Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT.
The flagellar proteins FlgN and FliT have been proposed to act as substrate-specific export chaperones, facilitating incorporation of the enterobacterial hook-associated axial proteins (HAPs) FlgK/FlgL and FliD into the growing flagellum. In Salmonella typhimurium flgN and fliT mutants, the export of target HAPs was reduced, concomitant with loss of unincorporated flagellin into the surrounding medium. Gel filtration chromatography of wild-type S. typhimurium cell extracts identified stable pools of FlgN and FliT homodimers in the cytosol, but no chaperone-substrate complexes were evident. Nevertheless, stable unique complexes were assembled efficiently in vitro by co-incubation of FlgN and FliT with target HAPs purified from recombinant Escherichia coli. The sizes of the chaperone-substrate complexes indicated that, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP. Recombinant polypeptides spanning the potentially amphipathic C-terminal regions of FlgN or FliT could not complement in trans the chaperone deficiency of the respective flgN and fliT mutants, but efficient flagellar assembly was restored by homodimeric translational fusions of these domains to glutathione S-transferase, which bound FlgK and FlgL like the wild-type FlgN. These data provide further evidence for the substrate-specific chaperone function of FlgN and FliT and indicate that these chaperones comprise common N- and C-terminal domains mediating homodimerization and HAP substrate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N-terminal half of FliT and the C-terminal half of FlgN.
Bennett
J C
JC
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Thomas
J
J
Fraser
G M
GM
Hughes
C
C
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002863:Chromogenic Compounds; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D005407:Flagella / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D001426:Bacterial Proteins; D002863:Chromogenic Compounds; C087498:FlgN protein, Bacteria; D018832:Molecular Chaperones; C035832:hook protein, bacterial flagellum; C076735:fliT protein, Bacteria