scholarly journals Stoichiometry for binding and transport by the twin arginine translocation system

2012 ◽  
Vol 197 (4) ◽  
pp. 523-534 ◽  
Author(s):  
Jose M. Celedon ◽  
Kenneth Cline
Keyword(s):  
Kinetic Analysis ◽  
Protein Transport ◽  
Thylakoid Membranes ◽  
Oxygen Evolving Complex ◽  
Receptor Complex ◽  
Twin Arginine Translocation ◽  
Precursor Proteins ◽  
Folded Proteins ◽  
Membrane Components ◽  
Oxygen Evolving

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

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 Twin-arginine-dependent translocation of folded proteins

2012 ◽  
Vol 367 (1592) ◽  
pp. 1029-1046 ◽  
Author(s):  
Julia Fröbel ◽  
Patrick Rose ◽  
Matthias Müller
Keyword(s):  
Protein Transport ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Proton Motive Force ◽  
Signal Peptides ◽  
Transport Pathway ◽  
Twin Arginine Translocation ◽  
Precursor Proteins ◽  
Binding Pockets ◽  
Folded Proteins

Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.

scholarly journals TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

2010 ◽  
Vol 21 (23) ◽  
pp. 4151-4161 ◽  
Author(s):  
Carlo Maurer ◽  
Sascha Panahandeh ◽  
Anna-Carina Jungkamp ◽  
Michael Moser ◽  
Matthias Müller
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Membrane Vesicles ◽  
Twin Arginine Translocation ◽  
Amphipathic Helices ◽  
Collective Data ◽  
Close Proximity ◽  
Precursor Proteins ◽  
Cross Linker ◽  
Folded Proteins

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

scholarly journals Component Specificity for the Thylakoidal Sec and Delta Ph–Dependent Protein Transport Pathways

1999 ◽  
Vol 146 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Hiroki Mori ◽  
Elizabeth J. Summer ◽  
Xianyue Ma ◽  
Kenneth Cline
Keyword(s):  
Protein Transport ◽  
Signal Recognition Particle ◽  
Thylakoid Membranes ◽  
Transport Pathways ◽  
Sec Pathway ◽  
Evolutionarily Conserved ◽  
In Series ◽  
Dependent Protein ◽  
Membrane Components ◽  
Substrate Proteins

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in ∼10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

scholarly journals Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Iain D. Hay ◽  
Matthew J. Belousoff ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Protein Transport ◽  
Substrate Uptake ◽  
Inner Membrane ◽  
Protein Substrates ◽  
Folded Proteins ◽  
Membrane Components ◽  
Substrate Proteins ◽  
Vexing Question

ABSTRACT Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. Pseudomonas aeruginosa has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this Pseudomonas-type secretin, which varies greatly in sequence from the well-characterized Klebsiella-type and Vibrio-type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the Klebsiella-type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin. IMPORTANCE How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane. IMPORTANCE How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane.

scholarly journals Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

2021 ◽  
Author(s):  
Ankith Sharma ◽  
Rajdeep Chowdhury ◽  
Siegfried M Musser
Keyword(s):  
Single Molecule ◽  
Membrane Vesicles ◽  
Reaction Chamber ◽  
Dependent Manner ◽  
Receptor Complex ◽  
Twin Arginine Translocation ◽  
Receptor Complexes ◽  
Maximal Diameter ◽  
Wide Range ◽  
Folded Proteins

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable, but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typical low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

scholarly journals The Tat-dependent protein translocation pathway

2011 ◽  
Vol 2 (6) ◽  
pp. 507-523 ◽  
Author(s):  
Bo Hou ◽  
Thomas Brüser
Keyword(s):  
Protein Transport ◽  
Protein Translocation ◽  
Transport Systems ◽  
Binding Motif ◽  
Twin Arginine Translocation ◽  
Multiple Copies ◽  
Dependent Protein ◽  
Tat System ◽  
Folded Proteins ◽  
Polytopic Membrane Protein

AbstractThe twin-arginine translocation (Tat) pathway is found in bacteria, archaea, and plant chloroplasts, where it is dedicated to the transmembrane transport of fully folded proteins. These proteins contain N-terminal signal peptides with a specific Tat-system binding motif that is recognized by the transport machinery. In contrast to other protein transport systems, the Tat system consists of multiple copies of only two or three usually small (∼8–30 kDa) membrane proteins that oligomerize to two large complexes that transiently interact during translocation. Only one of these complexes includes a polytopic membrane protein, TatC. The other complex consists of TatA. Tat systems of plants, proteobacteria, and several other phyla contain a third component, TatB. TatB is evolutionarily and structurally related to TatA and usually forms tight complexes with TatC. Minimal two-component Tat systems lacking TatB are found in many bacterial and archaeal phyla. They consist of a ‘bifunctional’ TatA that also covers TatB functionalities, and a TatC. Recent insights into the structure and interactions of the Tat proteins have various important implications.

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