The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains
2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês
10.1074/jbc.m503035200
ISSN1083-351X
AutoresFranziska Ertel, Oliver Mirus, Rolf Bredemeier, Sunčana Moslavac, Thomas Becker, Enrico Schleiff,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoSeveral β-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the β-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the β-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution. Several β-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the β-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the β-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution. β-barrel-type channels are involved in the translocation of polypeptides (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar), the assembly of proteins in the outer membrane of endosymbiotic organelles (2.Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Crossref PubMed Scopus (347) Google Scholar, 3.Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 4.Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Crossref PubMed Scopus (314) Google Scholar), or in the assembly of proteins in the outer membrane of bacteria (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar, 6.Jacob-Dubuisson F. Locht C. Antoine R. Mol. Microbiol. 2001; 40: 306-313Crossref PubMed Scopus (212) Google Scholar). These proteins belong to one class, which can be termed polypeptide-transporting β-barrel channels (2.Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Crossref PubMed Scopus (347) Google Scholar, 4.Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Crossref PubMed Scopus (314) Google Scholar, 7.Yen M.R. Peabody C.R. Partovi S.M. Zhai Y. Tseng Y.H. Saier M.H. Biochim. Biophys. Acta. 2002; 1562: 6-31Crossref PubMed Scopus (155) Google Scholar). Four proteins are in the focus of recent investigation, namely the bacterial outer membrane proteins Omp85 and ShlB, the mitochondrial outer membrane protein Tob55/Sam50, and the chloroplast outer envelope protein Toc75. ShlB is an outer membrane protein involved in the secretion of hemolysins or adhesins in various Gram-negative pathogens (8.Schiebel E. Schwarz H. Braun V. J. Biol. Chem. 1989; 264: 16311-16320Abstract Full Text PDF PubMed Google Scholar, 9.Hertle R. Brutsche S. Groeger W. Hobbie S. Koch W. Konninger U. Braun V. Mol. Microbiol. 1997; 26: 853-865Crossref PubMed Scopus (43) Google Scholar). Omp85 is an essential component for outer membrane biogenesis in Neisseria meningitidis that might have two functions: the assembly of outer membrane proteins (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar) and the translocation of lipids (10.Genevrois S. Steeghs L. Roholl P. Letesson J.J. van der Ley P. EMBO J. 2003; 22: 1780-1789Crossref PubMed Scopus (97) Google Scholar). Recently, it was discussed that the effect on lipid transfer by Omp85 depletion might be indirect and explained by an assembly defect of the required outer membrane protein, suggesting a function of Omp85 in outer membrane protein assembly only (11.Voulhoux R. Tommassen J. Res. Microbiol. 2004; 155: 129-135Crossref PubMed Scopus (115) Google Scholar). As for ShlB, a β-barrel transmembrane structure was suggested for Omp85 (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar). Recently, a new polypeptide-transporting protein was identified in the outer membrane of mitochondria and termed Sam50 (3.Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), Tob55 (2.Paschen S.A. Waizenegger T. Stan T. Preuss M. Cyrklaff M. Hell K. Rapaport D. Neupert W. Nature. 2003; 426: 862-866Crossref PubMed Scopus (347) Google Scholar), or mitochondrial Omp85 homologue (4.Gentle I. Gabriel K. Beech P. Waller R. Lithgow T. J. Cell Biol. 2004; 164: 19-24Crossref PubMed Scopus (314) Google Scholar). This protein facilitates the assembly of proteins into the outer membrane of mitochondria. Tob55/Sam50 is found in a larger complex with Mas37 (3.Kozjak V. Wiedemann N. Milenkovic D. Lohaus C. Meyer H.E. