The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding

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.

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Stoichiometry for binding and transport by the twin arginine translocation system

The Journal of Cell Biology ◽

10.1083/jcb.201201096 ◽

2012 ◽

Vol 197 (4) ◽

pp. 523-534 ◽

Cited By ~ 33

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.

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C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding

Biological Chemistry ◽

10.1515/hsz-2014-0249 ◽

2015 ◽

Vol 396 (4) ◽

pp. 349-357

Author(s):

René Schlesier ◽

Ralf Bernd Klösgen

Keyword(s):

Membrane Transport ◽

Receptor Binding ◽

Signal Peptide ◽

Membrane Topology ◽

Twin Arginine Translocation ◽

Rate Limiting Step ◽

Limiting Step ◽

Chimeric Model ◽

Rate Limiting ◽

Passenger Protein

Abstract During thylakoid transport of the chimeric model twin-arginine translocation (Tat) substrate 16/23, two consecutive translocation intermediates with different membrane topology are observed. The early translocation intermediate Ti-1 is bound to the membrane such that almost half of the protein is protected against proteolysis and it was concluded that not only the signal peptide but also part of the passenger protein participates in membrane binding. However, topology studies using a membrane-impermeable thiol-reactive reagent show that most of the passenger remains accessible from the stromal side in Ti-1 conformation. Establishment of such Ti-1 topology at the membrane apparently requires the fully folded passenger protein, as it was not observed with 16/23 truncation derivatives lacking the C-terminal 20, 40, 60, or 88 residues. Thylakoid transport of these mutants, which depends on a fully functional Tat machinery, is progressively reduced with increasing size of the truncated passenger polypeptide. The same holds true also for the interaction with the thylakoidal TatBC complexes, suggesting that in this case receptor binding, which is apparently impaired by extended unfolded or malfolded passenger polypeptides, is the rate-limiting step of Tat-dependent membrane transport.

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Twin-arginine-dependent translocation of folded proteins

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0202 ◽

2012 ◽

Vol 367 (1592) ◽

pp. 1029-1046 ◽

Cited By ~ 61

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.

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Structural determinants of vascular endothelial growth factor-D receptor binding and specificity

Blood ◽

10.1182/blood-2010-08-301549 ◽

2011 ◽

Vol 117 (5) ◽

pp. 1507-1515 ◽

Cited By ~ 57

Author(s):

Veli-Matti Leppänen ◽

Michael Jeltsch ◽

Andrey Anisimov ◽

Denis Tvorogov ◽

Kukka Aho ◽

...

Keyword(s):

Receptor Binding ◽

Proteolytic Cleavage ◽

Tyrosine Kinase Receptors ◽

Vascular Endothelial ◽

Cleavage Sites ◽

Structural Determinants ◽

Precursor Proteins ◽

And Function ◽

Endothelial Growth Factor ◽

Endothelial Growth Factors

Abstract Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2–specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function.

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Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167299 ◽

1996 ◽

Vol 54 ◽

pp. 966-967

Author(s):

C.A. Mannella ◽

K.F. Buttle ◽

K.A. O‘Farrell ◽

A. Leith ◽

M. Marko

Keyword(s):

Liver Mitochondrion ◽

Adenine Nucleotide ◽

Protein Complexes ◽

Mitochondrial Membranes ◽

Electron Microscopic ◽

Contact Sites ◽

Transmission Electron ◽

Precursor Proteins ◽

Membrane Contacts ◽

And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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