scholarly journals Relaxed Specificity of the Bacillus subtilis TatAdCd Translocase in Tat-Dependent Protein Secretion

2008 ◽  
Vol 191 (1) ◽  
pp. 196-202 ◽  
Author(s):  
Robyn T. Eijlander ◽  
Jan D. H. Jongbloed ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Lipid Bilayer ◽  
Protein Secretion ◽  
Protein Translocation ◽  
Substrate Specificities ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Dependent Protein ◽  
Hydrophobic Lipid

ABSTRACT Protein translocation via the twin arginine translocation (TAT) pathway is characterized by the translocation of prefolded proteins across the hydrophobic lipid bilayer of the membrane. In Bacillus subtilis, two different Tat translocases are involved in this process, and both display different substrate specificities: PhoD is secreted via TatAdCd, whereas YwbN is secreted via TatAyCy. It was previously assumed that both TatAy and TatCy are essential for the translocation of the YwbN precursor. Through complementation studies, we now show that TatAy can be functionally replaced by TatAd when the latter is offered to the cells in excess amounts. Moreover, under conditions of overproduction, TatAdCd, in contrast to TatAyCy, shows an increased tolerance toward the acceptance of various Tat-dependent proteins.

scholarly journals Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1776-1785 ◽  
Author(s):  
Robyn T. Eijlander ◽  
Magdalena A. Kolbusz ◽  
Erwin M. Berendsen ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Protein Secretion ◽  
Transmembrane Domain ◽  
Protein Translocation ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Twin Arginine Translocation ◽  
Secretion Efficiency ◽  
Dependent Protein ◽  
Tat System

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

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 Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

2021 ◽  
Author(s):  
Umesh K Bageshwar ◽  
Antara DattaGupta ◽  
Siegfried M Musser
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Binding Interaction ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Hand Off ◽  
Dependent Protein ◽  
Tat System ◽  
Folded Proteins ◽  
High Concentrations

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitrotransport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 M), and this monomer binds reversibly to spTorA (KD 1 M). While TorD binds to membranes (KD 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

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 TatABC Overexpression Improves Corynebacterium glutamicum Tat-Dependent Protein Secretion

2008 ◽  
Vol 75 (3) ◽  
pp. 603-607 ◽  
Author(s):  
Yoshimi Kikuchi ◽  
Hiroshi Itaya ◽  
Masayo Date ◽  
Kazuhiko Matsui ◽  
Long-Fei Wu
Keyword(s):  
Corynebacterium Glutamicum ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Threefold Increase ◽  
Tat Pathway ◽  
Dependent Protein ◽  
Tat System ◽  
Streptomyces Mobaraensis

ABSTRACT The twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum has been described previously. The minimal functional Tat system in C. glutamicum required TatA and TatC but did not require TatB, although this component was required for maximal efficiency of Tat-dependent secretion. We previously demonstrated that Chryseobacterium proteolyticum pro-protein glutaminase (pro-PG) and Streptomyces mobaraensis pro-transglutaminase (pro-TG) could be secreted via the Tat pathway in C. glutamicum. Here we report that the amounts of pro-PG secreted were more than threefold larger when TatC or TatAC was overexpressed, and there was a further threefold increase when TatABC was overexpressed. These results show that the amount of TatC protein is the first bottleneck and the amount of TatB protein is the second bottleneck in Tat-dependent protein secretion in C. glutamicum. In addition, the amount of pro-TG that accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum was larger than the amount that accumulated via the Sec pathway. We concluded that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum.

Identification of Streptomyces lividans proteins secreted by the twin-arginine translocation pathway following growth with different carbon sources

2008 ◽  
Vol 54 (7) ◽  
pp. 549-558 ◽  
Author(s):  
Julien Guimond ◽  
Rolf Morosoli
Keyword(s):  
Signal Sequence ◽  
Streptomyces Coelicolor ◽  
Carbon Sources ◽  
Streptomyces Lividans ◽  
Extracellular Proteins ◽  
Soil Extracts ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Close Proximity ◽  
Dependent Protein

Genome-based signal peptide predictions classified Streptomyces coelicolor as the microorganism that secretes the most proteins through the twin-arginine translocation (Tat)-dependent secretion pathway. Availability of a ΔtatC mutant of the closely related strain Streptomyces lividans impaired Tat-dependent protein secretion and enabled identification of many extracellular proteins that are secreted via the Tat pathway. Proteomic techniques were applied to analyze proteins from the supernatants of log-phase cultures. Since the bacterial secretome depends mainly on the carbon sources available during growth, xylose, glucose, chitin, and soil extracts were used. A total of 63 proteins were identified, among which 7 were predicted by the TATscan program, and 20 were not predicted but contained a potential Tat signal motif. Thirteen proteins having no signal sequence could be co-transported by Tat-dependent proteins because the genes that encode these proteins are in close proximity in the genome. Finally, the presence of 23 proteins lacking signal peptides was difficult to explain. More secreted proteins could be identified as Tat substrates in varying carbon sources.

Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion

2002 ◽  
Vol 98 (2-3) ◽  
pp. 243-254 ◽  
Author(s):  
Jan Maarten van Dijl ◽  
Peter G. Braun ◽  
Colin Robinson ◽  
Wim J. Quax ◽  
Haike Antelmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Secretion ◽  
Genomic Analysis ◽  
Functional Genomic ◽  
Functional Genomic Analysis ◽  
Tat Pathway

scholarly journals Phage Shock Protein PspA of Escherichia coli Relieves Saturation of Protein Export via the Tat Pathway

2004 ◽  
Vol 186 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Matthew P. DeLisa ◽  
Philip Lee ◽  
Tracy Palmer ◽  
George Georgiou
Keyword(s):  
Genomic Library ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Protein A ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Protein Precursor ◽  
Twin Arginine Translocation ◽  
Tat Pathway

ABSTRACT Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.

scholarly journals The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

2008 ◽  
Vol 74 (24) ◽  
pp. 7507-7513 ◽  
Author(s):  
Marc A. B. Kolkman ◽  
René van der Ploeg ◽  
Michael Bertels ◽  
Maurits van Dijk ◽  
Joop van der Laan ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Heterologous Proteins ◽  
Independent Manner ◽  
Sec Pathway ◽  
Tat Pathway ◽  
Cargo Proteins ◽  
Dependent Protein ◽  
Application Potential ◽  
Tat System

ABSTRACT Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.

scholarly journals Novel Tat-Dependent Protein Secretion

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Volkmar Braun ◽  
Klaus Hantke
Keyword(s):  
Outer Membrane ◽  
Protein Secretion ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Cytoplasmic Membrane ◽  
Twin Arginine Translocation ◽  
New Class ◽  
Ecf Sigma Factor ◽  
Dependent Protein ◽  
Tat System

ABSTRACT The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of Journal of Bacteriology, I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.

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