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2003; 278: 48520-48523Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 12.Gratzer S. Lithgow T. Bauer R.E. Lamping E. Paltauf F. Kohlwein S.D. Haucke V. Junne T. Schatz G. Horst M. J. Cell Biol. 1995; 129: 25-34Crossref PubMed Scopus (152) Google Scholar) and Tob38/Sam35 (13.Waizenegger T. Habib S.J. Lech M. Mokranjac D. Paschen S.A. Hell K. Neupert W. Rapaport D. EMBO Rep. 2004; 5: 704-709Crossref PubMed Scopus (104) Google Scholar, 14.Milenkovic D. Kozjak V. Wiedemann N. Lohaus C. Meyer H.E. Guiard B. Pfanner N. Meisinger C. J. Biol. Chem. 2004; 279: 22781-22785Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15.Ishikawa D. Yamamoto H. Tamura Y. Moritoh K. Endo T. J. Cell Biol. 2004; 166: 621-627Crossref PubMed Scopus (131) Google Scholar). The fourth investigated β-barrel-type polypeptide transporter is the 75-kDa subunit of the translocon of the outer envelope of chloroplasts, Toc75. Toc75 forms a complex with Toc34, Toc64, and Toc159 (16.Soll J. Schleiff E. Nat. Rev. Mol. Cell Biol. 2004; 5: 198-208Crossref PubMed Scopus (334) Google Scholar). In contrast to the other identified polypeptide transporters, such as Omp85, the translocation of proteins through Toc75 requires the action of assisting proteins, such as Toc159 (17.Schleiff E. Jelic M. Soll J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4604-4609Crossref PubMed Scopus (104) Google Scholar), but still Toc75 seems to contain a preprotein-binding site as determined by electrophysiological measurements (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar). Topological modeling of Toc75 from Pisum sativum (18.Sveshnikova N. Grimm R. Soll J. Schleiff E. Biol. Chem. 2000; 381: 687-693Crossref PubMed Scopus (61) Google Scholar) or Toc75-V from Arabidopsis thaliana (19.Schleiff E. Eichacker L.A. Eckart K. Becker T. Mirus O. Stahl T. Soll J. Protein Sci. 2003; 12: 748-759Crossref PubMed Scopus (99) Google Scholar) suggests a β-barrel-type structure. Previously, it was proposed that Toc75 might have evolved from the ShlB (20.Bölter B. Soll J. Schulz A. Hinnah S. Wagner R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15831-15836Crossref PubMed Scopus (139) Google Scholar, 21.Reumann S. Davila-Aponte J. Keegstra K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 784-789Crossref PubMed Scopus (161) Google Scholar) or from the Omp85 class (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar). This relationship to prokaryotic proteins is in line with the theory that chloroplasts have evolved from the ancestor of cyanobacteria (22.Martin W. Rujan T. Richly E. Hansen A. Cornelsen S. Lins T. Leister D. Stoebe B. Hasegawa M. Penny D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12246-12251Crossref PubMed Scopus (908) Google Scholar, 23.Timmis J.N. Ayliffe M.A. Huang C.Y. Martin W. Nat. Rev. Genet. 2004; 5: 123-135Crossref PubMed Scopus (1030) Google Scholar). Here we identified the Nostoc PCC7120 homologue of Toc75/Omp85. To understand the evolutionary relationship between Toc75 and alr 2269, we investigated the specific properties of the two proteins. We here present experimental evidence that the N-terminal domain of β-barrel-type polypeptide transporters is involved in the recognition of substrates and complex assembly, whereas the C-terminal domain assembles the poreforming β-barrel. Construct Generation, Expression, and in Vitro Translation—Translation or expression of Toc34ΔTM, pSSU 1The abbreviations used are: pSSU/mSSU, precursor/mature form of the small subunit of Rubisco; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; MOPS, 4-morpholinepropanesulfonic acid; aa, amino acids; Ni-NTA, nickel-nitrilotriacetic