Protein Export Apparatus Research Articles

2I1436 べん毛輸送装置近傍の局所pHに対するFlil ATPaseの効果(細胞生物的課題II:Prokaryotes,口頭発表,日本生物物理学会第50回年会(2012年度))

2I1436 べん毛輸送装置近傍の局所pHに対するFlil ATPaseの効果(細胞生物的課題II:Prokaryotes,口頭発表,日本生物物理学会第50回年会(2012年度))

Functional Defect and Restoration of Temperature-Sensitive Mutants of FlhA, a Subunit of the Flagellar Protein Export Apparatus

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhAC) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone–substrate complexes. FlhAC is composed of a flexible linker region and four compact domains (ACD1–ACD4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhAC blocks the export process after the FliH–FliI–FliJ–substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhAC(G368C), and its pseudorevertant variants FlhAC(G368C/L359F), FlhAC(G368C/G364R), FlhAC(G368C/R370S), and FlhAC(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhAC occurs in a single step, FlhAC(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhAC(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of ACD2 and ACD1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhAC(G368C) and that the loss of function is caused by denaturation of ACD2. We suggest that ACD2 is directly involved in the translocation of export substrates.

An energy transduction mechanism used in bacterial flagellar type III protein export

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton–protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH–FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus.

Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA

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.

Assembly and stability of flagellar motor in Escherichia coli

Bacterial flagellar motor is a highly ordered and complex supramolecular structure that powers rotation of flagella and serves as a type III export apparatus for flagellar assembly. Motor biogenesis represents a formidable example of self-assembly, but little is known about early steps of the motor structure formation. Here we used a combination of fluorescence microscopy techniques to dissect the order of the motor assembly in Escherichia coli cells, to map in vivo the underlying protein interactions and to investigate dynamics of protein exchange in the assembled motor structure. Our data suggest that motor self-assembly is initiated by oligomerization of the membrane export apparatus protein FlhA, which is followed by the recruitment of the MS ring component FliF and by the ordered association of other motor proteins. The assembly process combines the hierarchy with cooperativity, whereby the association of each subsequent motor structure stabilizes the growing assembly. Our results provide a novel and so far the most complete view of the early steps in flagellar motor assembly and improve understanding of the motor structure and regulation.

Common architecture of the flagellar type III protein export apparatus and F- and V-type ATPases

The proteins that form the bacterial flagellum are translocated to its distal end through the central channel of the growing flagellum by the flagellar-specific protein export apparatus, a family of the type III protein secretion system. FliI and FliJ are soluble components of this apparatus. FliI is an ATPase that has extensive structural similarity to the α and β subunits of F(o)F(1)-ATP synthase. FliJ is essential for export, but its function remains obscure. Here we show that the structure of FliJ derived from Salmonella enterica serovar Typhimurium is remarkably similar to that of the two-stranded α-helical coiled-coil part of the γ subunit of F(o)F(1)-ATP synthase and that FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring. These results suggest that the type III protein export system and F- and V-type ATPases share a similar mechanism and an evolutionary relationship.

1K1324 細菌べん毛タンパク質輸送装置近傍の局所pHの測定(細胞生物的課題1,第49回日本生物物理学会年会)

1K1324 細菌べん毛タンパク質輸送装置近傍の局所pHの測定(細胞生物的課題1,第49回日本生物物理学会年会)

Interactions among FliN, FliH and FliI of the Salmonella enterica flagellar export apparatus govern secretion

Using two optical biosensing methods, surface plasmon resonance and biolayer interferometry, a study of the interactions among proteins involved in flagellar type III secretion was undertaken to understand the mechanism of substrate delivery to the membrane‐embedded export complex. An interaction between C‐ring protein FliN and export apparatus protein FliH has been proposed to be a mode of targeting substrates via a complex that is necessarily dynamic as the position of FliN at the base of the C‐ring is too far from the membrane to allow the complex to remain associated during export. Both techniques allow quantitative measurement of association and dissociation. Rate and affinity constants are reported for pairwise interactions; both FliN‐FliH and FliH‐FliI have submicromolar KDs. Experiments investigating the conditions necessary to dissociate FliN from FliH or FliH from FliI are described. Substrates were screened for binding to FliH and FliI in the presence and absence of FliN. Kinetic analysis will allow us to rank affinities, determine how binding changes in the presence of other participants, search for export signals within substrates etc., developing a better understanding of type III secretion. This work was supported by grants from the Public Health Service (R15GM080701), NSF (CHE 0922699), the Research Corporation (CC6900) and a KSU Mentor‐Protégé Grant (to J.L.W.).

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export

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 (FlhA(TM)) and a C-terminal cytoplasmic domain (FlhA(C)). FlhA(C) 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 FlhA(C) at 2.8 A resolution. FlhA(C) consists of four subdomains (A(C)D1, A(C)D2, A(C)D3 and A(C)D4) and a linker connecting FlhA(C) to FlhA(TM). 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 suggested 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 A(C)D2 and A(C)D4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhA(C) is essential for protein export.

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.

2P169 部位特異的架橋法によるべん毛III型輸送装置へのFlIHのターゲティング機構の解析(分子モーター,第48回日本生物物理学会年会)

2P169 部位特異的架橋法によるべん毛III型輸送装置へのFlIHのターゲティング機構の解析(分子モーター,第48回日本生物物理学会年会)

2P036 べん毛III型蛋白質輸送装置におけるATP加水分解反応の制御機構(蛋白質-構造機能相関,第48回日本生物物理学会年会)

2P036 べん毛III型蛋白質輸送装置におけるATP加水分解反応の制御機構(蛋白質-構造機能相関,第48回日本生物物理学会年会)

Roles of the extreme N‐terminal region of FliH for efficient localization of the FliH–FliI complex to the bacterial flagellar type III export apparatus

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliHDelta1 (missing residues 2-10) but the complex did not show any localization. FliHDelta1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate.

Interaction of FliK with the bacterial flagellar hook is required for efficient export specificity switching

FliK-FlhB interaction switches export specificity of the bacterial flagellar protein export apparatus to stop hook protein export at an appropriate timing for hook length control. The hook structure is required for the productive FliK-FlhB interaction to flip the switch but it remains unknown how it works. Here, we characterize the role of FliK in the switching probability in the absence of the hook. When RflH/Flk was missing in the hook mutants, the switching occurred at a low probability. Overproduction of FliK significantly increased the switching probability although not at the wild-type level. An in-frame deletion of residues 129 through 159 of FliK weakened the interaction with the hook protein but not with the hook-capping protein, producing polyhooks with filaments attached. We suggest that temporary association of FliK with the inner surface of the hook during FliK secretion results in a pause in the secretion process to allow the C-terminal switch domain of FliK to be positioned and appropriately oriented near FlhB for catalysing the switch and that RflH/Flk interferes with premature switch by preventing access of cytoplasmic FliK to FlhB and even that of FliK during its secretion until hook length reaches 55 nm; only then FliK(C) passes the RflH/Flk block.

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors sigma(54) (RpoN), employed for expression of genes required midway through flagellar assembly, and sigma(28) (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhB(C)). The hook length control protein FliK interacts with FlhB(C) to modulate the substrate specificity of the export apparatus. FlhB(C) undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the DeltaflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the DeltaflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhB(N265A), expression of RpoN- and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhB(P266G). H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhB(C) (FlhB(CC)) and can substitute for FlhB(CC) in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the DeltafliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhB(CC) in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.

3P-070 べん毛タイプIII蛋白質輸送装置FliH/FliI複合体の構造解析に向けた試料調製(蛋白質-計測・解析の方法論,第47回日本生物物理学会年会)

3P-070 べん毛タイプIII蛋白質輸送装置FliH/FliI複合体の構造解析に向けた試料調製(蛋白質-計測・解析の方法論,第47回日本生物物理学会年会)

2P-142 細菌べん毛蛋白質輸送装置構成蛋白質FlhBのC末細胞質ドメインの機能解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

2P-142 細菌べん毛蛋白質輸送装置構成蛋白質FlhBのC末細胞質ドメインの機能解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

Crystallization and preliminary X-ray analysis of FliJ, a cytoplasmic component of the flagellar type III protein-export apparatus from Salmonella sp.

The axial component proteins of the bacterial flagellum are synthesized in the cytoplasm and then translocated into the central channel of the flagellum by the flagellar type III protein-export apparatus for self-assembly at the distal growing end of the flagellum. FliJ is an essential cytoplasmic component of the export apparatus. In this study, Salmonella FliJ with an extra three residues (glycine, serine and histidine) attached to the N-terminus as the remainder of a His tag (GSH-FliJ) was purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 300 as a precipitant. GSH-FliJ crystals grew in the hexagonal space group P6(1)22 or P6(5)22. While the native crystals diffracted to 3.3 A resolution, the diffraction resolution limit of mercury derivatives was extended to 2.1 A. Anomalous and isomorphous difference Patterson maps of the mercury-derivative crystal showed significant peaks in their Harker sections, indicating the usefulness of the derivative data for structure determination.

X-ray analysis of FliJ, a cytoplasmic component of the flagellar type III protein export apparatus

X-ray analysis of FliJ, a cytoplasmic component of the flagellar type III protein export apparatus

2P-041 サルモネラ菌べん毛タイプIII輸送装置構成蛋白質FliJの構造(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)

2P-041 サルモネラ菌べん毛タイプIII輸送装置構成蛋白質FliJの構造(蛋白質・構造機能相関(2),第46回日本生物物理学会年会)