Functional defect and restoration of temperature-sensitive mutants of FlhA, a subunit of the flagellar protein export apparatus

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhA_C) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone–substrate complexes. FlhA_C is composed of a flexible linker region and four compact domains (A_CD1–A_CD4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhA_C blocks the export process after the FliH–FliI–FliJ–substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhA_C(G368C), and its pseudorevertant variants FlhA_C(G368C/L359F), FlhA_C(G368C/G364R), FlhA_C(G368C/R370S), and FlhA_C(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhA_C occurs in a single step, FlhA_C(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhA_C(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of A_CD2 and A_CD1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhA_C(G368C) and that the loss of function is caused by denaturation of A_CD2. We suggest that A_CD2 is directly involved in the translocation of export substrates.