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 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 Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport

2001 ◽  
Vol 154 (4) ◽  
pp. 719-730 ◽  
Author(s):  
Kenneth Cline ◽  
Hiroki Mori
Keyword(s):  
Specific Binding ◽  
Signal Peptides ◽  
Hydrophobic Core ◽  
Precursor Proteins ◽  
Peptide Precursors ◽  
Folded Proteins ◽  
Dependent Transport ◽  
Dependent Pathway ◽  
Blue Native ◽  
Chemical Cross Linking

The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

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 Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

2009 ◽  
Vol 187 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ljiljana Milenkovic ◽  
Matthew P. Scott ◽  
Rajat Rohatgi
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Primary Cilia ◽  
Protein Transport ◽  
Transmembrane Protein ◽  
Signaling Proteins ◽  
Lateral Transport ◽  
Transport Pathway ◽  
Ciliary Membrane ◽  
Specific Proteins

The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration.

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 The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial β-Lactamases

2005 ◽  
Vol 187 (22) ◽  
pp. 7667-7679 ◽  
Author(s):  
Justin A. McDonough ◽  
Kari E. Hacker ◽  
Anthony R. Flores ◽  
Martin S. Pavelka ◽  
Miriam Braunstein
Keyword(s):  
Mycobacterium Smegmatis ◽  
Signal Sequence ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Cellular Functions ◽  
Signal Sequences ◽  
Twin Arginine Translocation ◽  
Folded Proteins ◽  
Export System

ABSTRACT The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of ΔtatA and ΔtatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active β-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active ′BlaC. Thus, ′BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.

scholarly journals Rapid diffusion of large TatA complexes detected using single particle tracking microscopy

2020 ◽  
Author(s):  
Aravindan Varadarajan ◽  
Felix Oswald ◽  
Holger Lill ◽  
Erwin J.G. Peterman ◽  
Yves J. M. Bollen
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Transmembrane Protein ◽  
Single Particle Tracking ◽  
Fast Diffusion ◽  
Translocation Event ◽  
Twin Arginine Translocation ◽  
Rhomboid Proteases ◽  
Folded Proteins ◽  
Membrane Spanning

AbstractThe twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of most bacteria and archaea. TatA, which contains a single membrane-spanning helix, is believed to be responsible for the actual translocation. According to the prevalent model, multiple TatA subunits form a transient protein-conducting pore, which disassembles after each translocation event. An alternative model exists, in which TatA proteins locally weaken the lipid bilayer to translocate folded proteins. Here, we imaged eGFP-fused TatA expressed from the genome in live E. coli cells. Images showed TatA occuring both in highly mobile monomers or small oligomers and in large, stable complexes that do not dissociate. Single-particle tracking revealed that large TatA complexes switch between fast and slow diffusion. The fast diffusion is too fast for a transmembrane protein complex consisting of multiple TatA monomers. In line with recent data on rhomboid proteases, we propose that TatA complexes switch between a slowly diffusing transmembrane conformation and a rapidly diffusing membrane-disrupting state that enables folded proteins to cross the membrane, in accordance with the membrane-weakening model.

scholarly journals Functional Tat Transport of Unstructured, Small, Hydrophilic Proteins

2007 ◽  
Vol 282 (46) ◽  
pp. 33257-33264 ◽  
Author(s):  
Silke Richter ◽  
Ute Lindenstrauss ◽  
Christian Lücke ◽  
Richard Bayliss ◽  
Thomas Brüser
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Hydrophobic Surface ◽  
Physiological Data ◽  
Nuclear Pore ◽  
Hydrophobic Core ◽  
Twin Arginine Translocation ◽  
Surface Patches ◽  
Tat System ◽  
Folded Proteins

The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.

scholarly journals Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation

2001 ◽  
Vol 183 (2) ◽  
pp. 604-610 ◽  
Author(s):  
Natascha Blaudeck ◽  
Georg A. Sprenger ◽  
Roland Freudl ◽  
Thomas Wiegert
Keyword(s):  
Fusion Protein ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Signal Peptides ◽  
E Coli ◽  
Amino Terminal ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Dependent Protein ◽  
Folded Proteins

ABSTRACT The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) ofZymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC andtatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

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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