (pH 7.8) at a protein concentration of 50–60 mg/ml. Eight milliliters of 0.1 m sodium carbonate was added, and the membrane suspension was stirred for 30 min on ice. The mixture was centrifuged at 125,000 × g for 40 min. The supernatant was removed, and the pellet was extracted with 0.1 m sodium carbonate for another 30 min. The pellet that resulted after the centrifugation was washed twice with 40 mm Tris to remove excess sodium carbonate. The final pellet of the membrane was resuspended in 0.25 mm sucrose and 5 mm potassium phosphate (pH 7.8). Protein concentration was determined according to Peterson (14.Peterson G.L. A simplification of the protein assay method of Lowry et al. which is more generally applicable.Anal. Biochem. 1977; 83: 346-356Google Scholar) using bovine serum albumin as a standard. Plasma membrane proteins were precipitated with methanol/chloroform according to the method described by Wessel (15.Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Google Scholar). The material precipitated from 1–1.5 mg of membrane proteins was solubilized in 250 μl of an electrofocusing solution containing 7 m urea, 2 m thiourea, 1% (w/v) ASB-14, 2 mm tributylphosphine, and 0.5% (v/v) immobilized pH gradient buffer, pH 3–10 (Amersham Biosciences). The mixture was incubated at room temperature for 1 h and then sonicated in the presence of protease inhibitor mixture (Sigma). After centrifugation at 9,000 × g for 3 min the supernatant was applied onto a linear immobilized pH gradient strip (pH 4–7, 13 cm). The strip was rehydrated overnight at 20 °C. The isoelectric focusing was performed at the same temperature, and the running conditions were 300 V for 40 min, 500 V for 40 min, 1,000 V for 1 h, and 8,000 V until a total of 100,000 V-h was reached. The strip was equilibrated in a buffer described by Nouwens et al. (16.Nouwens A.S. Cordwell S.J. Larsen M.R. Molloy M.P. Gillings M. Willcox M.D. Walsh B.J. Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.Electrophoresis. 2000; 21: 3797-3809Google Scholar) for 20 min and then loaded on the top of an SDS-polyacrylamide gel (12.5% polyacrylamide) prepared according to Laemmli (17.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Google Scholar) and sealed with 0.8% agarose. The electrophoresis was carried out at 5–6 °C and 5 mA/gel for 1 h and then 20 mA/gel using a Hoefer SE 600 apparatus (Amersham Biosciences). Proteins were detected by Coomassie Brilliant Blue G-250 according to Nouwens et al. (16) and then scanned using an image scanner and evaluated with the Image Master 2D Elite software (Amersham Biosciences). MALDI-TOF analysis was performed in reflector mode on a Voyager-DE STR MALDI-TOF mass spectrometer from Applied Biosystems (Foster City, CA). In-gel trypsin digestion and sample preparation for MALDI-TOF analysis was done manually in a way similar to that described by Fulda et al. (18.Fulda S. Huang F. Nilsson F. Hagemann M. Norling B. Proteomics of Synechocystis sp. PCC 6803: identification of periplasmic proteins in cells grown at low and high salt concentrations.Eur. J. Biochem. 