FIG. 1.
TolC assembly assay. (A) Cartoon showing the TolC assembly assay. The treatment of outer membrane (OM)-permeabilized cells with proteinase K (PK) results in the complete degradation of soluble TolC assembly intermediates, while assembled TolC molecules produce an OM-anchored 46-kDa fragment. The C-terminal 5-kDa fragment is either completely degraded or not detected by SDS-PAGE. (B) An autoradiograph of an SDS-polyacrylamide gel carrying immunoprecipitated samples from pulse-chase labeling experiments. Cells expressing wild-type TolC were labeled with [35S]methionine-[35S]cysteine as described in Materials and Methods. Half of the labeled samples was treated with proteinase K. TolC and MBP were immunoprecipitaed from protein extracts and analyzed by SDS-PAGE. Positions of TolC and MBP are shown. Note that TolC from proteinase K-treated samples runs faster (46 kDa) than that from untreated samples (51 kDa). Chase times are shown below the gel. (C) TolC levels, relative to MBP levels, were quantified and graphed. Total TolC (♦), proteinase K-resistant TolC (▪), and proteinase K-sensitive TolC (▴) results are shown.