Figure 7.

In vitro translocation assay of TatA variants with an extended TMH. (A) The presence of TatA, TatB, and TatC in the INVs is monitored using western blots with the corresponding antibodies. In all INVs sufficient amounts of the Tat components were present. However, the expression rate of the TatA variants with a massively extended TMH was slightly reduced. (B) The transport of the natural E. coli Tat substrate AmiC into INVs was monitored. Radioactively labeled AmiC was synthesized by means of in vitro transcription/translation in the presence or absence of INVs. After PK treatment to digest all untransported substrate, only AmiC that has been transported into the lumen of the INVs is detectable in the subsequent SDS-PAGE analysis. A transport efficiency (TE) was quantified on the basis of three independent experiments by comparison of the synthesized amount of AmiC with the amount that has been transported into the lumen. INVs containing the wild-type Tat components (TatA wt) show considerable transport into the vesicles (TE = 35%). It is seen that the transport efficiency is gradually reduced with increasing TMH length (TatA LAL = 16%; TatA LALA = 3%; TatA LALALAL = 1%). This observation proves that the unusually short length of the TMH of TatA plays an important role in the translocation process.