Twin arginine translocation (Tat)-dependent protein transport: the passenger protein participates in the initial membrane binding step

2010 ◽  
Vol 391 (12) ◽  
Author(s):  
René Schlesier ◽  
Ralf Bernd Klösgen
Keyword(s):  
Protein Transport ◽  
Membrane Binding ◽  
Transit Peptide ◽  
Initial Step ◽  
Mature Protein ◽  
Twin Arginine Translocation ◽  
Protected Fragment ◽  
Initial Membrane ◽  
Dependent Protein ◽  
Passenger Protein

Abstract The initial step in twin arginine translocation (Tat)-dependent thylakoid transport of the 16/23 chimera is the interaction of the protein with the lipid bilayer. It results in the formation of the early translocation intermediate Ti-1, which is represented by a protease-protected fragment of 14 kDa. Cys-scanning mutagenesis in combination with in thylakoido and liposome insertion assays was used to precisely map this membrane-interacting and protease-protected fragment within the 16/23 chimera. The fragment comprises 124 residues, which are provided both by the transit peptide (31 residues) and the mature protein (93 residues), demonstrating that the passenger protein directly participates in membrane binding. The implications of this finding on the mechanism of Tat-dependent protein transport are discussed.

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.

Unassisted Membrane Insertion as the Initial Step in ΔpH/Tat-dependent Protein Transport

2006 ◽  
Vol 355 (5) ◽  
pp. 957-967 ◽  
Author(s):  
Bo Hou ◽  
Stefan Frielingsdorf ◽  
Ralf Bernd Klösgen
Keyword(s):  
Protein Transport ◽  
Initial Step ◽  
Membrane Insertion ◽  
Dependent Protein

scholarly journals Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

2003 ◽  
Vol 185 (9) ◽  
pp. 2811-2819 ◽  
Author(s):  
Natascha Blaudeck ◽  
Peter Kreutzenbeck ◽  
Roland Freudl ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Alternative Possibilities ◽  
Amino Acid Residues ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Arginine Residues ◽  
Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

scholarly journals Twin-arginine translocation-arresting protein regions contact TatA and TatB

2014 ◽  
Vol 395 (7-8) ◽  
pp. 827-836 ◽  
Author(s):  
Johannes Taubert ◽  
Thomas Brüser
Keyword(s):  
Signal Peptide ◽  
Sequence Motif ◽  
Globular Domain ◽  
Mature Protein ◽  
Linker Region ◽  
Twin Arginine Translocation ◽  
C Terminus ◽  
Translocation Efficiency ◽  
Folded Proteins

Abstract Tat systems translocate folded proteins across biological membranes of prokaryotes and plant plastids. TatBC complexes recognize N-terminal Tat signal peptides that contain a sequence motif with two conserved arginines (RR-motif), and transport takes place after a recruitment of TatA. Unfolded Tat substrate domains lower translocation efficiency and too long linkers lead to translocation arrest. To identify the components that interact with transported proteins during their passage through the translocon, we used a Tat substrate that arrests translocation at a long unfolded linker region, and we chose in vivo site-directed photo cross-linking to specifically detect the interactions of this linker region. For comparison, we included the interactions of the signal peptide and of the folded domain at the C-terminus of this construct. The data show that the linker contacts only two, structurally similar Tat components, namely TatA and TatB. These contacts depend on the recognition of the Tat-specific signal peptide. Only when membrane translocation of the globular domain was allowed – i.e., in the absence of the linker – we observed the same TatAB-contacts also to the globular domain. The data thus suggest that mature protein domains are translocated through a TatAB environment.

Dual targeting ability of targeting signals is dependent on the nature of the mature protein

2003 ◽  
Vol 30 (7) ◽  
pp. 805 ◽  
Author(s):  
Orinda Chew ◽  
James Whelan
Keyword(s):  
Alternative Oxidase ◽  
Small Subunit ◽  
Trna Synthetase ◽  
Mature Protein ◽  
Bisphosphate Carboxylase ◽  
Dual Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Targeting Ability ◽  
Passenger Protein

The targeting ability of three signals previously shown to support the import of passenger proteins into both mitochondria and chloroplasts was investigated with authentic mitochondrial or chloroplastic proteins. An in vitro dual import assay that maintained import specificity showed that the ability of dual signals to support mitochondrial and chloroplastic import depended on the nature of the passenger protein. All dual targeting signals supported import of their native mature protein as a passenger into both mitochondria and chloroplasts. However the glutathione reductase targeting signal only supported mitochondrial import with the mitochondrial protein alternative oxidase, and chloroplast import with the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase. The Arabidopsis histidyl-tRNA synthetase targeting signal only supported mitochondrial import with the alternative oxidase as a passenger, but the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase was imported into both mitochondria and chloroplasts. The Arabidopsis asparaginyl-tRNA synthetase supported import of alternative oxidase and the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase into both mitochondria and chloroplasts. Analysis of the targeting signals of all known dual targeted proteins using targeting predictions indicates that most of them are more strongly predicted to be chloroplast-targeted. Secondary structure predictions indicate the ability of most dual targeted signals to form both α-helical and β-sheet-type structures, a feature of mitochondrial and plastid targeting signals, respectively. Thus, it appears that a major determinant of dual targeting ability is the nature of the mature or passenger protein.

scholarly journals Stromal Processing Peptidase Binds Transit Peptides and Initiates Their Atp-Dependent Turnover in Chloroplasts

1999 ◽  
Vol 147 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Stefan Richter ◽  
Gayle K. Lamppa
Keyword(s):  
Binding Assay ◽  
Specific Binding ◽  
Transit Peptide ◽  
Binding Motif ◽  
Mature Protein ◽  
Sequence Of Events ◽  
Mitochondrial Processing Peptidase ◽  
Transit Peptides ◽  
Processing Peptidase ◽  
Zinc Binding Motif

A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

scholarly journals Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

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