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 A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase

2002 ◽  
Vol 157 (2) ◽  
pp. 205-210 ◽  
Author(s):  
Hiroki Mori ◽  
Kenneth Cline
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Permeability Barrier ◽  
Synthetic Signal ◽  
Tat Pathway ◽  
Precursor Proteins ◽  
Varied Size ◽  
Tat System ◽  
Folded Proteins ◽  
Chemical Cross Linking

The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

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 The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e34159 ◽  
Author(s):  
Anitha Shanmugham ◽  
Adil Bakayan ◽  
Petra Völler ◽  
Joost Grosveld ◽  
Holger Lill ◽  
...  
Keyword(s):  
Specific Binding ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Tat Pathway

scholarly journals Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

2001 ◽  
Vol 12 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Doron Rapaport ◽  
Rebecca D. Taylor ◽  
Michael Käser ◽  
Thomas Langer ◽  
Walter Neupert ◽  
...  
Keyword(s):  
Intermediate Stage ◽  
In Vivo Studies ◽  
Mitochondrial Outer Membrane ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Blue Native

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

scholarly journals Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

1997 ◽  
Vol 136 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Mitsuru Akita ◽  
Erik Nielsen ◽  
Kenneth Keegstra
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
Protein Import ◽  
Cross Linking ◽  
Envelope Membrane ◽  
Precursor Proteins ◽  
Intact Chloroplasts ◽  
Outer Envelope Membrane ◽  
Envelope Membranes ◽  
Chemical Cross Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

Generation of the neutrophil-activating peptide-2 by cathepsin G and cathepsin G-treated human platelets

1992 ◽  
Vol 263 (2) ◽  
pp. L249-L256 ◽  
Author(s):  
A. B. Cohen ◽  
M. D. Stevens ◽  
E. J. Miller ◽  
M. A. Atkinson ◽  
G. Mullenbach
Keyword(s):  
Specific Binding ◽  
Human Platelets ◽  
Cathepsin G ◽  
Myocardial Infarctions ◽  
Activated Platelets ◽  
Pulmonary Capillaries ◽  
Precursor Proteins ◽  
Activated Neutrophils ◽  
Neutrophil Degranulation ◽  
Unique Mechanism

The neutrophil-activating peptide-2 (NAP-2) is a cytokine that is generated by the proteolytic cleavage of a precursor protein and that causes neutrophil degranulation and chemotaxis. NAP-2 precursors are produced in platelets and are normally found in the circulation. We showed that NAP-2 is generated by the action of neutrophil cathepsin G on two of the precursors, the connective tissue-activating peptide-III (CTAP-III) and beta-thromboglobulin (beta-TG). However, neutrophil elastase degraded the precursors to inactive peptides. The specific binding of cathepsin G to platelets caused the platelets to secrete NAP-2, and cathepsin G bound to the platelets could still generate NAP-2 from its precursor proteins. In addition, activated neutrophils in the presence of platelets generated NAP-2 from its precursors and caused platelets to secrete NAP-2. These studies demonstrate a unique mechanism for the activation of neutrophils through the interaction of neutrophils, platelets, and NAP-2 precursors that are released either by activated platelets or are present in circulation. It is therefore possible that NAP-2 may be generated at sites where aggregations of neutrophils and platelets occur in vessels such as pulmonary capillaries in patients with the adult respiratory distress syndrome and coronary arteries in patients with evolving myocardial infarctions.

scholarly journals Plastid chaperone HSP90C guides precursor proteins to the SEC translocase for thylakoid transport

2020 ◽  
Vol 71 (22) ◽  
pp. 7073-7087
Author(s):  
Tim Jiang ◽  
Bona Mu ◽  
Rongmin Zhao
Keyword(s):  
Protein Targeting ◽  
Fluorescent Protein ◽  
Heat Shock Protein 90 ◽  
Specific Targeting ◽  
In Organello ◽  
Precursor Proteins ◽  
Thylakoid Protein ◽  
Green Fluorescent ◽  
Dependent Transport

Abstract Chloroplast stromal factors involved in regulating thylakoid protein targeting are poorly understood. We previously reported that in Arabidopsis thaliana, the stromal-localized chaperone HSP90C (plastid heat shock protein 90) interacted with the nuclear-encoded thylakoid lumen protein PsbO1 (PSII subunit O isoform 1) and suggested a role for HSP90C in aiding PsbO1 thylakoid targeting. Using in organello transport assays, particularly with model substrates naturally expressed in stroma, we showed that light, exogenous ATP, and HSP90C activity were required for Sec-dependent transport of green fluorescent protein (GFP) led by the PsbO1 thylakoid targeting sequence. Using a previously identified PsbO1T200A mutant, we provided evidence that a stronger interaction between HSP90C and PsbO1 better facilitated its stroma–thylakoid trafficking. We also demonstrated that SecY1, the channel protein of the thylakoid SEC translocase, specifically interacted with HSP90C in vivo. Inhibition of the chaperone ATPase activity suppressed the association of the PsbO1GFP–HSP90C complex with SecY1. Together with analyzing the expression and accumulation of a few other thylakoid proteins that utilize the SRP, TAT, or SEC translocation pathways, we propose a model in which HSP90C forms a guiding complex that interacts with thylakoid protein precursors and assists in their specific targeting to the thylakoid SEC translocon.

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 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 The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex

2017 ◽  
Vol 292 (26) ◽  
pp. 10865-10882 ◽  
Author(s):  
Agnes Ulfig ◽  
Julia Fröbel ◽  
Frank Lausberg ◽  
Anne-Sophie Blümmel ◽  
Anna Katharina Heide ◽  
...  
Keyword(s):  
Signal Peptides ◽  
Receptor Complex ◽  
Precursor Proteins
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