2P-183 Functional analysis of conserved charged residues of FlhA, an export gate component of bacterial flagellar type III protein export appratus(Biol & Artifi memb.:Transport,The 47th Annual Meeting of the Biophysical Society of Japan) (original) (raw)
Related papers
Journal of Bacteriology, 2012
The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH EN ) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH EN with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.
The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H + –protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na + increased it dramatically. Phenamil, a blocker of Na + translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na + concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na + channel. In wild-type cells, however, neither Na + nor phenamil affected protein export, indicating that the Na + channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na + concentration. For construction of the bacterial flagellum beyond the inner and outer membranes, the fla-gellar type III export apparatus transports fourteen flagellar proteins with their copy numbers ranging from a few to tens of thousands to the distal growing end of the flagellar structure. The export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. Here, we show that the export engine of the flagellar type III export apparatus is robust in maintaining its export activity against internal and external perturbations arising from genetic variations and/or environmental changes. When the cytoplasmic ATPase complex is absent, the export gate complex is able to utilize
Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export
Molecular Microbiology, 2010
FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N-terminal transmembrane domain (FlhATM) and a C-terminal cytoplasmic domain (FlhAC). FlhAC is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhAC at 2.8 Å resolution. FlhAC consists of four subdomains (ACD1, ACD2, ACD3 and ACD4) and a linker connecting FlhAC to FlhATM. The sites of temperature-sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation sug-gested that FlhA C changes its conformation for its function. Molecular dynamics simulation demonstrated an open-close motion with a 5-10 ns oscillation in the distance between ACD2 and ACD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhAC is essential for protein export.
Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.
FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella
MicrobiologyOpen, 2016
For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of ...
Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus
Scientific reports, 2014
For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assemb...
FliI, a soluble ATPase component of the flagellar export apparatus; FliH and FliJ, soluble regulatory components of the export apparatus; FliC, flagellin, the major filament protein; FliS, chaperone for FliC; T3SS, type III secretion system; FlhA, FlhB, FliO, FliP, FliQ and FliR, membrane components of the flagellar export apparatus; FlgK and FlgL, hook-filament junction proteins; FlgN, chaperone for FlgK and FlgL; FliD, filament-cap protein; FliT, chaperone for FliD; FliN, a C-ring protein; Footnote: Complexes with defined stoichiometry (e. g. FliC:FliS) are indicated with a colon (:), while complexes with varying or unknown stoichiometry (e.g. FliI-FliJ-FliH) are indicated with hyphen (-).
Energy source of flagellar typeIII secretion
Nature, 2008
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.