acid; nS, nanosiemens; pS, picosiemens. , mSSU, pOE33, or pOE23 was described previously (24.Schleiff E. Motzkus M. Soll J. Plant Mol. Biol. 2002; 50: 177-185Crossref PubMed Scopus (21) Google Scholar, 25.Becker T. Jelic M. Vojta A. Radunz A. Soll J. Schleiff E. EMBO J. 2004; 23: 520-530Crossref PubMed Scopus (114) Google Scholar). psToc75 cDNA (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar) was used as the template for the generation of psA, psB, psC, and psD (Fig. 1) by PCR. The alr2269 cDNA was amplified from genomic Nostoc PCC7120 DNA. Constructs were generated by PCR, cloned into pTrcHis2 TOPO® TA (Invitrogen), and controlled by sequencing. BL21 cells (Novagen) transformed with plasmids were incubated in LB medium at 37 °C, and expression was induced by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested 2 h after induction and lysed using a French press (at 40 megapascals) in 50 mm NaPi, pH 8.0, 100 mm NaCl, and 2 mm β-mercaptoethanol (buffer A). Full-length proteins and C-terminal constructs (Fig. 1D) were accumulated in inclusion bodies. After centrifugation for 15 min at 25,000 × g at 4 °C, the pellet was resuspended in 50 mm NaPi, pH 8, 150 mm NaCl, 10 mm β-mercaptoethanol, 20 mm imidazole, and 5 m urea. The solution was centrifuged at 20,000 revolutions/min for 15 min at 4 °C, the supernatant was subjected to nickel-agarose (Qiagen, Hilden, Germany), and the protein was purified according to the manual. Soluble fragments (Figs. 1D and 2A) were loaded onto nickel-agarose after centrifugation of lysed cells for 15 min at 25.000 × g at 4 °C. After purification, proteins were dialyzed against 50 mm NaPi, pH8, 100 mm NaCl. Their concentration was determined by Lowry analysis. The rapid translation system RTS 100 Escherichia coli HY Kit (Roche Applied Science) was used for in vitro translation. Antibodies against alr2269 were produced as previously described (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar).Fig. 2The preprotein-binding region of psToc75. A, purified proteins representing the N-terminal region of psToc75 (lanes 1–4) or alr2269 (lanes 5–8) were separated by SDS-PAGE followed by Coomassie Blue staining. For purity control, 1 μl(lanes 1, 3, 5, and 7) or 5 μlof the indicated protein (lanes 2, 4, 6, and 8) were loaded. B, the far UV CD spectrum of anaA (1 μm, gray line) or anaB (5 μm, black line) was recorded as described under "Materials and Methods." C, pOE23 (lanes 1–3) or mSSU (lanes 4–6) were coupled to Toyopearl in equal molar amounts, as described, and incubated with 35S-labeled psA (upper panel) or psB (lower panel). A 20% translation product is shown (TP). Flow-through (F, lanes 1 and 4), wash (W, lanes 2 and 5), and elution (E, lanes 3 and 6) were collected and separated by SDS-PAGE, and radioactive labeled proteins were visualized. D, pOE23 (lanes 1–3), mSSU (lanes 4–6), or pOE33 (lanes 7–9) were coupled to Toyopearl (lanes 10–12) in equal molar amounts and incubated with expressed psA (L, upper panel) or psB (L, lower panel). Flow-through (F, lanes 1, 4, 7, and 10), wash (W, lanes 2, 5, 8, and 11), and elution (E, lanes 3, 6, 9, and 12) were collected and separated by SDS-PAGE followed by silver staining. E, the transit peptide of pSSU is shown as a bar diagram, and the positions of the synthetic peptides (E2, black; A1, white; B2, gray) are indicated. F, psB (1 μg) was incubated with A1 (lane 2)-, B2 (lane 3)-, E2 (lane 4)-, or bovine serum albumin (lane 5)-charged affinity matrix (charging was controlled by absorption spectroscopy of the flowthrough). Bound proteins were eluted and analyzed by immunodecoration using αToc75 antibodies. 10% input is shown (lane 1).View Large Image Figure ViewerDownload (PPT) Cell Fractionation of Nostoc PCC7120 —Nostoc PCC7120 was grown photoautotrophically at 30 °C in BG-11 medium (26.Allen M.M. J. Phycol. 