title: Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH.
abstract: The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.
authors.affiliation: Department of Pathology, Cambridge University, Tennis Court Road, CB2 1QP, UK.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Auvray
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Ozin
authors.forename: Laurent
authors.initials: L
authors.lastname: Claret
authors.forename: Colin
authors.initials: C
authors.lastname: Hughes
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000502:physiology; D003593:Cytoplasm / Q000502:physiology; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000378:metabolism; D005407:Flagella / Q000378:metabolism*; D017353:Gene Deletion; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000378:metabolism; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D012475:Salmonella / Q000166:cytology / Q000378:metabolism*; D000081416:SecA Proteins; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C489554:FliL protein, E coli; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C069250:fliH protein, Bacteria; C074426:FliL protein, Bacteria; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins