DnaK Plays a Pivotal Role in Tat Targeting of CueO and Functions beside SlyD as a General Tat Signal Binding Chaperone

Graubner, Wenke; Schierhorn, Angelika; Brüser, Thomas

doi:10.1074/jbc.m608235200

Cited by 78 publications

(86 citation statements)

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“…Thus, despite the Tat signal peptides from each of these complex enzymes having a final common purpose in interacting with the Tat translocase, the structures of the signal peptides must also be precision-tuned to fit only one type of binding protein. A future challenge will be to understand the molecular basis of how this exquisite chaperone specificity is balanced with a common transport function and, in some cases, the additional involvement of housekeeping chaperones (37,38).…”

Section: Discussionmentioning

confidence: 99%

Structural diversity in twin-arginine signal peptide-binding proteins

Maillard

Spronk

Buchanan

et al. 2007

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

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The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Escherichia coli, many Tat substrates bind redox-active cofactors in the cytoplasm before transport. Coordination of cofactor insertion with protein export involves a ''Tat proofreading'' process in which chaperones bind twin-arginine signal peptides, thus preventing premature export. The initial Tat signal-binding proteins described belonged to the TorD family, which are required for assembly of N-and S-oxide reductases. Here, we report that E. coli NapD is a Tat signal peptide-binding chaperone involved in biosynthesis of the Tatdependent nitrate reductase NapA. NapD binds tightly and specifically to the NapA twin-arginine signal peptide and suppresses signal peptide translocation activity such that transport via the Tat pathway is retarded. High-resolution, heteronuclear, multidimensional NMR spectroscopy reveals the 3D solution structure of NapD. The chaperone adopts a ferredoxin-type fold, which is completely distinct from the TorD family. Thus, NapD represents a new family of twin-arginine signal-peptide-binding proteins.metalloenzyme biosynthesis ͉ molecular chaperone ͉ NMR solution structure ͉ protein-protein interactions ͉ twin-arginine transport pathway

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Section: Discussionmentioning

confidence: 99%

Structural diversity in twin-arginine signal peptide-binding proteins

Maillard

Spronk

Buchanan

et al. 2007

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

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“…The cytoplasm contains a set of chaperones that is often essential for protein folding. An earlier work already indicated an essential role of the cytoplasmic chaperone DnaK, as CueO requires DnaK for being Tat-dependently translocated (19). The role of DnaK relates to the mature domain of CueO and not to the signal peptide (19).…”

Section: Discussionmentioning

confidence: 99%

“…An earlier work already indicated an essential role of the cytoplasmic chaperone DnaK, as CueO requires DnaK for being Tat-dependently translocated (19). The role of DnaK relates to the mature domain of CueO and not to the signal peptide (19). Proteome-wide analyses of the roles of the chaperones DnaK, trigger factor (TF), and GroEL identified CueO in aggregates that form in the absence of DnaK/DnaJ and TF (42).…”

Section: Discussionmentioning

confidence: 99%

“…Arguments for both scenarios have been reported (2,18). As CueO specifically serves to combat copper stress, it was suggested that sufficient copper reaches the cytoplasm under such conditions (2).However,CueOistransportedstrictlyTat-dependently also without copper stress (19), and a copper contentreducing mutation in either the type 1 or the type 2 copper binding site did not affect transport (20), which prompted us to analyze whether wild type CueO might actually be translocated as an apo-protein.…”

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confidence: 99%

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The Tat Substrate CueO Is Transported in an Incomplete Folding State

Stolle¹,

Hou²,

Brüser

2016

Journal of Biological Chemistry

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In Escherichia coli, cytoplasmic copper ions are toxic to cells even at the lowest concentrations. As a defense strategy, the cuprous oxidase CueO is secreted into the periplasm to oxidize the more membrane-permeable and toxic Cu(I) before it can enter the cytoplasm. CueO itself is a multicopper oxidase that requires copper for activity. Because it is transported by the twin-arginine translocation (Tat) pathway, which transports folded proteins, a requirement for cofactor assembly before translocation has been discussed. Here we show that CueO is transported as an apo-protein. Periplasmic CueO was readily activated by the addition of copper ions in vitro or under copper stress conditions in vivo. Cytoplasmic CueO did not contain copper, even under copper stress conditions. In vitro Tat transport proved that the cofactor assembly was not required for functional Tat transport of CueO. Due to the post-translocational activation of CueO, this enzyme contributes to copper resistance not only by its cuprous oxidase activity but also by chelation of copper ions before they can enter the cytoplasm. Apo-CueO was indistinguishable from holo-CueO in terms of secondary structural elements. Importantly, the binding of copper to apo-CueO greatly stabilized the protein, indicating a transformation from an open or flexible domain arrangement with accessible copper sites to a closed structure with deeply buried copper ions. CueO is thus the first example for a natural Tat substrate of such incomplete folding state. The Tat system may need to transport flexibly folded proteins in any case when cofactor assembly or quaternary structure formation occurs after transport.

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“…During the early steps in their biogenesis, proteins destined for Tat export interact with signal-sequencespecific (e.g., TorD) or general chaperones such as DnaK (Graubner et al 2007;Perez-Rodriguez et al 2007). There is no evidence that these cytoplasmic chaperones are exported together with their target polypeptide.…”

Section: Discussionmentioning

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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DnaK Plays a Pivotal Role in Tat Targeting of CueO and Functions beside SlyD as a General Tat Signal Binding Chaperone

Abstract: The Tat

Cited by 78 publications

References 43 publications

Structural diversity in twin-arginine signal peptide-binding proteins

Structural diversity in twin-arginine signal peptide-binding proteins

The Tat Substrate CueO Is Transported in an Incomplete Folding State

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

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