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Matthias Müller - One of the best experts on this subject based on the ideXlab platform.
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Unanticipated functional diversity among the TatA-type components of the Tat Protein Translocase.
Scientific Reports, 2018Co-Authors: Ekaterina Eimer, Wei-chun Kao, Julia Fröbel, Anne-sophie Blümmel, Carola Hunte, Matthias MüllerAbstract:Twin-arginine translocation (Tat) systems transport folded Proteins that harbor a conserved arginine pair in their signal peptides. They assemble from hexahelical TatC-type and single-spanning TatA-type Proteins. Many Tat systems comprise two functionally diverse, TatA-type Proteins, denominated TatA and TatB. Some bacteria in addition express TatE, which thus far has been characterized as a functional surrogate of TatA. For the Tat system of Escherichia coli we demonstrate here that different from TatA but rather like TatB, TatE contacts a Tat signal peptide independently of the proton-motive force and restricts the premature processing of a Tat signal peptide. Furthermore, TatE embarks at the transmembrane helix five of TatC where it becomes so closely spaced to TatB that both Proteins can be covalently linked by a zero-space cross-linker. Our results suggest that in addition to TatB and TatC, TatE is a further component of the Tat substrate receptor complex. Consistent with TatE being an autonomous TatAB-type Protein, a bioinformatics analysis revealed a relatively broad distribution of the tatE gene in bacterial phyla and highlighted unique Protein sequence features of TatE orthologs.
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mapping precursor binding site on tatc subunit of twin arginine specific Protein Translocase by site specific photo cross linking
Journal of Biological Chemistry, 2012Co-Authors: Stefan Zoufaly, Michael Moser, Julia Fröbel, Patrick Rose, Tobias Flecken, Carlo Maurer, Matthias MüllerAbstract:Abstract A number of secreted precursor Proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor Proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane Proteins, a hexahelical TatC Protein, and usually one or two single-spanning membrane Proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC Protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat Translocase.
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Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo
2012Co-Authors: Stefan Zoufaly, Michael Moser, Julia Fröbel, Patrick Rose, Tobias Flecken, Carlo Maurer, Matthias MüllerAbstract:Background: TatA, TatB, and TatC are subunits of the Tat Translocase allowing transport of folded pre-Proteins across cellular membranes Results: We identified TatC sites that interact with pre-Proteins, TatA, TatB, and TatC Conclusion: The cytosolic N terminus and first cytosolic TatC loop constitute part of a twin arginine recognition site Significance: We developed a working model of how twin arginine pre-Protein inserts into Tat Translocase. A number of secreted precursor Proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin argininecontaining sequence motif in their signal peptides. Many of these precursor Proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane Proteins, a hexahelical TatC Protein, and usually one or two single-spanning membrane Proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific crosslinking analysis of the Escherichia coli TatC Protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat Translocase.
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Genetic evidence for a TatC dimer at the core of the Escherichia coli twin arginine (Tat) Protein Translocase.
Journal of Molecular Microbiology and Biotechnology, 2011Co-Authors: Barbara Maldonado, Matthias Müller, Ben C. Berks, Grant Buchanan, Tracy PalmerAbstract:The twin arginine Protein transport (Tat) system transports folded Proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of plant chloroplasts. In Escherichia coli , the TatB and TatC components form a multivalent receptor complex that binds Tat substrates. Here, we have used a genetic fusion approach to construct covalent TatC oligomers in order to probe the organisation of TatC. A fused dimer of TatC supported Tat transport activity and was fully stable in vivo. Inactivating point mutations in one or other of the TatC units in the fused TatC dimer did not inactivate TatC function, indicating that only one TatC protomer in the TatC fused dimer needs to be active. Larger covalent fusions of TatC also supported Tat transport activity but were degraded in vivo to release smaller TatC forms. Taken together, these results strongly suggest that TatC forms a functional dimer, and support the idea that there is an even number of TatC protomers in the TatBC complex.
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In vitro analysis of the bacterial twin-arginine-dependent Protein export.
Protein Targeting Protocols, 2007Co-Authors: Michael Moser, Sascha Panahandeh, Eva Holzapfel, Matthias MüllerAbstract:Prokaryotic organisms possess a specialized Protein Translocase in their cytoplasmic membranes that catalyzes the export of folded preProteins. Substrates for this pathway are distinguished by a twin-arginine consensus motif in their signal peptides (twin-arginine translocation [Tat] pathway). We have compiled detailed protocols for the preparation and operation of a cell-free system by which the bacterial Tat pathway can be fully reproduced in vitro. This system has proven useful and is being further exploited for the study of precursor-Translocase interactions, assembly of the Translocase, and the mechanism of transmembrane passage.
Colin Robinson - One of the best experts on this subject based on the ideXlab platform.
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structure of tata paralog tate suggests a structurally homogeneous form of tat Protein Translocase that transports folded Proteins of differing diameter
Journal of Biological Chemistry, 2012Co-Authors: Jacopo Baglieri, Daniel O Beck, Nishi Vasisht, Corinne J Smith, Colin RobinsonAbstract:The twin-arginine translocation (Tat) system transports folded Proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA Protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat Translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.
