title: Functional defect and restoration of temperature-sensitive mutants of FlhA, a subunit of the flagellar protein export apparatus.
abstract: 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(C)D1-A(C)D4). 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(C)D2 and A(C)D1, 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(C)D2. We suggest that A(C)D2 is directly involved in the translocation of export substrates.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Shimada
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saijo-Hamano
authors.forename: Yukio
authors.initials: Y
authors.lastname: Furukawa
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
authors.forename: Katsumi
authors.initials: K
authors.lastname: Imada
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism*; D006358:Hot Temperature; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D021381:Protein Transport; D012475:Salmonella / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; D008565:Membrane Proteins; D018832:Molecular Chaperones