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Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

Hara N, Namba K, Minamino T - PLoS ONE (2011)

Bottom Line: The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation.Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR.Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

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Topology of FlhA.Cartoon showing the domain organization of Salmonella FlhA and the location of the highly conserved charged residues that have been genetically analyzed in this work. FlhA consists of an N-terminal transmembrane domain (FlhATM, TM1–TM8) and a C-terminal cytoplasmic domain (FlhAC) whose atomic structure was solved by X-ray crystallography [41]. FlhAC is involved in the early process of flagellar protein export along with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB. Closed circles in red and blue indicate invariant acidic and basic residues, respectively, which were identified by multiple sequence alignment of FlhA homologs (Figure S1).
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pone-0022417-g001: Topology of FlhA.Cartoon showing the domain organization of Salmonella FlhA and the location of the highly conserved charged residues that have been genetically analyzed in this work. FlhA consists of an N-terminal transmembrane domain (FlhATM, TM1–TM8) and a C-terminal cytoplasmic domain (FlhAC) whose atomic structure was solved by X-ray crystallography [41]. FlhAC is involved in the early process of flagellar protein export along with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB. Closed circles in red and blue indicate invariant acidic and basic residues, respectively, which were identified by multiple sequence alignment of FlhA homologs (Figure S1).

Mentions: FlhA is composed of an N-terminal integral membrane domain with eight predicted transmembrane (TM) helices (FlhATM, residues 1–327, 34.5 kDa) and a C-terminal cytoplasmic domain (FlhAC, residues 328–692, 40.5 kDa) (Figure 1) [26]. A well-conserved hydrophilic cytoplasmic loop between TM-4 and TM-5 is indispensable for FlhA function, but little is known about its role in flagellar protein export [27]. FlhAC interacts with FliH, FliI, FliJ, the C-terminal cytoplasmic domain of FlhB (FlhBC), and the FliS-FliC and FliT-FliD chaperone-substrate complexes and initiates the translocation of the substrates [10], [23], [28], [29]. FlhAC consists of four subdomains (D1, D2, D3, and D4) and a linker connecting FlhAC to FlhATM (Figure 1) [29]–[31]. The linker is involved in an interaction with FliJ [29]. The D2 subdomain is responsible for an interaction with the FliT-FliD and FliS-FliC complexes [29]. The D4 subdomain is dispensable for its function but is involved in the substrate specificity switching of the export apparatus [32]. Although FlhATM is required for the association of FlhA with the MS ring [12], it remains unknown whether it is directly involved in the PMF-driven protein export process.


Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

Hara N, Namba K, Minamino T - PLoS ONE (2011)

Topology of FlhA.Cartoon showing the domain organization of Salmonella FlhA and the location of the highly conserved charged residues that have been genetically analyzed in this work. FlhA consists of an N-terminal transmembrane domain (FlhATM, TM1–TM8) and a C-terminal cytoplasmic domain (FlhAC) whose atomic structure was solved by X-ray crystallography [41]. FlhAC is involved in the early process of flagellar protein export along with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB. Closed circles in red and blue indicate invariant acidic and basic residues, respectively, which were identified by multiple sequence alignment of FlhA homologs (Figure S1).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139655&req=5

pone-0022417-g001: Topology of FlhA.Cartoon showing the domain organization of Salmonella FlhA and the location of the highly conserved charged residues that have been genetically analyzed in this work. FlhA consists of an N-terminal transmembrane domain (FlhATM, TM1–TM8) and a C-terminal cytoplasmic domain (FlhAC) whose atomic structure was solved by X-ray crystallography [41]. FlhAC is involved in the early process of flagellar protein export along with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB. Closed circles in red and blue indicate invariant acidic and basic residues, respectively, which were identified by multiple sequence alignment of FlhA homologs (Figure S1).
Mentions: FlhA is composed of an N-terminal integral membrane domain with eight predicted transmembrane (TM) helices (FlhATM, residues 1–327, 34.5 kDa) and a C-terminal cytoplasmic domain (FlhAC, residues 328–692, 40.5 kDa) (Figure 1) [26]. A well-conserved hydrophilic cytoplasmic loop between TM-4 and TM-5 is indispensable for FlhA function, but little is known about its role in flagellar protein export [27]. FlhAC interacts with FliH, FliI, FliJ, the C-terminal cytoplasmic domain of FlhB (FlhBC), and the FliS-FliC and FliT-FliD chaperone-substrate complexes and initiates the translocation of the substrates [10], [23], [28], [29]. FlhAC consists of four subdomains (D1, D2, D3, and D4) and a linker connecting FlhAC to FlhATM (Figure 1) [29]–[31]. The linker is involved in an interaction with FliJ [29]. The D2 subdomain is responsible for an interaction with the FliT-FliD and FliS-FliC complexes [29]. The D4 subdomain is dispensable for its function but is involved in the substrate specificity switching of the export apparatus [32]. Although FlhATM is required for the association of FlhA with the MS ring [12], it remains unknown whether it is directly involved in the PMF-driven protein export process.

Bottom Line: The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation.Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR.Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

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Related in: MedlinePlus