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

Abstract

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.The bacterial flagellum, which is responsible for motility, is a supramolecular complex of about 30 different proteins, and it consists of at least three substructures: the basal body, the hook, and the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. Many of the flagellar component proteins are translocated into the central channel of the growing flagellar structure and then to the distal end of the structure for self-assembly by the flagellar type III protein export apparatus (11,16,22). This export apparatus consists of six integral membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and three soluble proteins, FliH, FliI, and FliJ (18,21). These protein components show significant sequence and functional similarities to those of the type III secretion systems of pathogenic bacteria, which directly inject virulence factors into their host cells (11,16).

Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

“…These observations suggest that FliI requires FliH to support the function of the FlhA mutants. and FlhB C provide binding sites for FliH, FliI, FliJ, flagellar chaperones, and export substrates (1,25,29,32,34,36,50). Because the reduced secretion activity of the fliH null mutant is significantly improved by the overexpression of FliI or extragenic suppressor mutations in FlhA or FlhB, FliH is thought to be required for the productive association of FliI with the FlhA-FlhB complex (25).…”

“…The export apparatus consists of a membrane-embedded export gate made of six membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and a water-soluble ATPase complex consisting of FliH, FliI, and FliJ (22,28). The C-terminal cytoplasmic domains of FlhA (FlhA C ) and FlhB (FlhB C ) provide binding sites for the ATPase complex, flagellar substrate-specific chaperone, and export substrates (1,25,29,32,34,36,50). FlhB C acts as the export specificity switch to mediate the ordered export of the axial flagellar components during flagellar assembly (19).…”

Interaction between FliJ and FlhA, Components of the Bacterial Flagellar Type III Export Apparatus

“…FlhA C consists of a compactly folded core domain (FlhA C 38K) formed by four subdomains-D1, D2, D3, and D4 -and a flexible linker region (FlhA L ), which connects FlhA C 38K to the N-terminal integral membrane domain (FlhA TM ) with eight predicted transmembrane helices (15,34,42). It has been shown that FliJ binds to FlhA L (16,27).…”

“…The C-terminal cytoplasmic domains of FlhA (FlhA C ) and FlhB (FlhB C ) provide the docking sites for FliH, FliI, FliJ, flagellar chaperones, and export substrates (13)(14)(15)(16)(17). FliI is a Walker-type ATPase (18) and forms a stable FliH 2 -FliI complex with FliH (19).…”

Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

“…FliJ also was proposed to recycle substrate-free flagellar chaperones at the export gate (24). FliI, FliH, and FliJ bind to FlhA and FlhB proteins at the export gate (11,25,26).…”