In vitro analysis of the stop-transfer process during translocation across the cytoplasmic membrane of Escherichia coli

Abstract

In this study, using a derivative of proOmpA containing an artificial stop-transfer sequence (proOmpA2xH1), we analyzed the process of stop- transfer during translocation across the cytoplasmic membrane of Escherichia coli. ProOmpA2xH1 did not interfere with the transit of wild-type proOmpA. When proOmpA2xH1 was anchored in the membrane, membrane-inserted SecA was deinserted with the reversion of the inverted topology of SecG. Cross- linking experiments revealed that the anchored proOmpA2xH1 that does not interact with either SecY or SecA. These results, taken together, suggest that proOmpA2xH1 leaves the translocation pathway by means of a specific interaction between the stop-transfer sequence and the translocational channel.