Figure 2.

Effect of TDG on NEM labeling of Cys-148 in single-Cys-148 permease or mutants E325Q/Cys-148, E325D/Cys-148, R302A/Cys-148, R302K/Cys-148, H322A/Cys-148, H322N/Cys-148, H322Q/Cys-148, and E269D/Cys-148 at pH 5.5, pH 7.5, and pH 9.5. (Left) Right-side-out membrane vesicles were incubated with 0.5 mM (at pH 5.5 and 7.5) or 0.22 mM (at pH 9.5) [¹⁴C]NEM at 25°C, for 15 min (pH 5.5), 5 min (pH 7.5), or 2 min (pH 9.5) in the absence or presence of given concentrations of TDG. Reactions were quenched with DTT, and biotinylated permease was solubilized and purified by affinity chromatography on monomeric avidin. Aliquots of protein were separated on a SDS/12% polyacrylamide gel, and [¹⁴C]-labeled permease was visualized by autoradiography. Although not shown, a fraction of the protein was analyzed by Western blotting to determine the amount of permease in each sample; no significant differences were observed. (Right) Incorporation of [¹⁴C]NEM was quantitated by a Storm 860 PhosphorImager, and labeling in the presence of various concentrations of TDG is expressed as % labeling observed in the absence of TDG. ○, pH 5.5; ■, pH 7.5; ●, pH 9.5.