Figure 3. Tse8 targets the transamidosome.
a-c, Tse8 is only toxic in bacteria which rely on the transamidosome for protein synthesis. Expression of Tse8 in A. tumefaciens is toxic but can be rescued by coexpression of Tsi8 ((-) no induction; (+) with induction) (a). Expression of Tse8 in E. coli is not toxic ((-) no induction; (+) with induction) (b), despite Tse8 being expressed (c). d-e, Proteins were added to His-Tag Dynabeads as indicated. d, Left panel, lane 1: His-GatB (as bait) interacts with GatC-HA and GatA-V5. Left panel, lane 2: Tse8 HA-Strep interacts with the GatCAB complex, but does not displace GatA-V5, even at a 15-fold molar excess. Right panel, lane 1: Tse8-HA-Strep alone does not interact with the Dynabeads. Right panel, lane 2: Tse8-HA-Strep does not interact with a different His-tagged bait protein, CcmE. e, Left panel, lane 1: His-GatB (as bait) interacts with GatC-HA and GatA-V5. Left panel, lane 2: The presence of Tse8-HA-Strep leads to drastic increase of the amount of GatA-V5 interacting with His-GatB and GatC-HA. Right panel, lane 1: GatA-V5 alone does not interact with the Dynabeads. Right panel, lane 2: GatA-V5 does not interact with a different His-tagged bait protein, CcmE. f, Quantification of the amount of GatA-V5 bound to His-GatB and GatC-HA in the presence or absence of Tse8-HA-Strep by densitometry. For panels (c-e) molecular weight markers positions are indicated on the left in kDa. Statistical analyses: (a-b) Mean OD600 ± SD is plotted over time from three independent replicates; (c) Representative blot from three independent replicates (n=3); (d) Representative blots from one independent replicate (n=1); (e) Representative blots from three independent replicates (n=3) for the left panel and one independent replicate (n=1) for the right panel; (f) Mean densitometry ± SEM from three independent replicates (n=3). Two-tailed student’s t-test, *** P<0.001 for GatBC+GatA compared to GatBC+GatA+Tse8.