No crosstalk between the Sec and Tat translocases during Rieske assembly. (A) Cartoon representation of the chimeric constructs used in these experiments. (B) E. coli strain HS3018-A (deleted for malE) and an otherwise isogenic tatABC mutant strain containing pBAD24 (vector) or pBAD24, producing the Sco2149-MBP fusion protein (TM123-MBP), the first two TMs of MtlA fused to the Sco2149-MBP fusion protein (MtlA(2TM)-TM123-MBP), and MBP fused to TM3 of Sco2149 (130-MBP) or the first two TMs of MtlA fused to the Sco2149 TM3-MBP construct (MtlA(2TM)-130-MBP). These were cultured on maltose indicator broth containing bromocresol purple. (C) Crude membranes were prepared from E. coli strains HS3018-A and HS3018-A ΔtatABC producing MtlA(2TM)-TM123-MBP or MtlA(2TM)-130-MBP followed by recovery of the membrane pellet by ultracentrifugation. The presence of the fusion protein in whole cells (WC), the wash supernatant (S), and pelleted membrane (P) was analyzed by immunoblotting using anti-MBP antiserum.