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Characterisation of an Arabidopsis thaliana cDNA encoding a novel thylakoid lumen Protein imported by the ΔpH-dependent pathway
Planta, 1999Co-Authors: Alexandra Mant, Thomas Kieselbach, Wolfgang P. Schröder, Colin RobinsonAbstract:An Arabidopsis thaliana (L.) Heynh. cDNA encoding a novel 16-kDa Protein (P16) of the chloroplast thylakoid lumen has been characterised. The function of the Protein is unknown but it shares some sequence similarity with alpha allophycocyanins. P16 is synthesised with a bipartite, lumen-targeting presequence, and import experiments demonstrated that this Protein follows the ΔpH-dependent pathway. Analysis of the thylakoid transfer peptide revealed two unusual features. Firstly, the key targeting determinant is predicted to be a twin-arginine followed by a highly hydrophobic residue two residues later, rather than at the third position as in most transfer peptides. Secondly, the C-terminal domain of the transfer peptide contains multiple charged residues which may help to prevent mistargeting by the Sec-type Protein Translocase.
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MULTIPLE PATHWAYS FOR THE TARGETING OF THYLAKOID ProteinS IN CHLOROPLASTS
Plant Molecular Biology, 1998Co-Authors: Colin Robinson, Peter J. Hynds, David Robinson, Alexandra MantAbstract:The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised Proteins and their correct targeting into or across the thylakoid membrane. Biochemical and genetic studies have revealed the operation of several targeting pathways for these Proteins, some of which are used for thylakoid lumen Proteins whereas others are utilised by membrane Proteins. Some pathways can be traced back to the prokarytoic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus. In this article we review recent findings in this field that point to the operation of a mechanistically unique Protein Translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor Proteins.
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a new type of signal peptide central role of a twin arginine motif in transfer signals for the delta ph dependent thylakoidal Protein Translocase
The EMBO Journal, 1995Co-Authors: A M Chaddock, Alexandra Mant, I Karnauchov, Susanne Brink, Reinhold G Herrmann, Ralf Bernd Klosgen, Colin RobinsonAbstract:The delta pH-driven and Sec-related thylakoidal Protein Translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane Protein does not affect the translocation pathway.
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A new type of signal peptide: central role of a twin‐arginine motif in transfer signals for the delta pH‐dependent thylakoidal Protein Translocase.
The EMBO Journal, 1995Co-Authors: A M Chaddock, Alexandra Mant, I Karnauchov, Susanne Brink, Reinhold G Herrmann, Ralf Bernd Klosgen, Colin RobinsonAbstract:The delta pH-driven and Sec-related thylakoidal Protein Translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane Protein does not affect the translocation pathway.
André Schneider - One of the best experts on this subject based on the ideXlab platform.
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a trypanosomal orthologue of an intermembrane space chaperone has a non canonical function in biogenesis of the single mitochondrial inner membrane Protein Translocase
PLOS Pathogens, 2017Co-Authors: Christoph Wenger, Silke Oeljeklaus, André Schneider, Bettina Warscheid, Anke Judith HarsmanAbstract:Mitochondrial Protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric Protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic Proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim Proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim Proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three Proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17.
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TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane Protein Translocase of Trypanosoma brucei.
Molecular Microbiology, 2016Co-Authors: Silvia Franziska Desy, Jan Mani, Anke Judith Harsman, Sandro Käser, André SchneiderAbstract:TbLOK1 has previously been characterized as a trypanosomatid-specific mitochondrial outer membrane Protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial Protein import, both in vivo and in vitro. Co-immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic Protein Translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial Protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial Protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.
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tblok1 atom19 is a novel subunit of the noncanonical mitochondrial outer membrane Protein Translocase of trypanosoma brucei
Molecular Microbiology, 2016Co-Authors: Silvia Franziska Desy, Jan Mani, Anke Judith Harsman, Sandro Käser, André SchneiderAbstract:TbLOK1 has previously been characterized as a trypanosomatid-specific mitochondrial outer membrane Protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial Protein import, both in vivo and in vitro. Co-immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic Protein Translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial Protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial Protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.
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Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase NEW PERSPECTIVES FROM COMPARATIVE SINGLE CHANNEL ELECTROPHYSIOLOGY
Journal of Biological Chemistry, 2012Co-Authors: Anke Judith Harsman, André Schneider, Moritz Niemann, Mascha Pusnik, Oliver Schmidt, Chris Meisinger, Bjoern M. Burmann, Sebastian Hiller, Richard WagnerAbstract:Mitochondria are of bacterial ancestry and have to import most of their Proteins from the cytosol. This process is mediated by Tom40, an essential Protein that forms the Protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic Translocase of the outer mitochondrial membrane (ATOM), a Protein that shows similarities to both eukaryotic Tom40 and bacterial Protein Translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded Proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other Protein Translocases reveals that ATOM closely resembles bacterial-type Protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like Protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes.
