FIGURE 1.

The absence of PpiD does not cause a significant protein transport defect in vitro. A, INV of wild type and a ΔppiD strain were separated on SDS-PAGE and after Western transfer decorated with the indicated antibodies. B, YidC and OmpA were in vitro synthesized using a coupled transcription/translation system in the presence of inner membrane vesicles (INV), derived from either a wild type E. coli strain or a ΔppiD strain. After 30 min of synthesis at 37 °C, the samples were split in half, and one part was directly precipitated with TCA (−). The other half was first treated for 20 min at 25 °C with 0.5 mg/ml proteinase K (Prot. K) and only then TCA-precipitated. pOmpA corresponds to the signal sequence containing pro-OmpA and OmpA to the mature version. YidC and its membrane-protected fragment (YidC-MPF) are indicated. The YidC-MPF corresponds to the first two transmembrane domains of YidC plus the connecting 325-amino acid-long periplasmic loop (66, 67).