title: Interaction between FliJ and FlhA, components of the bacterial flagellar type III export apparatus.
abstract: A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH(X)-FliI(6) ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues-Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83-of FliJ. A structural model of the FliI(6)-FliJ ring complex suggests that they extend out of the FliI(6) ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ibuki
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Uchida
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hironaka
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Katsumi
authors.initials: K
authors.lastname: Imada
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D019779:Salmonella enterica / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; D008565:Membrane Proteins; C069253:fliJ protein, Bacteria