An escort mechanism for cycling of export chaperones during flagellum assembly

Evans, Lewis; Stafford, Graham P.; Ahmed, Sangita; Fraser, Gillian M.; Hughes, Colin

doi:10.1073/pnas.0605197103

Cited by 95 publications

(146 citation statements)

References 29 publications

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“…During F-T3SS protein export, the chaperone-substrate complexes dock at the membrane ATPase (Gauthier & Finlay, 2003;Thomas et al, 2004), which facilitates the release of the chaperone from the cognate secreted substrate in an ATP-dependent manner (Akeda & Galán, 2005). After release by an escort mechanism in F-T3SS protein export, free chaperones can be cycled (Evans et al, 2006). Thus, chaperone transition between cytosol and membrane compartments facilitates F-T3SS export of the flagellar component and, by extension, facilitates secretion of NF-T3SS proteins.…”

Section: Discussionmentioning

confidence: 99%

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Wáng

Mao

et al. 2009

Microbiology

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Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and coimmunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD 50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

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Section: Discussionmentioning

confidence: 99%

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Wáng

Mao

et al. 2009

Microbiology

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“…FlgN, a flagellum-specific chaperone for the hook-filament junction proteins FlgK and FlgL, has been reported to bind to two soluble components of the flagellar type III export apparatus, FliI and FliJ (17,18). Because FliT is required for efficient export of FliD, FliT also is expected to bind to the soluble components of the export apparatus.…”

Section: Interactions Of Flit With Flagellar Export Apparatus Proteinmentioning

confidence: 99%

“…They bind not only to their cognate substrates to prevent premature aggregation in the cytoplasm (15,16) but also to the soluble export component proteins FliI and FliJ (17,18). These chaperones also play other distinct roles in the flagellar construction process.…”

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confidence: 99%

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Structural insight into the regulatory mechanisms of interactions of the flagellar type III chaperone FliT with its binding partners

Imada

Minamino

Kinoshita

et al. 2010

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

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For self-assembly of the bacterial flagellum, most of the flagellar component proteins synthesized in the cytoplasm are exported by the flagellar type III export apparatus to the growing, distal end. Flagellar protein export is highly organized and well controlled in every step of the flagellar assembly process. Flagellar-specific chaperones not only facilitate the export of their cognate proteins, as well as prevent their premature aggregation in the cytoplasm, but also play a role in finetuning flagellar gene expression to be coupled with the flagellar assembly process. FliT is a flagellar-specific chaperone responsible for the export of the filament-capping protein FliD and for negative control of flagellar gene expression by binding to the FlhDC complex. Here we report the crystal structure of Salmonella FliT at 3.2-Å resolution. The structural and biochemical analyses clearly reveal that the C-terminal segment of FliT regulates its interactions with the FlhDC complex, FliI ATPase, and FliJ (subunits of the export apparatus), and that its conformational change is responsible for the switch in its binding partners during flagellar protein export.bacterial flagellum | crystal structure | flagellar gene expression | molecular chaperone | type III protein export

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“…To examine the FliI requirement more closely we 15,16 . The MIC measured for the DfliHIJ strain was 12 mg ml 21 , reproducibly larger than that of a negative-control strain lacking the MS-ring gene fliF (,3 mg ml 21 ) or a strain with all the flagellar genes repressed by downregulation of the master regulators flhDC (,3 mg ml 21 ) (Table 1).…”

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confidence: 99%

Energy source of flagellar type III secretion

Paul

Erhardt

Hirano

et al. 2008

Nature

290

317

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Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane 1 . This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion 2,3 . The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export 1,4 . Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the nonflagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a protondriven protein exporter and that ATP hydrolysis is not essential for type III secretion.Flagellar assembly begins with structures in the cytoplasmic membrane and proceeds through steps that add the exterior structures in a proximal-to-distal sequence (Fig. 1) 1 . Assembly of the rod, hook and filament requires the action of the secretion apparatus, which transports the needed subunits into a central channel through the structure that conducts them to their site of incorporation at the tip ( Fig. 1). Flagellar export is notably fast: in the early stages of filament growth flagellin is delivered at a rate of several 55 kDa subunits per second 5 .ATP hydrolysis by FliI was thought to provide the energy for export because mutations that delete or reduce the activity of FliI block flagellar synthesis at the stage of rod assembly 1,4,6 (Fig. 1). Homologues of FliI also occur in the type III secretion apparatus of injectisomes and are usually assumed to energize export in those systems as well. Some evidence for a different view has also been reported: it was observed that type III secretion in Yersinia enterocolitica was prevented by the protonophore CCCP 7 , and it was shown that the secretion ATPase InvC of Salmonella functions to dissociate export substrate from the chaperone 8 , a role distinct from transport itself. The energy source for type III secretion thus remains uncertain.To address the energy requirements for type III secretion, we first measured the effect of the uncoupler CCCP on flagellar export in S. enterica, assayed by accumulation of the export substrate FlgM in the medium. FlgM export was prevented by 10 mM or more CCCP (Fig. 2a). Overall cellular energy levels seemed unaffected, because cells grew normally in 10 mM CCCP (growth data not shown) and ATP levels were unchanged ( Supplementary Fig. 1). The effect was reversible: FlgM export was largely re...

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An escort mechanism for cycling of export chaperones during flagellum assembly

Cited by 95 publications

References 29 publications

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Structural insight into the regulatory mechanisms of interactions of the flagellar type III chaperone FliT with its binding partners

Energy source of flagellar type III secretion

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