Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB.
FlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella. It permits export of rod/hook-type proteins before hook completion, whereupon it switches specificity to recognize filament-type proteins. The cytoplasmic C-terminal domain of FlhB (FlhBC) is cleaved between Asn-269 and Pro-270, defining two subdomains: FlhBCN and FlhBCC. Here, we show that subdomain interactions and cleavage within FlhB are central to substrate-specificity switching. We found that deletions between residues 216 and 240 of FlhBCN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn-269 and Pro-270 abolished both. The mutation N269A prevented cleavage at the FlhBCN-FlhBCC boundary. Cells producing FlhB(N269A) exported the same amounts of hook-capping protein as cells producing wild-type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament-type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook-type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.
Fraser
Gillian M
GM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Hirano
Takanori
T
Ferris
Hedda U
HU
Devgan
Lara L
LL
Kihara
May
M
Macnab
Robert M
RM
D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins; D005407:Flagella / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012475:Salmonella / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C085668:FlhB protein, Bacteria; D008565:Membrane Proteins; D015534:Trans-Activators; C090552:flhC protein, E coli