Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria

DeLisa, Matthew P.; Samuelson, Patrik; Palmer, Tracy; Georgiou, George

doi:10.1074/jbc.m201956200

Cited by 126 publications

(131 citation statements)

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“…1A) and that YidC co- purifies with overproduced SecDFyajC, but not with overproduced SecYEG (Nouwen and Driessen, 2002). It has also been known that the YidC pathway has a threading mechanism for unfolded proteins (DeLisa et al, 2002), YidC can interact directly with a Sec-independent membrane protein (Chen et al, 2002), and E. coli YidC is essential for viability (Samuelson et al, 2000). These results demonstrated the presence of the novel Yid pathway (named for its association with the YidCrelated pathway), differing from the Sec pathway and possessing a SecDFyajC translocon.…”

Section: Resultsmentioning

confidence: 99%

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Lee

Han

Kim

et al. 2011

Molecules and Cells

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Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔG RNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.

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Section: Resultsmentioning

confidence: 99%

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Lee

Han

Kim

et al. 2011

Molecules and Cells

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“…2A2). Substitution of the twin arginine dipeptide with a pair of lysines completely abolishes Tat export (DeLisa et al 2002;Ize et al 2002): Cells expressing the ssTorA(KK)-wbJun (pEMS16) gave rise to yellow-orange colonies (Fig. 2A1), indicating that the mal + phenotype shown in Figure 2A2 was dependent on Tat export.…”

Section: Resultsmentioning

confidence: 99%

A bacterial two‐hybrid system based on the twin‐arginine transporter pathway of E. coli

Strauch

Georgiou

2007

Protein Science

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We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide (''hitchhiker export''). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA À mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.Keywords: protein interaction; Tat pathway; protein export; two-hybrid system; folding; fusion protein; maltose-binding protein; DsbA Supplemental material: see www.proteinscience.orgIn recent years, the numbers of genes identified in a broad spectrum of organisms has grown exponentially. A major challenge will be categorizing the corresponding proteins into their functional units within the cell. Several approaches have been used to assign newly identified proteins into cellular networks (Galperin and Koonin 2000; Bork et al. 2004;de Lichtenberg et al. 2005). In vitro, interacting proteins can be detected by mass spectrometry or by chromatographic techniques, typically following genetic fusion with an appropriate affinity tag (Rigaut et al. 1999;Gavin et al. 2002;Butland et al. 2005). In vivo, genetic techniques for the identification of interacting proteins rely mainly on two-hybrid systems and protein complementation assays. A number of such techniques have been developed and used extensively in Escherichia coli (Hu 2001;Karimova et al. 2002Karimova et al. , 2005. So far, the detection of protein interactions in bacteria has capitalized on fusions to transcriptional repressors such as lcI, LexA, or AraC, transcriptional activators (involving the recruitment of RNA polymerase or the dimerization of the Vibrio cholera ToxR), complementation of biosynthetic Reprint requests to: George Georgiou, Chemical Engineering/UT Austin, 1 University Station C0400, Austin, TX 78712-0231, USA; e-mail: gg@che.utexas.edu; fax: (512) 471-7963.Abbreviations: Tat, twin-arginine translocation; Sec, general secretory pathway; TorA, trimethylamine N-oxide reductase; AP, alkaline phosphatase; MBP, maltose-binding protein; Im2, immunity protein 2; E2, endonuclease domain of colici...

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“…GFP, tagged on the C terminus with the SsrA peptide, is targeted for rapid degradation by the powerful ClpXP proteolytic machinery of the bacterial cytoplasm (35). Fusion of GFP-SsrA to the ssTorA leader that directs protein export via twin arginine transporter pathway enables export of ssTorA-GFP-SsrA protein into the periplasm where it sequestered away from the ClpXP protease and rescued from proteolytic degradation (36). As a result, cells expressing ssTorA-GFP-SsrA exhibit high fluorescence.…”

Section: Fluorescent Cell Labeling Using Endogenously Expressed Antigmentioning

confidence: 99%

“…Even though the fluorescence signal obtained was not as high compared with that obtained with exogenous fluorescent antigens, it is sufficient for library screening purposes. A higher signal may be obtained in strains that afford more efficient degradation of GFP-SsrA in the cytoplasm or by using leader peptides optimized for GFP export (36).…”

Section: Fluorescent Cell Labeling Using Endogenously Expressed Antigmentioning

confidence: 99%

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli -expressed libraries

Harvey

Georgiou

Hayhurst

et al. 2004

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

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Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final KD of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display͞APEx strategies without the need for further subcloning.

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Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria

Cited by 126 publications

References 41 publications

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

A bacterial two‐hybrid system based on the twin‐arginine transporter pathway of E. coli

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli -expressed libraries

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