1968; 4: 1-4Crossref PubMed Scopus (1021) Google Scholar) under constant illumination at 70 μmol of photons m–2 s–1 with aeration by air containing 1% CO2. Cells were harvested at OD750 = 1.0 by centrifugation at 4,000 × g for 10 min and then washed and lysed by French press at 40 megapascals. Broken cells were centrifuged at 48,000 × g for 45 min at 4 °C. The pellet was resuspended in 55% sucrose solution containing 20 mm HEPES, pH 8.0, and 0.2 mm phenylmethylsulfonyl fluoride. Floating sucrose density gradients were prepared by overlaying the cell/sucrose mixture with 40, 30, and 10% sucrose solutions and centrifuged at 130,000 × g for 16 h at 4 °C. Plasma membrane, thylakoid membrane, and outer membrane were collected, diluted in 20 mm HEPES, pH 8.0, and 1 mm phenylmethylsulfonyl fluoride and collected by centrifugation at 380,000 × g for 1 h at 4 °C and resuspended and stored at –20 °C. Binding Analysis—The peptides A1, B2, and E2 were synthesized at the Department of Peptide and Protein Chemistry, Charite (Berlin, Germany). Binding experiments using nickel-agarose or Toyopearl-AF-Tresyl 650 m (TosoHaas Corporation, Tokyo, Japan) were performed as described previously (24.Schleiff E. Motzkus M. Soll J. Plant Mol. Biol. 2002; 50: 177-185Crossref PubMed Scopus (21) Google Scholar, 25.Becker T. Jelic M. Vojta A. Radunz A. Soll J. Schleiff E. EMBO J. 2004; 23: 520-530Crossref PubMed Scopus (114) Google Scholar). Here, for one experiment similar molar amounts of preproteins (Fig. 2), synthetic peptides (Fig. 2), or constructs of alr2269 or Toc75 (Figs. 3 and 4) were coupled to the material. The coupled amount was controlled by adding defined amounts for coupling and analyzing the concentration of proteins or peptides in the flow-through not associated with the matrix. For cross-linking analysis, wheat germ-translated 35S-labeled pSSU was incubated with psToc75 or alr2269 constructs at 1 μm in 50 mm NaPi, pH 8.0, and 100 mm NaCl in 50 μl of bis(sulfosuccinimidyl)suberate (1 mm final, BS3; Perbio, Bonn, Germany) was added and quenched after 45 min of incubation at 4 °C by the addition of glycine (100 mm final). The binding efficiency was determined using AIDA software, expressed to the percent of loaded material and normalized to the indicated binding reaction.Fig. 4The N-terminal domain of the proteins is involved in complex formation. A, 35S-labeled Toc34ΔTM was incubated with psA, psB (upper panel), anaA, or anaB (lower part) coupled to Ni-NTA. Flow-through (FT, lanes 2 and 4) and elution (E, lane 3, 5) were collected, separated by SDS-PAGE, and radioactivity-visualized. 100% of the translation product is shown (TP, lane 1). B, interaction between the fragments of psToc75 or alr2269 and Toc34ΔTM was quantified and normalized to the interaction efficiency of psA and Toc34ΔTM. The average of at least three experiments is shown. C, 35S-labeled pSSU was incubated with psA (lanes 1–3) or anaA (lanes 4–6) coupled to Ni-NTA in the presence of expressed Toc34ΔTM (T34, lanes 2 and 5) or the transit peptide of pSSU (P, lanes 3 and 6). Bound protein was collected and visualized by phosphorimaging. 100% of the translation product is shown (TP). D, 35S-labeled anaA was incubated with psA, psB (upper panel), anaA, or anaB (lower panel) coupled to Ni-NTA (empty). Flow-through (FT, lanes 2, 4, and 6) and elution (E, lanes 3, 5, and 7) were collected and radioactivity-visualized. 100% of the translation product is shown (TP, lane 1). E, the interaction between the fragments of psToc75 or alr2269 and anaA (black bars) or anaB (gray bars) was quantified and normalized to the interaction efficiency of anaA dimerization. The average of at least three experiments is shown. F, a model of the interacting regions and the recognized targets for alr2269 and psToc75 is shown.View Large Image Figure ViewerDownload (PPT) Liposome Swelling Assay and Transport-specific Fractionation—Lipids for reconstitution were supplied by Nutfield Nurseries (Surrey, UK). Liposomes were prepared and proteins were reconstituted as described previously (17.Schleiff E. Jelic M. Soll J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4604-4609Crossref PubMed Scopus (104) Google Scholar). The insertion was controlled by extraction. Swelling analysis was performed as previously established (27.Luckey M. