Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

DeLisa, Matthew P.; Tullman, Danielle; Georgiou, George

doi:10.1073/pnas.0937838100

Cited by 279 publications

(403 citation statements)

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“…In the absence of the assembly partner or when cofactor assembly has been impaired by mutation, protein export through Tat does not take place (Rodrigue et al 1996;Halbig et al 1999). These observations together with in vitro and genetic evidence point to a protein folding quality control mechanism that is intrinsic to Tat translocation (DeLisa et al 2003). As a result of the folding quality control feature of the Tat pathway, only folded proteins are accepted for translocation across the membrane.…”

mentioning

confidence: 71%

“…We then sought to develop an enzymatic assay for the export of the ssTorAbait:prey-reporter complex into the periplasm. However, we could not take advantage of the widely used periplasmic reporter alkaline phosphatase (AP) because it has to fold in the cytoplasm before it can be exported via Tat (DeLisa et al 2003). Cytoplasmic folding of AP can be accomplished only in specialized oxidizing mutant strains (Bessette et al 1999).…”

Section: Resultsmentioning

confidence: 99%

“…Rodrigue et al (1999) first coined the term ''hitchhiker export'' after observing that the large subunit HybC of the E. coli hydrogenase 2, which does not contain a signal peptide, was translocated as a complex with HybO, which contains a 37-aminoacid-long Tat signal peptide. More recent studies demonstrated that dimeric F AB antibody fragments can also be exported by a hitchhiker mechanism via a signal peptide on only one of the two polypeptides that comprise the F AB protein (DeLisa et al 2003). In our approach, one protein is fused to a Tat signal peptide and the second protein to a protein reporter that can confer a phenotype only upon export into the periplasmic space.…”

Section: Discussionmentioning

confidence: 99%

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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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confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Strauch

Georgiou

2007

Protein Science

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“…37 Blots were probed with either anti-MBP antibodies conjugated with horseradish peroxidase (HRP) (New England Biolabs) or hR6P antiserum that is specific for the C. jejuni heptasaccharide (kindly provided by Dr. Brendan Wren and Dr. Markus Aebi). In the case of hR6P, anti-rabbit IgG-HRP (Promega) was used as the secondary antibody.…”

Section: Western Blot Analysismentioning

confidence: 99%

A filamentous phage display system for N‐linked glycoproteins

et al. 2010

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We have developed a filamentous phage display system for the detection of asparaginelinked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X 1 -N-X 2 -S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 3 10 5 per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phagedisplayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the 22 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.

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“…Proteins that utilize the Tat pathway are characterized by two defining features: (i) they contain a consensus S/T-R-R-x-F-L-K twin arginine (RR) motif in their N-terminal leader peptide sequence (RR-leader); and (ii) they are usually translocated across the cytoplasmic membrane as an active and folded holoenzyme (8)(9)(10). The Tat translocon consists of the TatABC subunits and the current model identifies TatA as the homo-oligomeric pore subunit, whereas TatB and TatC act in substrate recognition and delivery (11,12).…”

Section: So Dmso] (4)mentioning

confidence: 99%

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

Cherak

Turner

2017

Biomolecular Concepts

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Protein folding and assembly into macromolecule complexes within the living cell are complex processes requiring intimate coordination. The biogenesis of complex iron sulfur molybdoenzymes (CISM) requires use of a system specific chaperone -a redox enzyme maturation protein (REMP) -to help mediate final folding and assembly. The CISM dimethyl sulfoxide (DMSO) reductase is a bacterial oxidoreductase that utilizes DMSO as a final electron acceptor for anaerobic respiration. The REMP DmsD strongly interacts with DMSO reductase to facilitate folding, cofactor-insertion, subunit assembly and targeting of the multi-subunit enzyme prior to membrane translocation and final assembly and maturation into a bioenergetic catalytic unit. In this article, we discuss the biogenesis of DMSO reductase as an example of the participant network for bacterial CISM maturation pathways.

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Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

Cited by 279 publications

References 44 publications

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

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

A filamentous phage display system for N‐linked glycoproteins

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

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