Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen
Fig 1
Typhoid toxin traffics to the ER by retrograde transport.
(A) Tracking typhoid toxin (TT) transport by immunofluorescence microscopy. HEK293T cells were incubated with Oregon Green-488-labeled typhoid toxin (green) at 4°C for 30 min, washed, and fixed with 4% paraformaldehyde (binding). Alternatively, after 30 min incubation at 4°C, cells were washed, and then switched to 37°C, incubated for 0.5, 2, and 8 hs and fixed as indicated above. Fixed cells were stained with an anti-GM130 antibody (red) and visualized by fluorescence microscopy. Scale bar, 5 μm. (B and C) Typhoid toxin undergoes retrograde transport to the trans-Golgi network (TGN). (B) Schematic representation of the assay to detect typhoid toxin transport through the Golgi. (C) HEK293T cells transiently expressing myc epitope tagged SNAP-Galactosyl transferase 1 (Myc-SNAP-GalT) were treated with BG-NHS-labeled (BG-TT) or unlabeled (TT) typhoid toxin for 6 hr at 37°C. BG-labeled toxin molecules that were “captured” by SNAP-GalT formed chimeric protein complexes (indicated as "TT-SNAP-GalT") that were detected by Western blot analysis with an antibody directed to the Myc epitope. Dotted lines indicate places where the experimentally relevant lanes were spliced together (all lanes originate from a single gel). (D and E) Typhoid toxin transport to the endoplasmic reticulum (ER). (D) Schematic representation of the typhoid toxin-disassembly assay in the ER. (E) HEK293T cells were treated with purified typhoid toxin for 30 min at 37°C and lysed at the indicated time points. The mobility of typhoid toxin in SDS-PAGE in the presence or absence of DTT (as indicated) was then analyzed by Western blot with an antibody to CdtB. The positions of CdtB and the CdtB-PltA heteromeric complex are indicated. * denotes the migration of a non-specific cross-reacting protein (F) Typhoid toxin retro-translocation from the ER to the cell cytosol. HEK293T cells were incubated with purified typhoid toxin at 37°C and then harvested at the indicated time points. Cells were selectively permeabilized with digitonin and the presence of typhoid toxin in the cytosolic fraction was detected by Western blot analysis with an antibody to CdtB.