The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles
- Tobias Wunder†1,
- Roman Martin†1,
- Wolfgang Löffelhardt2,
- Enrico Schleiff1, 3Email author and
- Jürgen M Steiner2Email author
© Wunder et al; licensee BioMed Central Ltd. 2007
Received: 31 July 2007
Accepted: 28 November 2007
Published: 28 November 2007
Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems.
To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon.
The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported.
The plastids of glaucophytes, rhodophytes, green algae and higher plants are surrounded by an envelope consisting of two membranes. These "primary" plastids are thought to have originated from a single primary endosymbiotic event [e.g. ]. Cyanelles (muroplasts) are the peptidoglycan-armored plastids of glaucocystophyte algae which represent the first diverging phototrophic eukaryotes, on the earliest branch after initial endosymbiosis. Cyanelles can be envisaged as the closest cousins to free-living cyanobacteria among plastids [2–4]. The unique murein layer serving as "organelle wall" is modified through amidation of the free C-1 carboxylic group of the D-isoglutamyl moiety with N-acetylputrescine , which is unusual in the eubacterial kingdom and reduces the negative net charge of the murein layer that might interfere with protein import. Cyanelle (and likely the red algal) protein import machineries in plastids should be considered as prototypes of translocation systems that later underwent substantial modifications. These primitive translocons strictly require phenylalanine in the N-terminal domain of the transit peptide, even when they fulfil their function in a secondary plastid derived from a red alga . A key event for transition of the "primordial plastids" to the chloroplasts of green algae and higher plants is the gain of (additional?) receptors in the Toc complex conferring less stringent and overlapping specificities finally leading to dispensability of the once crucial phenylalanine .
Recent reports confirmed the dependence of protein translocation across the outer membrane of cyanelles or the third outermost membrane of plastids derived from secondary endosymbiosis through a red alga, on the presence of a phenylalanine within the transit sequence [8–10]. In the absence of import experiments with red algae, the similarity of the N-terminal phenylalanine motif of Porphyra yezoensis plastid precursor proteins  to their counterparts from C. paradoxa is striking. However, the translocation of precursor proteins across the outer membrane of higher plant and green algal chloroplasts does not show such dependence . Thereby, the phenylalanine represents a key for the understanding of the development of translocation machinery and their receptors. Yet, one question has not been explored satisfactorily, namely, what the phenylalanine is needed for. Current models favour an interpretation where this amino acid interacts with the ancestral translocon built up by endosymbiont-derived Toc75 [7, 12]. However, other models on the evolutionary development and mechanism of translocation focused on an initial specific interaction between the precursor protein and the organellar membrane [e.g. [13, 14]]. We reconsidered both possibilities, since phenylalanine is an aromatic amino acid known to exhibit pronounced hydrophobic properties , and it is known to insert more deeply into the membrane than other less hydrophobic amino acids like alanine . Furthermore, phenylalanine is one important component in "aromatic belts" demarcating the hydrophobic surface immersed in the lipid bilayer . It is noteworthy that all bacterial outer membrane protein structures analyzed so far show an interaction between their ultimate amino- and carboxyterminal domains. In some cases this contact is closing the β-barrel structure completely (e.g. E. coli OmpC ) or in addition forming a hydrophobic interaction between a phenylalanine in the N-terminal domain and the C-terminal phenylalanine (e.g. E. coli OmpG ). These observations would favour a role of phenylalanine in membrane association of the cytosolic precursor as first contact with the cyanelle.
The importance of aromatic amino acids in protein-protein interactions or substrate recognition is manifold documented. For example, aromatic residues are found in the substrate-binding site of the AAA+ chaperone ClpB that are located at the central pore of the first AAA domain. These aromatic residues may act as a molecular clamp by binding and releasing substrates in a nucleotide-dependent manner . Translocation of exposed segments enriched in aromatic residues (phenylalanine, tyrosine and tryptophane) of a protein aggregate by ClpB would lead to the continuous extraction of unfolded polypeptides from an aggregate. Therefore hydrophobic interactions between aromatic residues are a common principle of protein-protein cooperation. Furthermore, phenylalanine is reported to occupy prominent positions in the sequences of proteins targeted to the bacterial outer membrane , such as porins (C-terminal amino acid) and type IV pilins (N-terminal amino acid, after cleavage by prepilin peptidase). Recently it has been shown that the penultimate residue of bacterial outer membrane proteins is involved in the species specificity of Omp85 recognition . Interestingly, cyanobacterial outer membrane proteins show the C-terminal consensus sequence FxF . Thereby, it is tempting to speculate that a phenylalanine (an aromatic) residue is important for the interaction of precursor proteins with the Toc translocon of primitive plastids.
