TMRM labeling of cytoplasmic S67C single-Cys LacY. (A) SDS/PAGE gels showing TMRM labeling in RSO membrane vesicles or with purified protein (fluorescence above dotted line; protein below). (B) Time course of TMRM labeling in RSO membrane vesicles. (C) Time course of TMRM labeling of purified mutant S67C in DDM. RSO vesicles with S67C LacY were labeled with 40 μM TMRM or purified S67C LacY in DDM with 4 μM TMRM for times indicated at 0 °C in the absence of TDG (−TDG), were preincubated with TDG for 10 min before addition of TMRM (+TDG), or TMRM and TDG were added simultaneously [+(TDG+TMRM)]. Purified proteins were subjected to SDS/PAGE; fluorescent and silver-stained protein bands were measured as described in Fig. 2 and Experimental Procedures. The data are plotted relative to the 20-s points in the absence of TDG. Blue ◆, no sugar added; green ▲, preincubated with TDG before addition of TMRM; pink ■, TDG and TMRM added simultaneously.