scholarly journals Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

2021 ◽  
Vol 118 (22) ◽  
pp. e2026587118
Author(s):  
Tohru Minamino ◽  
Yusuke V. Morimoto ◽  
Miki Kinoshita ◽  
Keiichi Namba
Keyword(s):  
Ion Channel ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Protein Export ◽  
Membrane Voltage ◽  
Activation Mechanism ◽  
Dependent Manner ◽  
Concentration Difference ◽  
Flagellar Protein ◽  
External Ph

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

scholarly journals Membrane voltage-dependent activation of the flagellar protein export engine

2020 ◽  
Author(s):  
Tohru Minamino ◽  
Yusuke V. Morimoto ◽  
Miki Kinoshita ◽  
Keiichi Namba
Keyword(s):  
Electric Potential ◽  
Cytoplasmic Membrane ◽  
Protein Export ◽  
Ion Concentration ◽  
Activation Mechanism ◽  
Concentration Difference ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Flagellar Protein ◽  
External Ph

AbstractIon motive force (IMF) consists of the electric potential difference (ΔΨ) and the ion concentration difference (ΔpI) across the cytoplasmic membrane. The flagellar protein export machinery is an ion/protein antiporter utilizing IMF to drive ion-coupled protein export, but it remains unknown how. Here, we report a ΔΨ-dependent activation mechanism of the transmembrane export gate complex. Depletions of both H+ and Na+ gradients nearly diminished flagellar protein export in the absence of the cytoplasmic ATPase complex, but an increase in ΔΨ by an upward shift of external pH from 7.5 to 8.5 dramatically recovered it. An increase in the cytoplasmic level of export substrates and gain-of-function mutations in FlhA enhanced protein export at external pH 7.5 in the absence of Na+ in a similar manner to ΔΨ increase. We propose that the export gate complex has a voltage-gated mechanism to activate the ion/protein antiporter of the flagellar protein export engine.

scholarly journals In VitroReconstitution of Functional Type III Protein Export and Insights into Flagellar Assembly

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Akihiro Kawamoto ◽  
Chinatsu Tatsumi ◽  
Keiichi Namba ◽  
Tohru Minamino ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Secretion System ◽  
Protein Export ◽  
Type Iii ◽  
Transport Assay ◽  
Flagellar Protein

ABSTRACTThe type III secretion system (T3SS) forms the functional core of injectisomes, protein transporters that allow bacteria to deliver virulence factors into their hosts for infection, and flagella, which are critical for many pathogens to reach the site of infection. In spite of intensive genetic and biochemical studies, the T3SS protein export mechanism remains unclear due to the difficulty of accurate measurement of protein exportin vivo. Here, we developed anin vitroflagellar T3S protein transport assay system using an inverted cytoplasmic membrane vesicle (IMV) for accurate and controlled measurements of flagellar protein export. We show that the flagellar T3SS in the IMV fully retains export activity. The flagellar hook was constructed inside the lumen of the IMV by adding purified component proteins externally to the IMV solution. We reproduced the hook length control and export specificity switch in the IMV consistent with that seen in the native cell. Previousin vivoanalyses showed that flagellar protein export is driven by proton motive force (PMF) and facilitated by ATP hydrolysis by FliI, a T3SS-specific ATPase. Ourin vitroassay recapitulated these previousin vivoobservations but furthermore clearly demonstrated that even ATP hydrolysis by FliI alone can drive flagellar protein export. Moreover, this assay showed that addition of the FliH2/FliI complex to the assay solution at a concentration similar to that in the cell dramatically enhanced protein export, confirming that the FliH2/FliI complex in the cytoplasm is important for effective protein transport.IMPORTANCEThe type III secretion system (T3SS) is the functional core of the injectisome, a bacterial protein transporter used to deliver virulence proteins into host cells, and bacterial flagella, critical for many pathogens. The molecular mechanism of protein transport is still unclear due to difficulties in accurate measurements of protein transport under well-controlled conditionsin vivo. We succeeded in developing anin vitrotransport assay system of the flagellar T3SS using inverted membrane vesicles (IMVs). Flagellar hook formation was reproduced in the IMV, suggesting that the export apparatus in the IMV retains a protein transport activity similar to that in the cell. Using this system, we revealed that ATP hydrolysis by the T3SS ATPase can drive protein export without PMF.

scholarly journals FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella

2021 ◽  
Author(s):  
Tohru Minamino ◽  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Akio Kitao ◽  
Keiichi Namba
Keyword(s):  
Closed Form ◽  
Protein Transport ◽  
Building Blocks ◽  
Protein Export ◽  
Side Chain ◽  
Transport Activity ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Domain Motions

