scholarly journals Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway

2017 ◽  
Author(s):  
Johann Habersetzer ◽  
Kristoffer Moore ◽  
Jon Cherry ◽  
Grant Buchanan ◽  
Phillip Stansfeld ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Tat Protein ◽  
Disulfide Crosslinking ◽  
Additional Sequence ◽  
Tat System ◽  
Folded Proteins ◽  
Related Proteins

AbstractThe twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the ‘polar cluster’ site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

scholarly journals Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli

Open Biology ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 170091 ◽  
Author(s):  
Johann Habersetzer ◽  
Kristoffer Moore ◽  
Jon Cherry ◽  
Grant Buchanan ◽  
Phillip J. Stansfeld ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Protein Transport ◽  
Cytoplasmic Membrane ◽  
Polar Cluster ◽  
Additional Sequence ◽  
Tat System ◽  
Folded Proteins ◽  
Related Proteins

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

scholarly journals Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

2020 ◽  
Author(s):  
Qianqian Ma ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Structural Model ◽  
Substrate Recognition ◽  
Transmembrane Helix ◽  
Complex Function ◽  
Transmembrane Helices ◽  
Translocation Event ◽  
Active Substrate ◽  
Tat System ◽  
Folded Proteins ◽  
Transport Complex

AbstractThe Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

scholarly journals TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli

2021 ◽  
Author(s):  
Denise Mehner-Breitfeld ◽  
Michael T. Ringel ◽  
Daniel Alexander Tichy ◽  
Laura J. Endter ◽  
Kai Steffen Stroh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transmembrane Helix ◽  
Length Variation ◽  
Hydrophobic Mismatch ◽  
Hydrophobic Residues ◽  
Transmission Electron ◽  
Absolute Requirement ◽  
Bacterial Model ◽  
Folded Proteins ◽  
Dynamics Simulations

The Tat system translocates folded proteins across energy-transducing prokaryotic membranes. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was tolerated. Length-variation in TatB clearly destabilized TatBC-containing complexes, indicating that the 12-residues-length is crucial for Tat component interactions and translocon assembly. Metal tagging transmission electron microscopy revealed the dimensions of TatA assemblies, which prompted molecular dynamics simulations. These showed that interacting TMHs of larger TatA assemblies can thin the membrane together with laterally aligned tilted APHs that generate a deep V-shaped groove. The conserved hydrophobic mismatch may thus be important for membrane destabilization during Tat transport, and the exact length of 12 hydrophobic residues could be a compromise between functionality and proton leakage minimization.

scholarly journals Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Ian J. Passmore ◽  
Jennifer M. Dow ◽  
Francesc Coll ◽  
Jon Cuccui ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Sigma Factor ◽  
Signal Sequence ◽  
Transmembrane Helix ◽  
Ferric Citrate ◽  
Inner Membrane ◽  
Content Type ◽  
Signal Cascade ◽  
Tat System

ABSTRACT In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence. IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli. The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence.

scholarly journals Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

2008 ◽  
Vol 105 (40) ◽  
pp. 15376-15381 ◽  
Author(s):  
Mark C. Leake ◽  
Nicholas P. Greene ◽  
Rachel M. Godun ◽  
Thierry Granjon ◽  
Grant Buchanan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protonmotive Force ◽  
Oligomeric State ◽  
Essential Components ◽  
Tat System ◽  
Folded Proteins

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of ≈25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains ≈15 mobile TatA complexes and a pool of ≈100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

scholarly journals Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System

2009 ◽  
Vol 20 (7) ◽  
pp. 2060-2069 ◽  
Author(s):  
Carole Dabney-Smith ◽  
Kenneth Cline
Keyword(s):  
Protein Transport ◽  
Transmembrane Domain ◽  
Protein Translocation ◽  
Proton Gradient ◽  
Receptor Complex ◽  
Tat Protein ◽  
Twin Arginine Translocation ◽  
Conducting Component ◽  
Cross Links ◽  
Folded Proteins

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

scholarly journals Assembling the Tat protein translocase

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Felicity Alcock ◽  
Phillip J Stansfeld ◽  
Hajra Basit ◽  
Johann Habersetzer ◽  
Matthew AB Baker ◽  
...  
Keyword(s):  
Structural Model ◽  
Protein Translocation ◽  
Transmembrane Helix ◽  
Tat Protein ◽  
Polar Cluster ◽  
Evolution Analysis ◽  
Protein Interfaces ◽  
Multiple Copies ◽  
Folded Proteins ◽  
Combine Sequence

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.

Oligomeric Properties and Signal Peptide Binding by Escherichia coli Tat Protein Transport Complexes

2002 ◽  
Vol 322 (5) ◽  
pp. 1135-1146 ◽  
Author(s):  
Erik de Leeuw ◽  
Thierry Granjon ◽  
Ida Porcelli ◽  
Meriem Alami ◽  
Stephen B. Carr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Protein Transport ◽  
Peptide Binding ◽  
Tat Protein

Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure

2001 ◽  
Vol 268 (12) ◽  
pp. 3361-3367 ◽  
Author(s):  
Frank Sargent ◽  
Ulrich Gohlke ◽  
Erik de Leeuw ◽  
Nicola R. Stanley ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Protein Transport ◽  
Ring Structure ◽  
Tat Protein ◽  
System Form

The Twin-Arginine Translocation System: A Novel Means of Transporting Folded Proteins in Chloroplasts and Bacteria

2000 ◽  
Vol 381 (2) ◽  
pp. 89-93 ◽  
Author(s):  
C. Robinson
Keyword(s):  
Escherichia Coli ◽  
Transport Proteins ◽  
Membrane Systems ◽  
Related System ◽  
Twin Arginine Translocation ◽  
System A ◽  
Unfolded State ◽  
Tat System ◽  
Folded Proteins ◽  
Unique Ability

Abstract Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fullyfolded proteins across a tightly sealed energytransducing membrane. A related system, (the twinarginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle.

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