Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli

Barrett, Claire M.L.; Ray, Nicola; Thomas, Joanne; Robinson, Colin; Bolhuis, Albert

doi:10.1016/s0006-291x(03)00583-7

Cited by 79 publications

(77 citation statements)

References 21 publications

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“…2A, wild type panel), GFP is found as mature size protein in the periplasm (P) when expressed in wild type cells. Some mature size protein is also found in the cytoplasm, but we have observed this phenomenon before and concluded that it is due to proteolytic clipping of the precursor form (31). The TorA-GFP accumulates exclusively in the cytoplasm when expressed in a tat null mutant (⌬tat panel), again mostly as mature size protein.…”

Section: Overexpressed Tatadcd Forms An Active Translocationmentioning

confidence: 82%

“…The cell fractions were loaded and separated on a 10% native polyacrylamide gel that was subsequently assayed for TMAO reductase activity as described previously. For TorA-GFP export assays, a construct comprising the TorA signal peptide linked to GFP (30) was expressed using the pBAD-24 plasmid as previously described (31). For these experiments, TatAdCd was expressed from the compatible pEXT22 vector.…”

Section: Methodsmentioning

confidence: 99%

“…We first tested whether the cells can export a fusion protein comprising the TorA signal peptide linked to GFP. This fusion is exported by the Tat pathway in E. coli (30,31). We expressed TorA-GFP in E. coli ⌬ABCDE cells and coexpressed tatAdCd or E. coli tatABC on the compatible pEXT22 plasmid FIGURE 1.…”

Section: Overexpressed Tatadcd Forms An Active Translocationmentioning

confidence: 99%

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A Minimal Tat System from a Gram-positive Organism

Barnett

Eijlander

Kuipers

et al. 2008

Journal of Biological Chemistry

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The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, a TatABC substrate-binding complex and separate TatA complex are believed to coalesce to form an active translocon, with all three subunits essential for translocation. Most Gram-positive organisms lack a tatB gene, indicating major differences in organization and possible differences in mode of action. Here, we have studied Tat complexes encoded by the tatAdCd genes of Bacillus subtilis. Expression of tatAdCd in an Escherichia coli tat null mutant results in efficient export of a large, cofactorcontaining E. coli Tat substrate, TorA. We show that the tatAd gene complements E. coli mutants lacking either tatAE or tatB, indicating a bifunctional role for this subunit in B. subtilis. Second, we have identified and characterized two distinct Tat complexes that are novel in key respects: a TatAdCd complex of ϳ230 kDa that is significantly smaller than the analogous E. coli TatABC complex (ϳ370 kDa on BN gels) and a separate TatAd complex. The latter is a discrete entity of ϳ270 kDa as judged by gel filtration chromatography, very different from the highly heterogeneous E. coli TatA complex that ranges in size from ϳ50 kDa to over 600 kDa. TatA heterogeneity has been linked to the varying size of Tat substrates being translocated, but the singular nature of the B. subtilis TatAd complex suggests that discrete TatAC and TatA complexes may form a single form of translocon.The Tat (twin arginine translocation) pathway is involved in the transport of proteins across the chloroplast thylakoid membrane and the plasma membranes of a wide range of bacteria (1-6). It differs fundamentally from the other export pathway, the Sec pathway, in its ability to translocate prefolded proteins over the plasma membrane. This process appears not to require energy in the form of ATP hydrolysis but is instead dependent on the presence of the proton motive force across the membrane (1,7,8

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Section: Overexpressed Tatadcd Forms An Active Translocationmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Overexpressed Tatadcd Forms An Active Translocationmentioning

confidence: 99%

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A Minimal Tat System from a Gram-positive Organism

Barnett

Eijlander

Kuipers

et al. 2008

Journal of Biological Chemistry

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“…S1a), confirming that detection of this interaction depended on the Tat pathway. We suspected that the relatively low MIC in the case of WT cells was due to inefficient Tat export, which can often be alleviated by increasing the copy number of the TatABC proteins (40). Indeed, increased expression of TatABC from plasmid pBRTatABC resulted in an MIC of Ͼ400 g/mL Amp for WT cells coexpressing ssTorA-JunLZ-FLAG and FosLZ-Bla ( Fig.…”

Section: Conversion Of the Hitchhiker Mechanism Into A Genetic Selectmentioning

confidence: 99%

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Waraho

DeLisa

2009

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a reliable genetic selection strategy for isolating interacting proteins based on the ''hitchhiker'' mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an Nterminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 ␤-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer ␤-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.ligand binding proteins ͉ protein folding quality control ͉ signal peptide ͉ twin-arginine translocation ͉ 2-hybrid system P rotein-protein interactions are key molecular events that integrate multiple gene products into functional complexes in virtually every cellular process. Because such interactions mediate numerous disease states and biological mechanisms underlying the pathogenesis of bacterial and viral infections, identification of protein-protein interactions remains one of the most important challenges in the postgenomics era. The yeast 2-hybrid (Y2H) system (1) has been the tool of choice for revealing numerous protein-protein interactions, underlying diverse protein networks and complex protein machinery inside living cells. To date, Y2H has been used to generate protein interaction maps for humans (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), Saccharomyces cerivisiae (5, 6), vaccinia virus (7), and Escherichia coli bacteriophage T7 (8). Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (9, 10), and in certain instances ablate protein functi...

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“…In E. coli, even native Tat substrate proteins saturate the export process when overexpressed from strong promoters (36,68,134). Optimization of the growth conditions or coexpression of REMPs, PspA, or the TatABC proteins partially alleviates the saturation of the export apparatus (7,36,81). Still, protein yields for Tat-exported proteins are substantially lower than those obtained via Sec export, which often exceed 1 g liter −1 .…”

Section: Biotechnological Applicationsmentioning

confidence: 99%

The Bacterial Twin-Arginine Translocation Pathway

Lee

Tullman‐Ercek

Georgiou

2006

Annu. Rev. Microbiol.

294

241

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The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria. Substrates for the Tat pathway include redox enzymes requiring cofactor insertion in the cytoplasm, multimeric proteins that have to assemble into a complex prior to export, certain membrane proteins, and proteins whose folding is incompatible with Sec export. These proteins are involved in a diverse range of cellular activities including anaerobic metabolism, cell envelope biogenesis, metal acquisition and detoxification, and virulence. The Escherichia coli translocase consists of the TatA, TatB, and TatC proteins, but little is known about the precise sequence of events that leads to protein translocation, the energetic requirements, or the mechanism that prevents the export of misfolded proteins. Owing to the unique characteristics of the pathway, it holds promise for biotechnological applications.

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Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli

Cited by 79 publications

References 21 publications

A Minimal Tat System from a Gram-positive Organism

A Minimal Tat System from a Gram-positive Organism

Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

The Bacterial Twin-Arginine Translocation Pathway

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