Pre-SufI signal peptide insertion requires the RR-motif, TatB, and TatC. (A) Signal peptide insertion requires the RR-motif. The membrane-inserted nitroxide quenching signatures observed for the V21C and L23C BODIPY-tagged pre-SufI mutants under Tat++ conditions (ABC) were lost when the RR-motif was replaced by KK. Instead, the observed signature resembles the lipid-bound signature obtained under ΔTat (ΔABC) conditions. (B) Signal peptide insertion requires TatB and TatC. The nitroxide quenching signatures for the V21C and L23C BODIPY-tagged pre-SufI mutants were examined in the presence of various combinations of overproduced TatA, TatB, and TatC. The DOXYL quenching diagnostic for membrane insertion required TatB and TatC. However, full protection from 3-CP for the L23C mutant also required TatA.