Figure 3.

Topology analysis of YidC in the reconstituted proteoliposomes. Purified YidC protein was mixed with DOPC lipids and unilamellar proteoliposomes were generated with an extruder. The topology of YidC can be analyzed by protease accessibility by adding trypsin (A) (9). The proteolytic fragments were detected using immunoblotting by either an antiserum directed to the large periplasmic domain (left panel) or to the cytoplasmic tail (right panel). The topology of YidC modified with a fluorophore can be determined by the relative amount that is protected from the quencher added to the outside of the proteoliposome (B). The periplasmic domain of YidC was labeled with Atto520. For both cases (A and B), >80% of the YidC periplasmic domain was found in the liposomal lumen in the case of DOPC-reconstituted YidC.