Abstract
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.
Highlights
The bacterial flagellum is a tubular organelle extending out from the cell surface, and is rotated by a nano-scale rotary motor embedded in the cytoplasmic membrane
We demonstrated that the flagellar protein export apparatus in the inverted membrane vesicles (IMVs) maintains the export function for rod/hook-type proteins at a level similar to that in a living cell, and that ATP hydrolysis by FliI ATPase dramatically accelerates the export of rod/hook-type proteins
We previously showed that FlgE, FlgD, and FliK, which belong to the rod/hook-type substrate class, are transported into the IMVs through the flagellar protein export apparatus to form the hook on the endogenous rod in the basal body inside the IMVs [29]
Summary
The bacterial flagellum is a tubular organelle extending out from the cell surface, and is rotated by a nano-scale rotary motor embedded in the cytoplasmic membrane. BiomoHleceurlees, 2w02e0,s1h0o, 1w26ed that the filament-type substrates (FlgK, FlgL, FliC, FliD) in complex with t2hoefi1r4 cognate chaperones are transported into the interior of the IMVs after completion of the hook. We showed that the filament-type substrates (FlgK, FlgL, FliC, FliD) in complex with their cognate chaperones are transported into the interior of the IMVs after completion of the hook These transported filament-type proteins assemble into the filament at the tip of the hook. Even when the hook-type proteins (FlgD, FlgE, FliK) and filament-type proteins in complex with substrate-specific flagellar chaperones were simultaneously added to the in vitro transport assay solutions, these proteins autonomously and sequentially assembled into the normal flagellar structure, indicating that the coupling of flagellar gene expression with assembly is not really required for well-ordered flagellar formation
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