2000; 267: 5900-5907Google Scholar). Internal mass calibration was performed using trypsin autodigestion products (842.5094 and 2211.1046 Da). The proteins were identified as the highest ranking result by searching in the National Center for Biotechnology Information (NCBI) database among all species using MS-Fit (prospector.ucsf.edu/ucsfhtml4.0u/msfit.htm). The search parameters allowed for oxidation of methionine, carbamidomethylation of cysteine, one miscleavage of trypsin, and 30 ppm mass accuracy. At least 50% of the measured masses must match the theoretical masses. Measured peptides masses could be excluded if their isotopic patterns were clearly atypical or if their masses corresponded to those of trypsin autolysis products or adjacent identified proteins on the gel. The identification of the proteins was repeated at least once using spots from different gels. The presence of putative signal peptides and their cleavage sites were predicted using the SignalP program (www.cbs.dtu.dk/services/SignalP-2.0). The prediction of transmembrane helices in identified proteins was performed using the TMHMM program (www.cbs.dtu.dk/services/TMHMM-2.0). The lipoproteins were predicted using PROSITE (us.expasy.org/prosite). Plasma membrane was purified from total membranes of Synechocystis by a combination of sucrose density centrifugation and aqueous two-phase partitioning (10.Norling B. Zak E. Andersson B. Pakrasi H. 2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis PCC680.FEBS Lett. 1998; 436: 189-192Google Scholar). Plasma membranes collected from several cyanobacterial preparations were used in producing four two-dimensional gel maps from which spots were excised and trypsinized before subjection to MALDI-TOF analysis. In the first dimension an immobilized linear pH gradient pH 4–7 was used applying the nonionic detergent ASB-14, and in the second dimension SDS-PAGE (12.5%) was used. The four gels showed identical visual patterns after Coomassie Brilliant Blue staining revealing ∼200 spots. One of these gels is shown in Fig. 1. From each of two of these four gels about 170 stained spots were analyzed. Proteins could be identified from about 110 spots, all together corresponding to 63 different gene products (Table I). Fig. 2 shows spectra of two identified proteins. In spectrum A (spot 28) a total of nine masses were used in the MS-Fit search and all of them fitted to Sll0617, Vipp1 (vesicle-inducing protein in plastids). In spectrum B (spot 47) five of a total of eight masses fitted to Sll1324, the b-subunit of the F0 membrane part of ATP synthase.Table IProteins identified in plasma membranes of SynechocystisNo.ORFGene productMatched peptides/totalMature proteinpI predict./exper.Mass theor./exper.1Sll1021Hypothetical7/115.1/5.2–5.474.4/802Slr1841Putative porin4/74.4/4.565.0/703Sll041660-kDa chaperonin 2 GroEL-24/64.9/5.157.8/664Sll1053Membrane fusion protein MtrC6/74.9/5.156.3/645Sll1699Periplasmic oligopeptide-binding protein7/115.2/5.3–5.664.5/636Slr1624Hypothetical8/144.2/4.346.2/607Slr1270TolC10/144.7/4.7,4.853.6/608Sll1326ATP synthase α chain5/75.0/5.1,5.254.0/609Sll0141Membrane fusion protein9/145.0/5.247.9/6010Slr1506Hypothetical8/105.1/5.261.1/6011Sll0180Membrane fusion protein10/125.3/5.3–5.652.0/5912Slr1908Putative porin8/104.9/4.9–5.261.6/5513Slr1220Hypothetical5/64.2/4.436.0/5414Slr0872Hypothetical3/45.3/5.341.5/5415Sll1099EF-Tu11/155.2/5.543.7/5416Sll0606Hypothetical6/75.8/5.950.7/5017Slr0447Putative periplasmic binding protein4/74.7/4.845.5/4618Slr0040Bicarbonate transporter substrate-binding protein CmpA6/95.1/4.9–5.246.8/4619Slr1751Carboxyl-terminal protease CtpC16/174.9/5.244.4/4620Sll0752Hypothetical7/94.8/4.8–5.031.4/4521Slr1897SrrA protein7/75.0/5.0,5.143.7/4222Slr1274Membrane protein PilM10/114.5/4.740.9/4223Slr1275PilN3/44.5/4.530.0/4124Slr0151Hypothetical6/75.0/5.134.9/4125Slr1276PilO6/74.2/4.330.1/3926Slr1295Periplasmic iron-binding protein FutA16/74.8/4.836.3/3927Sll0034Hypothetical4/65.8/6.0,6.328.6/3928Sll0617Vipp1 Im309/95.0/5.0–5.128.9/3829Slr0513Periplasmic iron-binding