8.A.153. The Regulator of the Flagellar Protein Export Apparatus, FliK (FliK) Family.

FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella. As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. Minamino et al. 2020 reported the role of FlhBC cleavage in the switching mechanism. FlhB(N269A) and FlhB(P270A) mutations do not affect the binding affinity of FlhBC for FliK, but the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The FlhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. Possibly the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC, responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export.

The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, and an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation (Minamino et al. 2020).

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 (Minamino et al. 2021). 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, Minamino et al. 2021 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 suggested 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. Minamino et al. 2021 proposed 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.