FIGURE 3.

Inhibition of RSO transporters reveals the distribution of transmembrane orientation. A, strategy for thrombin cleavage of His-tagged Glt_Ph reconstituted in 3:1 E. coli polar lipid extract:trimethyl PE. The hexahistidine tag is susceptible to thrombin cleavage (scissors). Thrombin (10 units/mg of protein) was added to liposomes containing His-Glt_Ph (20 μg/mg of lipid). At the indicated time points, the digest was stopped with 1 mm AEBSF and 10 mm EDTA. Samples were run on a 10–20% SDS-polyacrylamide gel. B, percentage of transport remaining after 1 mm MTSET (in 100 mm KCl, 20 mm HEPES-Tris, pH 7.5) incubation of A364C reconstituted in liposomes. Data were normalized to transport of untreated liposomes in each lipid composition. Data represent the mean of experiments performed in triplicate, and error bars indicate S.E.