In here we describe the influence of the phenylalanine on the interaction with lipids and lipid surfaces represented by liposomes and proteoliposomes. The obtained specificity for proteoliposomes is subsequently discussed in the context of the two "initial receptor models" and the evolutionary development of the chloroplast translocon.
The phenylalanine defines lipid specificities of pFNR
To explore the influence of the phenylalanine on this interaction, we used the two types of mutants previously generated . In a first set, the phenylalanine was replaced either by a tyrosine or by glycine. In a second set, the phenylalanine itself or with the two neighbouring amino acids were removed. When binding of the mutants to lipids was analyzed we did obtain the highest affinity for PC as seen before for pFNR. However, some changes in lipid binding can be found. The most conservative mutant F→Y parallels the feature of wild type in terms of not interacting with mono – or digalactosyldiaclglycerol. (Fig 1B). It still binds to SQDG, but not to PG, suggesting that its interaction to SQDG is not driven by electrostatic interactions. In contrast, the exchange of the phenylalanine by glycine introduces marked alterations (Fig 1B) as this mutant shows no binding to PG but to all galactolipids. The two deletion mutants reveal a reduced but still recognizable binding to PC and SQDG, but not to the other lipids (Fig 1C). Hence, we obtained a chain of alterations of lipid specificities or binding capacities, which somewhat, but not fully parallel the import behaviour observed .
The aromatic properties of the phenylalanine define the lipid specificity
Binding of pFNR to liposomes
Summarizing, we document that the alteration of the phenylalanine modifies the lipid binding behavior of the precursor protein to lipids immobilized to a surface or present in liposomes. In both cases, deletion of the phenylalanine results in a reduction of the association (Fig 1C; 3A). The exchange of the phenylalanine to tyrosine somewhat changes the specificity for the lipids, but does not significantly change the affinity to phosphatidylcholine (Fig 1B; 2B, D; 3A). In contrast, exchange of phenylalanine to glycine on one hand enhances the association to phosphatidylcholine and changes the specificity of the protein from preferred interaction with phosphatidylglycerol to preferred interaction with galactolipids (Fig 1B; 2C, D; 3A, D). This discrepancy can not be explained by a difference of the hydrophobicity within the mutants. Considering the first seven amino acids, the wild type protein shows a mean hydrophobicity of 0.31 according to the Eisenberg scale [e.g.  and references therein], and the F → Y and F → G mutants a hydrophobicity of 0.22 and 0.24, respectively.
Binding of cyanelle pFNR to Omp85 from Anabaena or Toc75 from pea
Previous reports implicated a monogalactosyldiacylglycerol dependent interaction of the transit peptide of tobacco pSSU with liposomes  and a preference of the transit peptide of ferredoxin from Silene pratensis for 1,2-dioleoyl-sn-glycero-3-phosphoglycerol and sulfoquinovosyl-diacylglycerol . These findings are in contrast to our observations for the ferredoxin:NADP+-oxidoreductase (pFNR) from Cyanophora, which binds strongest to phophatidylcholine (Fig 1, 2, 3). The exchange of phenylalanine to glycine within the cyanelle precursor causes the most significant alteration of lipid binding, especially with respect to the affinity and lipid preference. For this mutant we obtained almost no binding to lipid mixtures with phosphatidylglycerol, but an enhanced interaction with liposomes containing galactosyldiacylglycerol (Fig 1, 2, 3). Hence, our results suggest that exchange of the phenylalanine by a non aromatic amino acid renders the physiochemical properties of the transit peptide from Cyanophora closer to those found for transit peptides of organisms with chloroplasts sensu stricto [29–32].