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 167-171Crossref PubMed Scopus (184) Google Scholar). The indicated amounts of NaCl or sucrose were added into liposome-containing solution, and the optical density at 500 nm (Fig. 5, B and C) or the integral of the optical density between 400–700 nm (D and E) was determined. Transport-specific fractionation was previously described (28.Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For visualization, 0.1 mol % of rhodamine-phosphatidylethanolamine (Avanti Inc., Alabaster) was added to the lipid mixture. Bioinformatic Tools—The alignment was conducted using ClustalW at the BCM search launcher server (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and visualized with Boxshade 3.21 (www.ch.embnet.org/software/BOX_form.html). Electrophysiological Measurements—Mega-9 (80 mm final) was added to purified psC, alr2269 or anaC. l-α-phosphatidylcholine (type IV-S, Sigma) was dissolved in 80 mm Mega-9, 10 mm MOPS/Tris (pH 7.0). Both samples were mixed (1 mg of protein/20 mg of lipid) and dialyzed against 2 liters of 10 mm KCl, 10 mm MOPS/Tris, pH 7.0, for 2 h at 25 °C and subsequently overnight at 4 °C. Planar lipid bilayers were produced using the painting technique (29.Mueller P. Rudin D. Tien R. Westcott W.C. J. Phys. Chem. 1963; 67: 534-535Crossref Scopus (394) Google Scholar). A solution of 75 mg/ml l-α-phosphatidylcholine in n-decan was applied to a hole in a Teflon septum, separating the two 3-ml chambers. To form a stable bilayer in 20 mm KCl, the 10 mm MOPS/Tris, pH 7.0, solution level was raised and lowered several times. The solution of the cis chamber was then exchanged to 250 mm KCl, 10 mm CaCl2, 10 mm MOPS/Tris, pH 7.0. Proteoliposomes were added to the cis chamber below the bilayer to allow the flow of the liposomes across the bilayer. The solution in the cis chamber was stirred to promote fusion. After fusion, the electrolytes in both compartments were changed to the final composition. Silver/AgCl electrodes were connected to the chambers through 2 m KCl-agar bridges. The electrode of the trans compartment was connected directly to the head stage of a current amplifier (GeneClamp500B, Axon Instruments, Union City, CA). The amplified currents were recorded using the pCLAMP9 software (Axon Instruments). Circular Dichroism Measurements—Circular dichroism spectra were recorded in 1-nm steps for 2 s of integration time, and a slit width of 2 nm in 10 mm HEPES/KOH, pH 7.6, 100 mm NaCl on a Jobin Yvon CD6 spectrometer (Division d'Instruments, SA) at 22 °C using a cuvette with a 1-mm path length. 15 spectra were recorded and averaged. The rough data were further manipulated as described previously (30.Schleiff E. Turnbull J.L. Biochemistry. 1998; 37: 13043-13051Crossref PubMed Scopus (35) Google Scholar). Previously, the protein slr1227 from Synechocystis PCC6803 was described as a protein related to Toc75 (20.Bölter B. Soll J. Schulz A. Hinnah S. Wagner R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15831-15836Crossref PubMed Scopus (139) Google Scholar, 21.Reumann S. Davila-Aponte J. Keegstra K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 784-789Crossref PubMed Scopus (161) Google Scholar). More recently, it was proposed that the Nostoc gene complement is most closely related to that of the ancestor of plastids based on the analysis of Nostoc punctiforme (22.Martin W. Rujan T. Richly E. Hansen A. Cornelsen S. Lins T. Leister D. Stoebe B. Hasegawa M. Penny