Tracy Palmer - One of the best experts on this subject based on the ideXlab platform.
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Assembling the Tat Protein Translocase
eLife, 2016Co-Authors: Felicity Alcock, Phillip J. Stansfeld, Hajra Basit, Johann Habersetzer, Matthew A. B. Baker, Tracy Palmer, Mark I. Wallace, Ben C. BerksAbstract:The twin-arginine Protein translocation system (Tat) transports folded Proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane Proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat Proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat Translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict Protein interfaces in multi-subunit complexes.
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The TatC component of the twin-arginine Protein Translocase functions as an obligate oligomer
Molecular Microbiology, 2015Co-Authors: François Cléon, Felicity Alcock, Phillip J. Stansfeld, Hajra Basit, Johann Habersetzer, Mark I. Wallace, Ben C. Berks, Holger Kneuper, Tracy PalmerAbstract:Summary The Tat Protein export system translocates folded Proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC Proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC Proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC Proteins produced smaller TatBC complexes than the wild-type TatC Protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.
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Characterisation of the membrane-extrinsic domain of the TatB component of the twin arginine Protein Translocase
FEBS Letters, 2011Co-Authors: Barbara Maldonado, Ben C. Berks, Frank Sargent, Holger Kneuper, Grant Buchanan, Kostas Hatzixanthis, Tracy PalmerAbstract:The twin arginine Protein transport (Tat) system transports folded Proteins across cytoplasmic membranes of bacteria and thylakoid membranes of plants, and in Escherichia coli it comprises TatA, TatB and TatC components. In this study we show that the membrane extrinsic domain of TatB forms parallel contacts with at least one other TatB Protein. Truncation of the C-terminal two thirds of TatB still allows complex formation with TatC, although Protein transport is severely compromised. We were unable to isolate transport-inactive single codon substitution mutations in tatB suggesting that the precise amino acid sequence of TatB is not critical to its function.
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Genetic evidence for a TatC dimer at the core of the Escherichia coli twin arginine (Tat) Protein Translocase.
Journal of Molecular Microbiology and Biotechnology, 2011Co-Authors: Barbara Maldonado, Matthias Müller, Ben C. Berks, Grant Buchanan, Tracy PalmerAbstract:The twin arginine Protein transport (Tat) system transports folded Proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of plant chloroplasts. In Escherichia coli , the TatB and TatC components form a multivalent receptor complex that binds Tat substrates. Here, we have used a genetic fusion approach to construct covalent TatC oligomers in order to probe the organisation of TatC. A fused dimer of TatC supported Tat transport activity and was fully stable in vivo. Inactivating point mutations in one or other of the TatC units in the fused TatC dimer did not inactivate TatC function, indicating that only one TatC protomer in the TatC fused dimer needs to be active. Larger covalent fusions of TatC also supported Tat transport activity but were degraded in vivo to release smaller TatC forms. Taken together, these results strongly suggest that TatC forms a functional dimer, and support the idea that there is an even number of TatC protomers in the TatBC complex.
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Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded Proteins.
FEMS Microbiology Letters, 2006Co-Authors: Frank Sargent, Ben C. Berks, Tracy PalmerAbstract:The twin-arginine (Tat) Protein Translocase is a highly unusual Protein transport machine that is dedicated to the movement of folded Proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In this minireview, we describe our current knowledge of the Tat system, paying particular attention to the function of the TatA Protein and to the often overlooked step of signal peptide cleavage.
Stefan Zoufaly - One of the best experts on this subject based on the ideXlab platform.
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mapping precursor binding site on tatc subunit of twin arginine specific Protein Translocase by site specific photo cross linking
Journal of Biological Chemistry, 2012Co-Authors: Stefan Zoufaly, Michael Moser, Julia Fröbel, Patrick Rose, Tobias Flecken, Carlo Maurer, Matthias MüllerAbstract:Abstract A number of secreted precursor Proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor Proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane Proteins, a hexahelical TatC Protein, and usually one or two single-spanning membrane Proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC Protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat Translocase.
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Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo
2012Co-Authors: Stefan Zoufaly, Michael Moser, Julia Fröbel, Patrick Rose, Tobias Flecken, Carlo Maurer, Matthias MüllerAbstract:Background: TatA, TatB, and TatC are subunits of the Tat Translocase allowing transport of folded pre-Proteins across cellular membranes Results: We identified TatC sites that interact with pre-Proteins, TatA, TatB, and TatC Conclusion: The cytosolic N terminus and first cytosolic TatC loop constitute part of a twin arginine recognition site Significance: We developed a working model of how twin arginine pre-Protein inserts into Tat Translocase. A number of secreted precursor Proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin argininecontaining sequence motif in their signal peptides. Many of these precursor Proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane Proteins, a hexahelical TatC Protein, and usually one or two single-spanning membrane Proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific crosslinking analysis of the Escherichia coli TatC Protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat Translocase.
