title: Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus.
abstract: For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assembly-disassembly cycle of FliI during flagellar assembly. We propose that the FliH2FliI complex and FliI6 ring function as a dynamic substrate carrier and a static substrate loader, respectively.
authors.affiliation: 1] Biodynamic Optical Imaging Center, Peking University, Beijing, 100871, China [2] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [3].
authors.forename: Fan
authors.initials: F
authors.lastname: Bai
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan [3].
authors.forename: Yusuke V
authors.initials: YV
authors.lastname: Morimoto
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shinsuke D J
authors.initials: SD
authors.lastname: Yoshimura
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hara
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Nobunori
authors.initials: N
authors.lastname: Kami-Ike
authors.affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
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
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005407:Flagella / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012475:Salmonella / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; C069251:fliI protein, bacteria; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases