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

Abstract

Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells. The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus. We have identified a stalled intermediate export complex in the flagellar type III export pathway of Salmonella typhimurium by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium. These chaperone variants bound their specific export substrates strongly and severely reduced their export. They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway. Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus. Membrane localization persisted in fliOPQR, flhB, flhA, fliJ, and fliH null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI. After expression of the variant chaperones in Salmonella, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated. Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate. Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

“…This finding is reminiscent of the function of the F-T3SS chaperone. During F-T3SS protein export, the chaperone-substrate complexes dock at the membrane ATPase (Gauthier & Finlay, 2003;Thomas et al, 2004), which facilitates the release of the chaperone from the cognate secreted substrate in an ATP-dependent manner (Akeda & Galán, 2005). After release by an escort mechanism in F-T3SS protein export, free chaperones can be cycled (Evans et al, 2006).…”

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

“…2 Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. FliH and FliI interact not only with FlhA and FlhB but also with flagellar chaperones such as FliJ and export substrates, leading to the proposal that the FliH-FliI complex is responsible for delivery of the substrates to the export gate 4,11,12,14 . To test this, gain-offunction mutants were isolated from the fliH fliI double null mutant by streaking an overnight culture out on soft agar plates, incubating at 30 uC for 2 days and looking for motility haloes emerging from the streak.…”

“…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 . FliI binds to FlgN (chaperone) and to a FlgN-FlgK (HAP1) complex 12 , suggesting that FliI has a critical role in substrate recognition as well. As Salmonella InvC-a virulence T3SS homologue of FliI-binds to chaperone-effector complexes and induces chaperone release from, and unfolding of, the effector to be secreted in an ATPase-dependent manner 13 , FliI has been thought to act in a similar manner.…”

Superchannel of Bacteria: Biological Significance and New Horizons

“…This evidence indicates that p5 and p6 are essential to the formation of the pit and the maintenance of cell surface structure and that they participate in the determination of cell destiny. 12) Flagellins are exported from the cytoplasm to the cell surface through the type-III exporter encoded by the flagellum cluster, 16) supporting the notion that the type-III secretion system evolved from the flagellar export apparatus, but the expression of p5 is suppressed in mutant cells with a disrupted peptidoglycan hydrolase gene, suggesting the presence of an alternative pathway to export flagellin to the cell surface and to localize it at the cell envelope. 12) If this is the case, flagellins destined for the outer membrane must cross the inner membrane and periplasm and be assembled in the outer membrane containing GSL, a more hydrophobic lipid molecule than LPS, in their appropriate conformation, possibly in the absence of a biochemical energy supply.…”