FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase activity.
Both FliH and the ATPase FliI are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N-terminus of FliI interacts with FliH and that this interaction is important for the ATPase function of the C-terminal domain of FliI. N-terminally histidine-tagged, wild-type FliI retarded untagged FliH in a Ni-NTA affinity chromatography assay, as did N-His-tagged versions of FliI carrying catalytic mutations. In contrast, N-His-tagged FliI carrying the double mutation R7C/L12P did not, further indicating that the N-terminus of FliI is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and FliI form a complex. Analytical gel filtration with in-line multiangle light scattering indicated that FliH alone forms a dimer, FliI alone remains as a monomer, and FliH and FliI together form a (FliH)2FliI complex. Ni-NTA affinity chromatography using N-His-tagged FliH and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH-FliI complex was about 10-fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.
Minamino
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 8114, USA.
MacNab
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002846:Chromatography, Affinity / Q000379:methods; D005407:Flagella / Q000378:metabolism*; D005799:Genes, Dominant; D008027:Light; D008565:Membrane Proteins; D009154:Mutation; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D012475:Salmonella / Q000166:cytology / Q000378:metabolism*; D012542:Scattering, Radiation
D001426:Bacterial Proteins; D008565:Membrane Proteins; C069250:fliH protein, Bacteria; C074426:FliL protein, Bacteria; D000255:Adenosine Triphosphate; D009571:Nitrilotriacetic Acid