Noritaka Hara | Osaka University (original) (raw)
Papers by Noritaka Hara
Seibutsu Butsuri
Flavoenzymes RebC and StaC are responsible for selective production of arcyriaflavin A and K252c ... more Flavoenzymes RebC and StaC are responsible for selective production of arcyriaflavin A and K252c in indolocarbazole biosynthesis, respectively. In this study, we have determined X-ray crystallographic structure of a mutant RebC F216V/R239N that has StaC-type activity at 2.4-Å resolution with reduced flavin. Compared to the putative substrate-bound WT RebC, the structural changes are confined to the active site. The side chain of Arg230, which are in between the two mutation sites, moves farther away from substrate binding site. As a result, the distance between substrate and FAD is longer than that in WT, which may not appropriate for flavin dependent monooxygenation in the mutant.
Seibutsu Butsuri
SflA from p]asmid repTessed motitity and biosynthesis of tlagellin. In addition, the A flhFG Asfi... more SflA from p]asmid repTessed motitity and biosynthesis of tlagellin. In addition, the A flhFG AsfiA triple deletion mutant represented the same phenotypeastheAflhFG-supstrain.Thus,weconc]udedthatmutationinsflAis responsible for suppressor phenotype of the AtlhFG-sup strain. We propose that sflA was a novel gene involved in the flagellation of V. algino]yticus.
Seibutsu Butsuri
BiosLtEncev Oyaka Llmve}sin i3 VlamadctoAa Suita Osaka j65 0S7f mpan ? LaboratoJre dL Chzmt(Baete... more BiosLtEncev Oyaka Llmve}sin i3 VlamadctoAa Suita Osaka j65 0S7f mpan ? LaboratoJre dL Chzmt(Baete"eitne On"ersik de ia MbditerranLe Aix Marseilte ll (VRS t PR9043 ldinrseilie CedeT 20 Fiance) ReLelltl) a ne" magnLLotac"c bactenurn caHed magneto oivoid gtrain
Seibutsu Butsuri
KeiLhi Naka]o (1} Maximilnn Ulbnch (2) Yoshihire Kube (1) Isacett E]]ucl (2) Ul) Nartenal Jnsttiu... more KeiLhi Naka]o (1} Maximilnn Ulbnch (2) Yoshihire Kube (1) Isacett E]]ucl (2) Ul) Nartenal Jnsttiute forPh)stotogt[aJScttncLg r2) Univ[rstA ofCaldernta Berkelex) see 2TP2 04 2P 180 fi.ptblkbltK+f"fiFJVOe-7(Ytieedieeut Gatmg mechansm ana]ysig in Noltage gensitive potassium channels Yuko Takeuchi (1) Mmako Hinne (]) Tik iaki Aeki (1) rushie Yinagida (1) loru lde {D UI)Giad"ateSchoeiofl'ronncrBiegetenceOsakaUntLeJstij) ConformaLional changes in the veltage se"soi domain ot K' channelg are slt11 m debate ln the paddlc modeT thL third and fouith transmernbrane gegment
MicrobiologyOpen, Jun 25, 2016
For construction of the bacterial flagellum, flagellar proteins are exported via its specific exp... more 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 ...
The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to t... more 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
Chemistry - A European Journal, 2010
Self-assembled monolayers of a series of tetraalkoxy-substituted octadehydrodibenzo[12]annulene (... more Self-assembled monolayers of a series of tetraalkoxy-substituted octadehydrodibenzo[12]annulene (DBA) derivatives 1c-g possessing butadiyne linkages were studied at the 1,2,4-trichlorobenzene (TCB) or 1-phenyloctane/graphite interface by scanning tunneling microscopy (STM). The purpose of this research is not only to investigate the structural variation of two-dimensional (2D) monolayers, but also to assess a possibility for peri-benzopolyacene formation by two-dimensionally controlled polymerization on a surface. As a result, the formation of three structures, porous, linear, and lamella structures, were observed by changing the alkyl chain length and the solute concentration. The formation of multilayers of the lamella structure was often observed for all compounds. The selection of molecular networks is basically ascribed to intermolecular and molecule-substrate interactions per unit area and network density. The selective appearance of the linear structure of 1d is attributed to favorable epitaxial registry matching between the substrate lattice and the overlayer lattice. Even though the closest interatomic distance between the diacetylenic units of the DBAs in the lamella structure (approximately 0.6 nm) is slightly larger compared to the typical distances necessary for topochemical polymerization, the reactivity toward external stimuli (electronic-pulse irradiation from an STM tip and UV irradiation) was investigated. Unfortunately, no evidence for polymerization of the DBAs on the surface was observed. The present results indicate the necessity for further designing a suitable system for the on-surface construction of structurally novel conjugated polymers, which are otherwise difficult to prepare.
PloS one, 2011
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal... more 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 ...
Nature Communications, 2011
Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a c... more 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.
Journal of Bacteriology, 2012
The flagellar type III protein export apparatus plays an essential role in the formation of the b... more 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.
Scientific reports, 2014
For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cyto... more 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...
Molecular microbiology, 2012
FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteol... more FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a ΔfliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A ΔfliH-fliI flhB(P28T) ΔflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues stil...
Molecular Microbiology, 2013
Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK ... more Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C-terminal cytoplasmic domain of FlhA (FlhAC ), a membrane component of the export apparatus, provides a binding-site for these chaperone-substrate complexes but it remains unknown how it co-ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhAC is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhAC for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14-fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhAC for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.