Summarizing, the following model for Toc translocon evolution can be assumed from the presented data. Primordial transit sequences evolved with a phenylalanine for two reasons; at first this amino acid attenuates the interaction with the cyanelle surface containing mono- and digalactosyldiacylglycerol. Interestingly, previous studies determined that diacylglycerol was needed to interfere with spontaneous insertion of proteins into liposomes using an E. coli in vitro system . In the absence of diacylglycerol spontaneous integration into phospholipids was observed even for multi-spanning membrane proteins. It was concluded that diacylglycerol seals the cytoplasmic membrane of E. coli against spontaneous insertion of hydrophobic proteins. Hence, the low affinity of pFNR for galactosyldiacylglycerol containing membranes might be essential to warrant the recognition by proteins present in the outer membrane. One such protein is Omp85 involved in outer membrane protein assembly in bacteria and predestined to interact with sequences composed like transit peptides . In a primitive translocon, this receptor pore likely is the only candidate to interact with precursor proteins whereas in the chloroplast system a whole set of receptors is available for binding and able to overrule precursor-lipid affinities. Secondly, the phenylalanine reflects a match to properties of bacterial Omp85 proteins, which are thought to recognize OMPs via their C-termini enriched in aromatic amino acids [e.g. [20, 21]]. In the course of evolution and paralleled by development of a more sophisticated translocation apparatus including cytosolic guidance complexes and regulatory receptor components like Toc34 and Toc159 [e.g. ], the importance of the N-terminal phenylalanine might have been gradually lost in the "green line", which allowed a more flexible and versatile (plastid type- and tissue-specific) regulation of the import process. Future research, especially on the translocon of cyanelles, will have to corroborate this model.
Construct generation, in vitro transcription and translation and protein expression and purification are described in [8, 27, 28]. Purified plant lipids were purchased from Nutfield Nurseries (Surrey, UK). Lipid A and fatty-acid free BSA was obtained from Sigma (Munich, G). The binding of precursor proteins to the N-terminal construct of psToc75 and alr2269 (anaOmp85) was analyzed as previously described .
FAT blot assay
Lipids were diluted in chloroform and indicated amounts spotted onto PVDF membranes . The membrane was subsequently saturated with 0,25% fatty-acid free BSA for 1 hour. Afterwards, 12,5 μl of the indicated translation product was diluted into 5 ml of 0,25% fatty-acid free BSA and 1 mM methionine and incubated for 1 hour at 20°C while rotating the blot. The blot was subsequently washed by 3 incubations with 1 mM methionine and 0,25% fatty-acid free BSA for at least 15 min, dried and binding quantified by phospho-imaging (Reader FLA 3000, Fuji-Film, Tokyo, Japan). The binding efficiency was determined by parallel detection of radioactivity in the added translation product using AIDA-Image Analyser software (Raytest, Isotopenmessgeräte GmbH, Steubenhard, G). In Figure 1, the binding was further normalized to the efficiency of the interaction between wild type pFNR and 300 pmol PC spotted to the PVDF membrane.
Liposome generation and protein reconstitution
For binding analysis liposomes with a 200 nm diameter were produced as described  in 20 mM Tris/HCl, pH 7,4 and 300 mM sucrose and diluted to a final concentration of 10 mM lipids. For reconstitution 4 mg phosphatidylcholine were resuspended in 400 μl 20 mM Tris/HCl pH 7,4 and 400 mM sucrose and Mega 9 was added to a final concentration of 80 mM. Proteins (0,2 mg; in 5 M urea, 50 mM NaPi pH 6.8, 150 mM NaCl, 10 mM β-Mercaptoethanol, 500 mM Imidazol) were also incubated with Mega 9 (80 mM final), subsequently added to the lipid mixture and dialyzed over night against a 2000 fold excess of 20 mM Tris/HCl, pH 7,4; 200 mM sucrose. The proteoliposomes were pelleted for 10 min at 80.000 × g, resuspended in 20 mM Tris/HCl, pH 7,4; 200 mM sucrose and stored at -80°C. Before use, liposomes were thawed and sonicated for 10 sec. Protein reconstitution was controlled by western blot analysis (not shown).
