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.

Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export

“…2B). Coomassie blue staining confirmed that outer membrane proteins located toward the bottom of the gradient, and immunoblotting revealed both the N⌬120-140 and N⌬130-140 in the inner-membrane fractions colocalizing with the NADH oxidase and flagellar C-ring component FliM (27,28).…”

“…E. coli total phospholipids (Avanti Polar Lipids) were prepared by sonication (27). Purified proteins (1-2 g) were mixed with 40 l of 10 mg⅐ml Ϫ1 lipid in Tris-buffered saline (20 mM Tris, pH 7.4͞150 mM NaCl͞TBS) (27) and incubated at room temperature for 15 min.…”

“…E. coli total phospholipids (Avanti Polar Lipids) were prepared by sonication (27). Purified proteins (1-2 g) were mixed with 40 l of 10 mg⅐ml Ϫ1 lipid in Tris-buffered saline (20 mM Tris, pH 7.4͞150 mM NaCl͞TBS) (27) and incubated at room temperature for 15 min. Sucrose was added to a final concentration of 50% (wt͞vol) in 1 ml of TBS (27), and samples were overlaid with 3.5 ml of 40% sucrose-TBS (wt͞vol) and 0.5 ml of TBS.…”

“…Purified proteins (1-2 g) were mixed with 40 l of 10 mg⅐ml Ϫ1 lipid in Tris-buffered saline (20 mM Tris, pH 7.4͞150 mM NaCl͞TBS) (27) and incubated at room temperature for 15 min. Sucrose was added to a final concentration of 50% (wt͞vol) in 1 ml of TBS (27), and samples were overlaid with 3.5 ml of 40% sucrose-TBS (wt͞vol) and 0.5 ml of TBS. After centrifugation at 75,000 ϫ g for 16 h at 16°C, 10 0.5-ml fractions were collected and precipitated with 10% (wt͞vol) TCA.…”

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

“…FliI shows extensive similarity to the a/b subunits of proton-driven F 0 F 1 -ATPase for its entire molecular structure 6 , although sequence similarity is limited to their respective ATPase domains 7,8 . Unlike F 1 -ATPase, however, which forms the a 3 b 3 hexameric ring, FliI self-assembles into a homohexamer 9,10 . When FliI oligomerization is suppressed by a small deletion in its amino-terminal region, flagellar protein export does not occur efficiently, suggesting that FliI hexamerizes on docking to the export gate made of the six integral membrane components, for which the cytoplasmic domains of FlhA and FlhB are thought to form the docking platform 11 .…”