protein FutA28/95.2/5.2,5.334.9/3830Sll1363Ketol-acid reductoisomerase IlvC3/45.0/5.140.0/3731Slr1319Iron(III) dicitrate periplasmic binding protein FecB4/54.8/4.933.0/3632Slr1106Prohibitin6/95.2/5.230.6/3533Slr1768Hypothetical7/95.6/5.0,5.231.8/3434Slr0875Large conductance mechanosensitive channel MscL3/54.7/4.615.8/3334Slr08754/54.7/4.615.8/1735Slr1377Leader peptidase type I LepB6/85.4/5.2,5.324.7/3336Sll1041ATP-binding protein of ABC transporter6/85.4/5.228.7/3037Sll0427PsbO3/44.7/4.826.9/3038Slr0431Hypothetical7/84.8/4.822.5/3039Slr1258Hypothetical4/74.8/5.2,5.423.7/2940Slr0677ExbB protein, TolQ3/55.1/5.222.1/2841Slr1128Stomatin-like protein4/65.7/5.433.8/2842Sll0947Light-repressed protein, LrtA6/66.1/6.621.9/2743Sll1835Hypothetical3/54.7/4.825.0/2744Slr1730Potassium-transporting ATPase C chain, KdpC4/64.5/4.817.8/2745Slr1881High affinity branched-chain amino acid transport ATP-binding protein BraG (LivF)6/75.7/5.825.7/2746Sll0749Hypothetical4/44.5/4.617.9/2547Sll1324ATP synthase subunit b AtpF5/85.1/5.219.8/1948Sll0813Cytochrome c oxidase subunit II CoxB4/76.0/6.2,6.629.5/1949Slr0695Hypothetical lipoprotein5/105.5/5.416.2/1850Sll1578Phycocyanin α chain4/55.3/5.217.6/1851Slr0737Photosystem I subunit II PsaD4/78.9/7.015.7/1952Sll1323ATP synthase subunit b′ AtpG5/85.2/5.116.2/1653Sll0819Photosystem I subunit III PsaF5/86.1/6.615.7/1654Slr0013Hypothetical8/96.5/5.613.7/1555Sll0565Hypothetical3/54.6/4.915.2/1556Sll1405ExbD protein, TolR3/49.0/5.615.6/1457Slr1513Hypothetical4/66.6/5.7–7.012.0/1458Sll1638Hypothetical lipoprotein4/55.6/5.1,5.414.2/1459Ssl0707Nitrogen regulatory protein P-II GlnB6/76.6/5.7,6.512.4/1260Sll1028Carbon dioxide-concentrating mechanism protein CcmK4/75.3/5.611.1/1261Sll1118Hypothetical6/65.6/5.911.5/1262Sll1029Carbon dioxide-concentrating mechanism protein CcmK5/66.1/6.512.1/1063Ssl0563PsaC2/35.7/6.08.8/9 Open table in a new tab Fig. 2MALDI-TOF mass spectra of spot numbers 28 and 47 from the two-dimensional gel map of Synechocystis plasma membrane proteins.A, the MALDI-TOF mass spectrum of peptides generated by tryptic digestion of protein spot 28. B, the MALDI-TOF mass spectrum of peptides generated by tryptic digestion of protein spot 47. The spectra were internally calibrated using the trypsin-autodigested peptides 842.5094 and 2211.1046. Spot 28 matched Vipp1, and spot 47 matched the b-subunit of the F0 membrane part of ATP synthase.View Large Image Figure ViewerDownload (PPT) Proteins of the cyanobacterial plasma membrane can be of three types: integral hydrophobic membrane proteins and peripheral (extrinsic) hydrophilic proteins located on the outer (periplasmic) or inner (cytoplasmic) surface of the membrane. Peripheral proteins located on the inner side do not need an amino-terminal signal sequence for their cellular targeting, whereas peripheral proteins located at the outer side contain such a signal (19.von Heijne G. Transcending the impenetrable: how proteins come to terms with membranes.Biochim. Biophys. Acta. 1988; 947: 307-333Google Scholar). In addition some integral membrane proteins have a signal peptide. Fig. 3 illustrates the distribution of the identified proteins according to membrane topology as will be discussed below. About two-thirds of the proteins identified in the present work have known functions as judged either by genetic or biochemical studies or annotated from high sequence similarity. For many of these proteins their cellular localization has not been previously established. About one-third of the proteins are annotated as hypothetical proteins due to lack of sequence similarity with any other protein with known function, thus their functional significance in the plasma membrane remains to be elucidated. In the present work the expression and the localization of these hypothetical proteins are determined. As can be seen in the gel map of Fig. 1 several proteins produced multiple spots probably due to post-translational modifications. In most cases the shift in position is horizontal, suggesting that the modification influences only the pI and leaves the molecular mass substantially unchanged. Thus, the modifications are in the side chains of the amino acids rather than due to differential processing of the precursor molecules. These types of protein modifications are currently under investigation. It is well known that there are limitations in the present technology for resolution and identification of integral membrane proteins by two-dimensional gel electrophoresis and MALDI-TOF analysis. Hydrophobic proteins are not easily solubilized in the nonionic detergents used for isoelectric focusing, and the hydrophobic fragments obtained after trypsin treatment are difficult to ionize for mass spectrometer analysis. By using the TMHMM program (20.Krogh A. Larsson B. von Heijne G. Sonnhammer E.L.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Google Scholar), currently the best performing transmembrane prediction program (21.Moller S. Croning M.D.R. Apweiler R. Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics. 2001; 17: 646-653Google Scholar), only 17 integral membrane proteins could be identified in the isolated plasma membranes (Table II). Notably as many as 12 of these proteins only possess one single transmembrane helix, and the rest have two or three transmembrane helices. This means that a majority of the resolved and identified proteins are peripheral plasma membrane proteins (Fig. 3).Table IIIntegral membrane proteins identified in the plasma membrane of SynechocystisORFGene productPosition of transmembrane helicesSlr0677ExbB protein, TolQ13–35, 111–135, 153–175Slr0875Large conductance mechanosensitive channel MscL30–52, 72–94Slr1106Prohibitin13–35Slr1275PilN39–61Slr1276PilO37–59Slr1377Leader peptidase type I LepB21–43Slr1730Potassium-transporting ATPase C chain KdpC13–35Slr1768Hypothetical1–20, 31–52Sll0034Hypothetical40–59Sll0606Hypothetical19–41Sll0813Cytochromec oxidase subunit II CoxB48–70, 91–113Sll0819Photosystem I subunit III PsaF88–110, 123–145Sll1021Hypothetical60–82Sll1053Membrane fusion protein MtrC40–59Sll1323ATP synthase subunit b′ AtpG10–27Sll1324ATP synthase subunit b AtpF30–50Sll1405ExbD protein, TolR21–43 Open table in a new tab By applying the SignalP program for Gram-negative bacteria (22.Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.Protein Eng. 1997; 10: 1-6Google Scholar) all identified proteins were analyzed, and for 25 of them a signal peptide could be predicted (Table III). Only two of the identified integral membrane proteins have a signal peptide, cytochrome c oxidase subunit II (CoxB) and photosystem I subunit III (PsaF). As summarized in Table III typical Sec signal amino-termini (23.von Heijne G. Signal sequences. The limits of variation.J. Mol. Biol. 1985; 184: 99-105Google Scholar, 24.Pugsley A.G. The complete general secretory pathway in Gram-negative bacteria.Microbiol. Rev. 1993; 57: 50-108Google Scholar) were found in 13 of the identified proteins, while three proteins have the twin arginine signal peptide motif TAT (25.Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. Overlapping functions of components of bacterial Sec-independent protein export pathway.EMBO J. 1998; 17: 3640-3650Google Scholar, 26.Weiner J.H. Bilous P.T. Shaw G.M. Lubitz S.P. Frost L. Thomas G.H. Cole J.A. Turner R.J. A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins.Cell. 1998; 93: 93-101Google Scholar). For one of the hypothetical proteins (Slr1506) we suggest that the methionine which is in position 46 is the actual start of the protein since then a typical signal peptide is clearly predicted in the N terminus. For another hypothetical protein (Slr0431) a 41-amino acid, or a 30-amino acid, signal peptide could be predicted by the SignalP program (22.Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.Protein Eng. 1997; 10: 1-6Google Scholar). Among the three proteins with TAT signals, two (Slr0447 and Slr0513) have a "Sec-avoidance" positive charge in the carboxyl-terminal region just upstream of the signal peptidase cleavage site (27.Bogsch E. Brink S. Robinson C. Pathway specificity for a pH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein.EMBO J. 1997; 16: 3851-3859Google Scholar, 28.Cristobal S. de Gier J.W. Nielsen H. von Heijne G. Competition between Sec- and Tat-dependent protein translocation in Escherichia coli.EMBO J. 1999; 18: 2982-2990Google Scholar). We found earlier that the periplasmic proteins of Synechocystis contain both Sec and TAT signals (18.Fulda S. Huang F. Nilsson F. Hagemann M. Norling B. Proteomics of Synechocystis sp. PCC 6803: identification of periplasmic proteins in cells grown at low and high salt concentrations.Eur. J. Biochem. 2000; 267: 5900-5907Google Scholar).Table IIIPrediction of signal peptides in proteins identified in the plasma membrane of SynechocystisORFGene productSignal sequenceaaSec signalSlr0013HypotheticalMKLIDSRGRIFGIVSLLDLGAALIILMVAVGIFVLPGSSGKSILAQANA-AS49Slr0431HypotheticalMRPKFFSRRPT41M*GISKLSKFSASVLLSGAILTTLPPSPLWA-NE30aAssuming that the second methionine represents the real translational starting point.Slr1258HypotheticalMSTIKALLPPKFPQLLTGLALLSLSLVVSTAIA-AK33Slr1270TolCMKSIHPLKFWSSSTLLLLLSTSVGVFLPGFSGGQGAIAVA-QS40Slr1624HypotheticalMLDRHWHNQNNCRPSYWSHVTTVLTICLLAIAMGLGGCQSLSA-SS43Slr1751Carboxyl-terminal protease CtpCMLKQKRSLILGTTALLLTTVAVT-GV23Slr1841Putative porinMLKLSWKSLLVSPAVIGAALVAGAASA-AP27Slr1908Putative porinMNKLTSHLLKLFPLALGSSLAIVPGAMA-QS28Sll0427PsbOMRFRPSIVALLSVCFGLLTFLYSGSAFA-VD28Sll0749HypotheticalMEKIMSEQKSSSSLTGFALAALMVALVGTGFAF-WT32Sll0813Cytochrome c oxidase subunit II CoxBMSRKNLILLAVYIVFTVGASLWLGQRAYQWLPPAAA-QE36Sll0819PsaFMKHLLALLLAFTLWFNFAPSASA-DD23Sll1835HypotheticalMATHNLDRVAAPLISKLFPFFLVLAGMFSGTLAAQA-QG36TAT signalSlr0447Putative periplasmic binding proteinMTNPFGRRKFLLYGSATLGASLLLKA-CG26Slr0513Periplasmic iron-binding protein FutA2MTTKISRRTFFVGGTALTALVVANLPRRASA-QS31Slr1506Hypothetical (Met-46 as start)MVTFPLNLRRWLQSVCLGALTAIA-VQ24LipoproteinSlr0695HypotheticalMRKRLTRFLSLALVLGLLWFGTAA-CASQP24Slr1295Periplasmic iron-binding protein FutA1MVQKLSRRLFLSIGTAFTVVVGSQLLSS-CGQSP28Slr1319Iron(III) dicitrate periplasmic binding protein FecBMKSKLIIFTFCLVLFG-CAKQV16Slr1897SrrAMVSWCRWRSPRRWFLFACLGLLLSGLIS-CQSNS28Sll0141Membrane fusion proteinMNKYIPHQRLRRQLSLLGLLSFSLMG-CSDLW26Sll0180Membrane fusion proteinMVRKRSQFPVIGSMVALALLNTA-CGGDK25Slr0040Bicarbonate transporter substrate-binding protein CmpAMGSFNRRKFLLTSAATATGALFLKG-CAGNP25Sll1638HypotheticalMSRLRSLLSLILVLVTTVLVS-CSSPQ22Sll1699Periplasmic oligopeptide-binding proteinMRWGNKVAM*SRVAGQRKTAIAREKNPGQQNYLSGRSWGQKLIS-57 ALLCCLALTFSLGG-CFSPE49aAssuming that the second methionine represents the
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