Liposome binding experiments
The protocol was adjusted according to . Liposomes and proteoliposomes at indicated concentrations were incubated for 10 min at 20°C with 5 μl of the radioactive labelled precursor proteins in 20 mM Tris/HCl pH 7,4 and 100 mM NaCl (100μl final). The mixture was laid on top of a sucrose cushion (1 ml 20 mM Tris/HCl, pH 7,4/200 mM sucrose, 100μl 20 mM Tris/HCl, pH 7,4/1 M sucrose), centrifuged for 45 min at 50000 × g at 4°C and liposomes were collected from the bottom. Fractions were subjected to SDS-PAGE and bound protein analyzed by phosphor-imaging.
The apparent dissociation constant for the FAT blot assay was determined according to
Binding % = 100% * [Lipids]/(KD(app) + [Lipids])
and the least square fit analysis was performed with Sigma Plot 7 (SPSS Inc.).
precursor of the ferredoxin NADP reductase
precursor for small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
We thank Oliver Mirus for support in bioinformatic approaches. The work was supported by grants from the Deutsche Forschungsgemeinschaft (SFBTR01-C7), the Volkswagenstiftung and by the Cluster of Excellence "Macromolecular Complexes" at the Goethe University Frankfurt (DFG Project EXC 115) to E.S.
- Rodriguez-Ezpeleta N, Brinkmann H, Burey SC, Roure B, Burger G, Löffelhardt W, Bohnert HJ, Philippe H, Lang BF: Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes. Curr Biol. 2005, 15: 1325-1330. 10.1016/j.cub.2005.06.040.View ArticlePubMedGoogle Scholar
- Martin W, Stoebe B, Goremykin V, Hapsmann S, Hasegawa M, Kowallik KV: Gene transfer to the nucleus and the evolution of chloroplasts. Nature. 1998, 393: 162-165. 10.1038/30234.View ArticlePubMedGoogle Scholar
- Löffelhardt W, Bohnert HJ: The cyanelle (muroplast) of Cyanophora paradoxa : a paradigm for endosymbiotic organelle evolution. Symbiosis. Edited by: Seckbach, J. 2001, Dordrecht: Kluwer Academic Publishers, 111-130.Google Scholar
- McFadden GI, van Dooren GG: Evolution: red algal genome affirms a common origin of all plastids. Curr Biol. 2004, 14: R514-R516. 10.1016/j.cub.2004.06.041.View ArticlePubMedGoogle Scholar
- Pfanzagl B, Allmaier G, Schmid ER, de Pedro MA, Löffelhardt W: N-acetylputrescine as a characteristic constituent of cyanelle peptidoglycan in glaucocystophyte algae. J Bacteriol. 1996, 178: 6994-6997.PubMed CentralPubMedGoogle Scholar
- Patron NJ, Waller RF: Transit peptide diversity and divergence: a global analysis of plastid targeting signals. Bioessays. 2007, 29: 1048-1058. 10.1002/bies.20638.View ArticlePubMedGoogle Scholar
- Steiner JM, Löffelhardt W: Protein translocation into and within cyanelles. Mol Membr Biol. 2005, 22: 123-132. 10.1080/09687860500041411.View ArticlePubMedGoogle Scholar
- Steiner JM, Yusa F, Pompe JA, Löffelhardt W: Homologous protein import machineries in chloroplasts and cyanelles. Plant J. 2005, 44: 646-652. 10.1111/j.1365-313X.2005.02559.x.View ArticlePubMedGoogle Scholar
- Gruber A, Vugrinec S, Hempel F, Gould SB, Maier UG, Kroth PG: Protein targeting into complex diatom plastids:functional characterization of a specific targeting motif. Plant Mol Biol. 2007, 64: 519-530. 10.1007/s11103-007-9171-x.View ArticlePubMedGoogle Scholar
- Gould SB, Sommer MS, Hadfi K, Zauner S, Kroth PG, Maier UG: Protein targeting into the complex plastid of cryptophytes. J Mol Evol. 2006, 62: 674-681. 10.1007/s00239-005-0099-y.View ArticlePubMedGoogle Scholar
- Nikaido I, Asamizu E, Nakajima M, Nakamura Y, Saga N, Tabata S: Generation of 10,154 Expressed Sequence Tags from a Leafy Gametophyte of a Marine Red Alga, Porphyra yezoensis. DNA Research. 2000, 7: 223-227. 10.1093/dnares/7.3.223.View ArticlePubMedGoogle Scholar
- Löffelhardt W, von Haeseler A, Schleiff E: The β-barrel shaped polypeptide transporter, an old concept for precursor protein transfer across membranes. Symbiosis. 2007, 44: 33-42.Google Scholar
- Bruce BD: The role of lipids in plastid protein transport. Plant Mol Biol. 1998, 38: 223-246. 10.1023/A:1006094308805.View ArticlePubMedGoogle Scholar
- Bruce BD: Chloroplast transit peptides: structure, function and evolution. Trends Cell Biol. 2000, 10: 440-447. 10.1016/S0962-8924(00)01833-X.View ArticlePubMedGoogle Scholar
- Ulmschneider MB, Sansom MS, Di Nola A: Properties of integral membrane protein structures: derivation of an implicit membrane potential. Proteins. 2005, 59: 252-265. 10.1002/prot.20334.View ArticlePubMedGoogle Scholar
- Victor K, Jacob J, Cafiso DS: Interactions controlling the membrane binding of basic protein domains: phenylalanine and the attachment of the myristoylated alanine-rich C-kinase substrate protein to interfaces. Biochem. 1999, 38: 12527-12536. 10.1021/bi990847b.View ArticleGoogle Scholar
- Yildiz O, Vinothkumar KR, Goswami P, Kühlbrandt W: Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation. EMBO J. 2006, 25: 3702-3713. 10.1038/sj.emboj.7601237.PubMed CentralView ArticlePubMedGoogle Scholar
- Basle A, Rummel G, Storici P, Rosenbusch JP, Schirmer T: Crystal structure of osmoporin OmpC from E. coli at 2.0 Å. J Mol Biol. 2006, 362: 933-942. 10.1016/j.jmb.2006.08.002.View ArticlePubMedGoogle Scholar
- Schlieker C, Weibezahn J, Patzelt H, Tessarz P, Strub C, Zeth K, Erbse A, Schneider-Mergener J, Chin JW, Schultz PG, Bukau B, Mogk A: Substrate recognition by the AAA+ chaperone ClpB. Nat Struct Mol Biol. 2004, 11: 607-615. 10.1038/nsmb787.View ArticlePubMedGoogle Scholar
- Struyve M, Moons M, Tommassen J: Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol. 1991, 218: 141-148. 10.1016/0022-2836(91)90880-F.View ArticlePubMedGoogle Scholar
- Robert V, Volokhina EB, Senf F, Bos MP, Van Gelder P, Tommassen J: Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol. 2006, 4: e377-10.1371/journal.pbio.0040377.PubMed CentralView ArticlePubMedGoogle Scholar
- Cavalier-Smith T: Membrane heredity and early chloroplast evolution. Trends Plant Sci. 2000, 5: 174-182. 10.1016/S1360-1385(00)01598-3.View ArticlePubMedGoogle Scholar
- McFadden GI: Endosymbiosis and evolution of the plant cell. Curr Opin Plant Biol. 1999, 2: 513-519. 10.1016/S1369-5266(99)00025-4.View ArticlePubMedGoogle Scholar
- Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M, Nishijima M: Molecular machinery for non-vesicular trafficking of ceramide. Nature. 2003, 426: 803-809. 10.1038/nature02188.View ArticlePubMedGoogle Scholar
- Eichacker LA, Granvogl B, Mirus O, Müller BC, Miess C, Schleiff E: Hiding behind hydrophobicity. Transmembrane segments in mass spectrometry. J Biol Chem. 2004, 279: 50915-50922. 10.1074/jbc.M405875200.View ArticlePubMedGoogle Scholar
- Ertel F, Mirus O, Bredemeier R, Moslavac S, Becker T, Schleiff E: The evolutionarily related beta-barrel polypeptide transporters from Pisum sativum and Nostoc PCC7120 contain two distinct functional domains. J Biol Chem. 2005, 280: 28281-28289. 10.1074/jbc.M503035200.View ArticlePubMedGoogle Scholar