D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12246-12251Crossref PubMed Scopus (908) Google Scholar). To understand the evolutionary development of the polypeptide-transporting β-barrel protein Toc75, we wanted to compare the properties of psToc75 from P. sativum and a protein of the genus Nostoc. We therefore analyzed the genome of the two species N. punctiforme and Nostoc PCC7120 for the presence of a homologue of the polypeptide transporters Omp85 from N. meningitidis (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar) and psToc75 (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar) (supplemental figure, panel A). We identified two proteins related to psToc75/Omp85, namely Npun02006512 and alr2269. Sequence alignment of these two proteins revealed a sequence identity of 71% (supplemental figure). Because the two proteins identified from species N. punctiforme and Nostoc PCC7120 share such high identity, it is reasonable to use the protein encoded by the Nostoc PCC7120 gene alr2269 for further analysis (Fig. 1A). The protein encoded by alr2269 shares 19.4% identity and 29.3% similarity with psToc75 and 15.8% identity and 25.9% similarity with nmOmp85. The similarity in regard to the proposed topologies of the proteins is very high (Fig. 1D) (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar, 19.Schleiff E. Eichacker L.A. Eckart K. Becker T. Mirus O. Stahl T. Soll J. Protein Sci. 2003; 12: 748-759Crossref PubMed Scopus (99) Google Scholar). In addition to alr2269, two further homologues to psToc75 and Omp85 can be found in the Nostoc PCC7120 proteome, namely alr4893 and alr0075 (31.Moslavac S. Mirus O. Bredemeier R. Soll J. von Haeseler A. Schleiff E. FEBS J. 2005; 272: 1367-1378Crossref PubMed Scopus (73) Google Scholar, 32.Inoue K. Potter D. Plant J. 2004; 39: 354-365Crossref PubMed Scopus (91) Google Scholar). The two latter proteins are shorter than Omp85 with predicted molecular mass values of 72 and 54 kDa, respectively. Further, these two gene products are not as similar to psToc75 and Omp85 as alr2269 (not shown). Finally, in mass spectrometric analysis of outer membranes, only alr2269 could be detected (33.Moslavac S. Bredemeier R. Mirus O. Granvogl B. Eichacker L.A. Schleiff E. J. Proteome Res. 2005; (in press)PubMed Google Scholar). Hence, we have focused in this study on the analysis of alr2269. The existence of the coding RNA was demonstrated by reverse transcription-mediated PCR (Fig. 1B). Immunodecoration of isolated fractions of Nostoc PCC7120 shows that the protein encoded by alr2269 is localized in the outer membrane (Fig. 1C, lane 3), but not in the thylakoid membrane (lane 1) or plasma membrane (lane 2). Topological modeling of alr2269 (Fig. 1D) revealed two dominant regions, an N-terminal mostly soluble part and a C-terminal portion mostly involved in transmembrane β-strand formation. The localization of the domains in regard to the two sides of the membrane cannot be defined, because further experimental data have to be accumulated, as has been done for Toc75 (18.Sveshnikova N. Grimm R. Soll J. Schleiff E. Biol. Chem. 2000; 381: 687-693Crossref PubMed Scopus (61) Google Scholar, 19.Schleiff E. Eichacker L.A. Eckart K. Becker T. Mirus O. Stahl T. Soll J. Protein Sci. 2003; 12: 748-759Crossref PubMed Scopus (99) Google Scholar). To test the predicted domain architecture, we constructed mutants of psToc75 and alr2269 comprising specific sections of these proteins. The first mutant (referred to as A in Fig. 1D, aa 161–470 of alr2269 and aa 149–440 of psToc75, empty box and box with bricks) was chosen in regard to the proposed polypeptide transport-associated domain (POTRA) (34.Sanchez-Pulido L. Devos D. Genevrois S. Vicente M. Valencia A. Trends Biochem. Sci. 2003; 28: 523-526Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The domain does not include the previously predicted transit peptide of psToc75 ending at amino acid 131 (35.Tranel P.J. Froehlich J. Goyal A. Keegstra K. EMBO J. 1995; 14: 2436-2446Crossref PubMed Scopus (189) Google Scholar). AnaB (aa 388–470) or psB (aa 365–440) (Fig. 1D, box with bricks) covers the third loop region in the topological model. The third construct (referred to as C in Fig. 1D, aa 469–833 of alr2269 and aa 439–809 of psToc75, box with crossed lines and box with transversal lines) represents the entire postulated pore-forming region (5.Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (586) Google Scholar, 19.Schleiff E. Eichacker L.A. Eckart K. Becker T. Mirus O. Stahl T. Soll J. Protein Sci. 2003; 12: 748-759Crossref PubMed Scopus (99) Google Scholar). The last mutant (referred to as D in Fig. 1D, aa 702–833 of alr2269 and aa 678–809 of psToc75, box with transversal lines) reflects the region with high similarity among the polypeptide transporters containing two structural motifs and eight proposed β-strands (31.Moslavac S. Mirus O. Bredemeier R. Soll J. von Haeseler A. Schleiff E. FEBS J. 2005; 272: 1367-1378Crossref PubMed Scopus (73) Google Scholar). The generated constructs of the N-terminal region (Fig. 1D, constructs A and B) were expressed in E. coli and purified as soluble proteins to high homogeneity (Fig. 2A). The constructs C and D (in Fig. 5A) were expressed as insoluble proteins and purified under denaturing conditions. To confirm the structural content of the N-terminal constructs CD spectroscopy was performed. All N-terminal constructs showed a defined spectrum in the far UV region (Fig. 2B) (not shown for psA and psB), accounting for a secondary structure content (30.Schleiff E. Turnbull J.L. Biochemistry. 1998; 37: 13043-13051Crossref PubMed Scopus (35) Google Scholar). Using a simple approach for the estimation of the secondary structure content (30.Schleiff E. Turnbull J.L. Biochemistry. 1998; 37: 13043-13051Crossref PubMed Scopus (35) Google Scholar, 36.Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3332) Google Scholar), we observed that the A constructs contained both helical (∼50%) and β-sheet (∼20%) content, whereas for the B constructs only helical content could be determined (∼65%). From the obtained result, we concluded that the soluble expressed N-terminal constructs can be used for in vitro interaction analysis in solution. psToc75 Contains an N-terminal Preprotein-binding Domain—Previously, it was suggested that psToc75 interacts with the transit peptide of chloroplast preproteins (1.Hinnah S.C. Hill K. Wagner R. Schlicher T. Soll J. EMBO J. 1997; 16: 7351-7360Crossref PubMed Scopus (189) Google Scholar). We now investigated whether the N-terminal region of psToc75 could mediate this interaction. When a matrix charged with the precursor of the subunit of the oxygen-evolving complex of 23 kDa (pOE23) was incubated with radioactive labeled psA or psB (Fig. 2C, lanes 3 and 6) or 2-fold molar excess of psA or psB (Fig. 2D, lanes 1–9), both domains of psToc75 were associated with the matrix charged with the preprotein (Fig. 2C, lane 3; 2D, lanes 3 and 9) but not with the empty matrix or a matrix charged with the mature form of the small subunit of Rubisco (Fig. 2C, lane 6; 2D, lanes 6 and 12). Interestingly, the results for the qualitative (radioactive labeled psToc75 constructs; Fig. 2C) or quantitative approach (expressed psToc75 constructs; Fig. 2D) are comparable. In addition, the two psToc75 constructs associated in a similar quantitative manner with the precursor of the 33- or 23-kDa subunit of the oxygen-evolving complex (pOE33/23) (Fig. 2D, lane 9). These results suggest that psToc75 contains a transit peptide-binding site in its N-terminal region. To confirm this notion, synthetic peptides representing the transit sequence of the model preprotein pSSU were coupled to an affinity matrix. Here, the E2 peptide re
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