In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564–5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position −10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.