Abstract The flagellar type III secretion system (fT3SS) transports flagellar building blocks from the cytoplasm to the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for efficient flagellar assembly. FlhAC adopts open and closed conformations, and the chaperones bind to the open form, allowing the fT3SS to transport the substrates to the cell exterior. To clarify the role of the closed form in flagellar protein export, we isolated pseudorevertants from the flhA(G368C/K549C) mutant, in which the closed conformation is stabilized to inhibit the protein transport activity of the fT3SS. Each of M365I, R370S, A446E and P550S substitutions in FlhAC identified in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhAC structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhAC is required for rapid and efficient flagellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhAC.

scholarly journals The FlgN chaperone activates the Na+-driven engine of the flagellar protein export apparatus

2020 ◽  
Author(s):  
Tohru Minamino ◽  
Miki Kinoshita ◽  
Yusuke V. Morimoto ◽  
Keiichi Namba
Keyword(s):  
Protein Transport ◽  
Protein Export ◽  
Gain Of Function ◽  
Export Activity ◽  
Flagellar Proteins ◽  
Flagellar Protein

AbstractThe bacterial flagellar protein export machinery promotes H+-coupled translocation of flagellar proteins to the cell exterior. When the cytoplasmic ATPase complex does not function, the transmembrane export gate complex opens its Na+ channel and continues protein transport. However, it remains unknown how. Here we report that the FlgN chaperone acts as a switch to activate a backup export mechanism for the ATPase complex by activating the Na+-driven engine. Impaired interaction of FlhA with the FliJ subunit of the ATPase complex increased Na+-dependence of flagellar protein export. Deletion of FlgN inhibited protein export in the absence of the ATPase complex but not in its presence. Gain-of-function mutations in FlhA restored not only the FlgN defect but also the FliJ defect. We propose that the interaction of FlgN with FlhA opens the Na+ channel in the export engine, thereby maintaining the protein export activity in the absence of the active ATPase complex.

scholarly journals In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 126
Author(s):  
Hiroyuki Terashima ◽  
Chinatsu Tatsumi ◽  
Akihiro Kawamoto ◽  
Keiichi Namba ◽  
Tohru Minamino ◽  
...  
Keyword(s):  
Protein Transport ◽  
Early Stage ◽  
Membrane Vesicles ◽  
Protein Export ◽  
Axial Component ◽  
Axial Structure ◽  
Inverted Membrane Vesicles ◽  
Flagellar Protein

The bacterial flagellum is a filamentous organelle extending from the cell surface. The axial structure of the flagellum consists of the rod, hook, junction, filament, and cap. The axial structure is formed by axial component proteins exported via a specific protein export apparatus in a well-regulated manner. Although previous studies have revealed the outline of the flagellar construction process, the mechanism of axial structure formation, including axial protein export, is still obscure due to difficulties in direct observation of protein export and assembly in vivo. We recently developed an in vitro flagellar protein transport assay system using inverted membrane vesicles (IMVs) and succeeded in reproducing the early stage of flagellar assembly. However, the late stage of the flagellar formation process remained to be examined in the IMVs. In this study, we showed that the filament-type proteins are transported into the IMVs to produce the filament on the hook inside the IMVs. Furthermore, we provide direct evidence that coordinated flagellar protein export and assembly can occur at the post-translational level. These results indicate that the ordered construction of the entire flagellar structure can be regulated by only the interactions between the protein export apparatus, the export substrate proteins, and their cognate chaperones.

scholarly journals The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Tohru Minamino ◽  
Yusuke V. Morimoto ◽  
Miki Kinoshita ◽  
Phillip D. Aldridge ◽  
Keiichi Namba
Keyword(s):  
Atp Hydrolysis ◽  
Protein Export ◽  
Flagellar Proteins ◽  
Flagellar Protein

scholarly journals Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures

1999 ◽  
Vol 112 (5) ◽  
pp. 589-600 ◽  
Author(s):  
T. Farmaki ◽  
S. Ponnambalam ◽  
A.R. Prescott ◽  
H. Clausen ◽  
B.L. Tang ◽  
...  
Keyword(s):  
Protein Transport ◽  
Protein Export ◽  
Sensitive Assay ◽  
Plasma Membrane Protein ◽  
Animal Cells ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Intermediate Compartment ◽  
First Time ◽  
Mitotic Cells

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

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