- Bredemeier R, Schlegel T, Ertel F, Vojta A, Borissenko L, Bohnsack MT, Groll M, von Haeseler A, Schleiff E: Functional and phylogenetic properties of the pore-forming beta-barrel transporters of the Omp85 family. J Biol Chem. 2007, 282: 1882-1890. 10.1074/jbc.M609598200.View ArticlePubMedGoogle Scholar
- Pinnaduwage P, Bruce BD: In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent. J Biol Chem. 1996, 271: 32907-32915. 10.1074/jbc.271.51.32907.View ArticlePubMedGoogle Scholar
- van 't Hof R, de Kruijff B: Transit sequence-dependent binding of the chloroplast precursor protein ferredoxin to lipid vesicles and its implications for membrane stability. FEBS Lett. 1995, 361: 35-40. 10.1016/0014-5793(95)00135-V.View ArticlePubMedGoogle Scholar
- Theg SM, Geske FJ: Biophysical characterization of a transit peptide directing chloroplast protein import. Biochem. 1992, 31: 5053-5060. 10.1021/bi00136a018.View ArticleGoogle Scholar
- Horniak L, Pilon M, van 't Hof R, de Kruijff B: The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids. FEBS Lett. 1993, 334: 241-246. 10.1016/0014-5793(93)81720-K.View ArticlePubMedGoogle Scholar
- Bos MP, Tommassen J: Biogenesis of the Gram-negative bacterial outer membrane. Curr Opin Microbiol. 2004, 7: 61061-6. 10.1016/j.mib.2004.10.011.View ArticleGoogle Scholar
- Schleiff E, Soll J: Membrane protein insertion: mixing eukaryotic and prokaryotic concepts. EMBO Rep. 2005, 6: 1023-1027. 10.1038/sj.embor.7400563.PubMed CentralView ArticlePubMedGoogle Scholar
- Gentle IE, Burri L, Lithgow T: Molecular architecture and function of the Omp85 family of proteins. Mol Microbiol. 2005, 58: 1216-1225.View ArticlePubMedGoogle Scholar
- Sanchez-Pulido L, Devos D, Genevrois S, Vicente M, Valencia A: POTRA: a conserved domain in the FtsQ family and a class of beta-barrel outer membrane proteins. Trends Biochem Sci. 2003, 28: 523-526. 10.1016/j.tibs.2003.08.003.View ArticlePubMedGoogle Scholar
- Inoue K, Keegstra K: A polyglycine stretch is necessary for proper targeting of the protein translocation channel precursor to the outer envelope membrane of chloroplasts. Plant J. 2003, 34: 661-669. 10.1046/j.1365-313X.2003.01755.x.View ArticlePubMedGoogle Scholar
- Moslavac S, Mirus O, Bredemeier R, Soll J, von Haeseler A, Schleiff E: Conserved pore-forming regions in polypeptide-transporting proteins. FEBS J. 2005, 272: 1367-1378. 10.1111/j.1742-4658.2005.04569.x.View ArticlePubMedGoogle Scholar
- Nishiyama K, Ikegami A, Moser M, Schiltz E, Tokuda H, Müller M: A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations. J Biol Chem. 2006, 281: 35667-35676. 10.1074/jbc.M608228200.View ArticlePubMedGoogle Scholar
- Soll J, Schleiff E: Protein import into chloroplasts. Nat Rev Mol Cell Biol. 2004, 5: 198-208. 10.1038/nrm1333.View ArticlePubMedGoogle Scholar
- Vojta A, Scheuring J, Neumaier N, Mirus O, Weinkauf S, Schleiff E: Determination of liposome size: a tool for protein reconstitution. Anal Biochem. 2005, 347: 24-33. 10.1016/j.ab.2005.09.003.View ArticlePubMedGoogle Scholar
- Schleiff E, Tien R, Salomon M, Soll J: Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7. Mol Biol Cell. 2001, 12: 4090-4102.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamada S, Gotoh O, Yamana H: Improvement in accuracy of multiple sequence alignment using novel group-to-group sequence alignment algorithm with piecewise linear gap cost. BMC Bioinformatics. 2006, 7: 524-10.1186/1471-2105-7-524.PubMed CentralView ArticlePubMedGoogle Scholar
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