Seibutsu Butsuri
Flavoenzymes RebC and StaC are responsible for selective production of arcyriaflavin A and K252c ... more Flavoenzymes RebC and StaC are responsible for selective production of arcyriaflavin A and K252c in indolocarbazole biosynthesis, respectively. In this study, we have determined X-ray crystallographic structure of a mutant RebC F216V/R239N that has StaC-type activity at 2.4-Å resolution with reduced flavin. Compared to the putative substrate-bound WT RebC, the structural changes are confined to the active site. The side chain of Arg230, which are in between the two mutation sites, moves farther away from substrate binding site. As a result, the distance between substrate and FAD is longer than that in WT, which may not appropriate for flavin dependent monooxygenation in the mutant.
Seibutsu Butsuri
SflA from p]asmid repTessed motitity and biosynthesis of tlagellin. In addition, the A flhFG Asfi... more SflA from p]asmid repTessed motitity and biosynthesis of tlagellin. In addition, the A flhFG AsfiA triple deletion mutant represented the same phenotypeastheAflhFG-supstrain.Thus,weconc]udedthatmutationinsflAis responsible for suppressor phenotype of the AtlhFG-sup strain. We propose that sflA was a novel gene involved in the flagellation of V. algino]yticus.
Seibutsu Butsuri
BiosLtEncev Oyaka Llmve}sin i3 VlamadctoAa Suita Osaka j65 0S7f mpan ? LaboratoJre dL Chzmt(Baete... more BiosLtEncev Oyaka Llmve}sin i3 VlamadctoAa Suita Osaka j65 0S7f mpan ? LaboratoJre dL Chzmt(Baete"eitne On"ersik de ia MbditerranLe Aix Marseilte ll (VRS t PR9043 ldinrseilie CedeT 20 Fiance) ReLelltl) a ne" magnLLotac"c bactenurn caHed magneto oivoid gtrain
Seibutsu Butsuri
KeiLhi Naka]o (1} Maximilnn Ulbnch (2) Yoshihire Kube (1) Isacett E]]ucl (2) Ul) Nartenal Jnsttiu... more KeiLhi Naka]o (1} Maximilnn Ulbnch (2) Yoshihire Kube (1) Isacett E]]ucl (2) Ul) Nartenal Jnsttiute forPh)stotogt[aJScttncLg r2) Univ[rstA ofCaldernta Berkelex) see 2TP2 04 2P 180 fi.ptblkbltK+f"fiFJVOe-7(Ytieedieeut Gatmg mechansm ana]ysig in Noltage gensitive potassium channels Yuko Takeuchi (1) Mmako Hinne (]) Tik iaki Aeki (1) rushie Yinagida (1) loru lde {D UI)Giad"ateSchoeiofl'ronncrBiegetenceOsakaUntLeJstij) ConformaLional changes in the veltage se"soi domain ot K' channelg are slt11 m debate ln the paddlc modeT thL third and fouith transmernbrane gegment
MicrobiologyOpen, Jun 25, 2016
For construction of the bacterial flagellum, flagellar proteins are exported via its specific exp... more 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 ...
The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to t... more 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
Chemistry - A European Journal, 2010
Self-assembled monolayers of a series of tetraalkoxy-substituted octadehydrodibenzo[12]annulene (... more Self-assembled monolayers of a series of tetraalkoxy-substituted octadehydrodibenzo[12]annulene (DBA) derivatives 1c-g possessing butadiyne linkages were studied at the 1,2,4-trichlorobenzene (TCB) or 1-phenyloctane/graphite interface by scanning tunneling microscopy (STM). The purpose of this research is not only to investigate the structural variation of two-dimensional (2D) monolayers, but also to assess a possibility for peri-benzopolyacene formation by two-dimensionally controlled polymerization on a surface. As a result, the formation of three structures, porous, linear, and lamella structures, were observed by changing the alkyl chain length and the solute concentration. The formation of multilayers of the lamella structure was often observed for all compounds. The selection of molecular networks is basically ascribed to intermolecular and molecule-substrate interactions per unit area and network density. The selective appearance of the linear structure of 1d is attributed to favorable epitaxial registry matching between the substrate lattice and the overlayer lattice. Even though the closest interatomic distance between the diacetylenic units of the DBAs in the lamella structure (approximately 0.6 nm) is slightly larger compared to the typical distances necessary for topochemical polymerization, the reactivity toward external stimuli (electronic-pulse irradiation from an STM tip and UV irradiation) was investigated. Unfortunately, no evidence for polymerization of the DBAs on the surface was observed. The present results indicate the necessity for further designing a suitable system for the on-surface construction of structurally novel conjugated polymers, which are otherwise difficult to prepare.
PloS one, 2011
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal... more 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 ...
Nature Communications, 2011
Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a c... more 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.
Journal of Bacteriology, 2012
The flagellar type III protein export apparatus plays an essential role in the formation of the b... more 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.
Scientific reports, 2014
For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cyto... more 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...
Molecular microbiology, 2012
FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteol... more FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a ΔfliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A ΔfliH-fliI flhB(P28T) ΔflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues stil...
Molecular Microbiology, 2013
Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK ... more Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C-terminal cytoplasmic domain of FlhA (FlhAC ), a membrane component of the export apparatus, provides a binding-site for these chaperone-substrate complexes but it remains unknown how it co-ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhAC is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhAC for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14-fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhAC for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.