Article Figures & Data
Figures
- FIG. 1.
(A) YopT fragments. The first and last amino acids of each fragment are indicated. N-terminal truncations were named Δ1-31, Δ1-74, Δ1-100, Δ1-121, and Δ301-322, whereas C-terminally truncated fragments were designated Δ301-322, Δ308-322, Δ315-322, and Δ319-322. All fragments contain the amino acids cysteine 139, histidine 258, and aspartate 274, which are crucial for the activity of full-length YopT. (B) Immunoblot of GST-YopT fragments. All of the fragments used in this study were expressed as GST fusion proteins in E. coli TG1. Expression of the different GST fusion proteins was checked by immunoblot analysis with a YopT antibody. wt, wild type; WB, Western blot.
- FIG. 2.
Microinjection of YopT into EBL cells. (A) Time course of EBL cells after microinjection of GST-YopT. (B) N-terminal (Δ1-31, Δ1-74, Δ1-100, and Δ1-121) and C-terminal (Δ301-322, Δ308-322, Δ315-322, and Δ319-322) GST-YopT fragments were microinjected into EBL cells. Shown are pictures of crucial GST-YopT fragments, which were taken 60 and 180 min after microinjection, respectively. Injected cells are indicated by arrowheads. The experiment was repeated at least three times with similar results. wt, wild type.
- FIG. 3.
Membrane release of RhoA. Isolated rat brain membranes were incubated with fragments (A) or mutant forms (B) of GST-YopT, as indicated. After separation of membranes and corresponding supernatants by ultracentrifugation, the samples were analyzed for RhoA by Western blotting (WB). Shown is a typical result of at least four independent experiments. wt, wild type.
- FIG. 4.
Rhotekin pulldown assay. Lysates of CNF-1-treated HeLa cells were incubated as indicated with wild-type (wt) GST-YopT and fragments (A) or mutant forms (B) thereof for 30 min at 37°C. After incubation with GST-YopT, 1/15 of the volume was taken as input. The rest of the lysates were incubated with GST-rhotekin beads for 60 min at 4°C. The beads were then washed, and the rhotekin-bound RhoA and the input control were separated by SDS-PAGE and detected by Western blotting (WB) with a RhoA-specific antibody. Shown is a typical result of at least four independent experiments.
- FIG. 5.
Influence of lovastatin on the effect of YopT. Subconfluent HeLa cells were treated with 30 μM lovastatin for 16 h and then incubated for 2 h with CNF-1 (400 ng/ml) or only treated with CNF-1. Cytosols were prepared and incubated with or without GST-YopT for 30 min at 37°C. After incubation with GST-YopT, 1/15 of the probes was taken as input while the rest was incubated with either GST-rhotekin or GST-GDI beads for 60 min at 4°C. The beads were then washed. Bound RhoA and the input control were separated by SDS-PAGE and detected by Western blotting (WB) with a RhoA-specific antibody. Shown is a typical result of at least four independent experiments.
- FIG. 6.
Binding of YopT fragments to RhoA. The indicated YopT C139A fragments were expressed as GST fusion proteins, and then equal amounts of the proteins were separated by SDS-PAGE and transferred onto PVDF membrane. The membrane was subsequently used for an overlay assay with isoprenylated, 125I-labeled RhoA that had previously been activated with GTPγS. After incubation with RhoA, the membrane was washed extensively and the bound RhoA was analyzed by phosphorimaging. Equal amounts of the GST-YopT proteins were checked by Ponceau S staining (bottom of panel A). In panel A, a typical experiment is shown. Panel B shows the quantification of three independent experiments as means plus standard deviations. fl, full length.
Tables
- TABLE 1.
Primers used in this study
Primer Sequence 5′ → 3′ YopTsens GGA TCC ATG GAC AGT ATT CAC GGA CAC TAC C YopTanti CCC GGG TTA AAC CTC CTT GGA GTC AAA TG YopTΔ1-31sens GGA TCC GGC GCA CAC CGA GTG AAA GT YopTΔ1-74sens GGA TCC GCT AAC CAA CGA AGC AGC TTT ACC T YopTΔ1-100sens GGA TCC GCG GTG AGA GAG TCT GTT GC YopTΔ1-121sens GGA TCC GGG GCT TTT CTT CAT CAA ATA ATA YopTΔ301-322anti CCC GGG AGA ATT TTC CAA GAA TGA GTT AGT AAA YopTΔ308-322anti CCC GGG CAC CCC CAA AGG ATA ATG ATA CAT A YopTΔ315-322anti CCC GGG TTA AAC GCT AAA ACT CTG CCC CAC CCC YopTΔ319-322anti CCC GGG TTA GTC AAA TGT AAA AAC GCT AAA ACT CTG C YopT C139Asens GGG GGT CGC TGA GGC TTT ATG YopTC139Aanti CAT AAA GCC TCA GCG ACC CCC YopTD274Nsens GTT ACT TTC TTC AAT CCC AAT TTC GGT G YopTD274Nanti CAC CGA AAT TGG GAT TGA AGA AAG TAA
