FIGURE 3.

The effect of cytoplasmic loops deletions in YidC on the membrane insertion of F₀c. A, a topology diagram of E. coli endogenous substrates F₀c. B and D, the YidC variant proteins was reconstituted into the E. coli liposomes and monitored by 12.5% SDS-PAGE, with empty liposomes (Empty) and proteoliposomes-reconstituted WT YidC as negative and positive controls, respectively. C and E, the in vitro synthesis and insertion of F₀c were carried out as described under “Experimental Procedures.” After conducting the synthesis/insertion at 37 °C for 20 min, 10% of the reaction volume was taken as a synthesis control, and the rest was subjected to proteinase K treatment and TCA precipitation. Buffer, buffer without liposomes; Empty, empty liposomes; WT, wild-type YidC-containing proteoliposomes; YidCΔ1–ΔC, YidC variants-containing proteoliposomes; Mw, molecular mass markers.