title: Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus.
abstract: Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliHDelta1 (missing residues 2-10) but the complex did not show any localization. FliHDelta1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
authors.forename: Shinsuke D J
authors.initials: SD
authors.lastname: Yoshimura
authors.forename: Yusuke V
authors.initials: YV
authors.lastname: Morimoto
authors.forename: Bertha
authors.initials: B
authors.lastname: González-Pedrajo
authors.forename: Nobunori
authors.initials: N
authors.lastname: Kami-Ike
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005407:Flagella / Q000737:chemistry; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012486:Salmonella typhimurium / Q000502:physiology*; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; C077389:FliN protein, Bacteria; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C069250:fliH protein, Bacteria; C069251:fliI protein, bacteria; D006180:Proton-Translocating ATPases