FIG 3.
The Rieske protein, QcrA, is integrated into S. coelicolor M145 membranes in the absence of the Tat machinery. Membrane fractions were prepared from hyphae of strains M145 and TP4 (Tat + and −, respectively) (A) and strain APH5-KK (ΔqcrCAB::Hygr ϕC31 PhrdB-qcrCAR161K R162KBHis10, producing a QcrA protein with the conserved twin arginines mutated to twin lysines [QcrAKK]) (B) as described in Materials and Methods. The membranes were incubated with either 0.2 M Na2CO3 or 4 M urea, followed by recovery of the membrane pellet by ultracentrifugation. The presence of QcrA in the wash supernatant (S) and pelleted membrane (P) was analyzed by immunoblotting using anti-QcrA antiserum.