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.

Fig. 1.

Identification of dominant-negative export-defective chaperone variants. (A) Representation of the 140-residue FlgN chaperone. In-frame deletions Δ80-100 to Δ130-140 were made in the substrate-binding C-terminal domain, in which the predicted amphipathic helix is shaded (residues 74-114). Vertical lines indicate intervals of 10 residues. The N-terminal domain determines homodimerization. (B) Motility (M) and flagellar protein export of the S. typhimurium flgN null mutant (flgN) and the wild-type (wt) carrying vector pBAD18 (-) or derivatives expressing the (His)₆-tagged FlgN wild type or a deleted variant. Motility was assessed on 0.3% soft tryptone agar after point inoculation and incubation at 37°C for 6-8 h. Secreted proteins from late-exponential-phase cultures were separated by SDS (12%)/PAGE and immunoblotted with FlgK, FlgL, or FliD antisera or stained with Coomassie blue (FliC). In the flgN mutant, low-level expression was induced by 0.01% l-arabinose; in the wild type, high-level expression was induced by 0.1% l-arabinose. (C) Interaction of FlgN variants with export substrate. Lysates of E. coli BL21 (DE3)-coexpressing substrates FlgK or FlgL with (His)₆-tagged wild-type or variant FlgN were incubated with Ni-NTA resin, and proteins were purified by affinity chromatography. Coeluted proteins were separated by SDS (15%)/PAGE and stained with Coomassie blue. Negative control (-) carries pBAD18.

Fig. 2.

Membrane localization of dominant-negative chaperone variants. (A) Whole-cell lysates (wc) and membrane/insoluble (m) and cytoplasmic fractions of the S. typhimurium flgN mutant expressing either wild-type FlgN or variants NΔ120-140 or NΔ130-140 were separated by SDS (15%)/PAGE and immunoblotted with anti-FlgN antiserum. (B) Membrane fractions of S. typhimurium flgN expressing NΔ120-140 or NΔ130-140 were harvested (100,000 × g, 1 h) and centrifuged through a 0.8-2.0 M sucrose gradient (75,000 × g, 16 h) to separate inner and outer membranes. Precipitated proteins were separated by SDS (12%)/PAGE and stained with Coomassie blue (Top) or blotted with anti-FliM or anti-FlgN antisera. Positions of inner-membrane-associated NADH oxidase and outer membrane proteins (OMPs) are indicated. (C) Purified wild-type or variant FlgN was incubated with E. coli total phospholipid liposomes and placed at the bottom of a three-step sucrose density gradient. After centrifugation (75,000 × g, 16 h), fractions were collected, and proteins precipitated from the top (T), middle (M), and bottom (B) of the gradient were separated by SDS (15%)/PAGE and visualized with Coomassie blue.

Fig. 3.

FliI-dependent membrane localization of dominant-negative variants. Whole-cell lysates (wc) and membrane/insoluble (m) and cytoplasmic (c) fractions of S. typhimurium null mutants (indicated on the right) expressing (His)₆-tagged NΔ120-140 (Left) or NΔ130-140 (Right) were separated by SDS (15%)/PAGE, and FlgN proteins were probed with Ni-NTA-horseradish peroxidase conjugate (Qiagen).

Fig. 4.

Intermediate export complexes stalled by dominant-negative variants. Cultures of the S. typhimurium flgN null mutant expressing FlgN wild type or variants were lysed in a French pressure cell. After removal of cell debris, soluble fractions were incubated with Ni-NTA, and eluted proteins were separated by SDS (12%)/PAGE and immunoblotted with anti-FlgN, -FlgK, -FlgL, -FliI, -FliH, -FliD, or -FliC antisera. Negative control (-) carries the vector pBAD18.

Fig. 5.

Interaction of chaperone-substrate complexes with the export ATPase FliI. (A) Purified FliI was preincubated with either FlgK substrate, FlgN wild type, or NΔ130-140 variant and added to E. coli total phospholipids liposomes. Sucrose was added to 55% (wt/vol) and overlaid with 40% (wt/vol) sucrose, and the protein-lipid vesicle mix was allowed to float through the density gradient during centrifugation (75,000 × g, 16 h). Proteins from the top (T), middle (M), and bottom (B) fractions of the gradient were precipitated and analyzed by SDS (15%)/PAGE before immunoblotting with anti-FliI or FlgK antisera. (B) Complex formation was assayed as described above after FliI was incubated with a preformed complex of FlgN-FlgK (Left) or NΔ130-140-FlgK (Right).