FIGURE 6.

Inhibition of heterodimer activity by a thiosulfonate reagent. Membranes from cells expressing CLA (untagged Cys-less EmrE) were solubilized with 0.8% DDM/Na buffer and mixed at 80 °C with solubilized membranes of E14C or G67C (∼100 ng of tagged protein per assay), inactive His-tagged mutants. The extract was centrifuged for 5 min at 14,000 × g to discard precipitates and allowed to bind to Ni-NTA beads. MTSET at a final concentration of 1 mm was added for 10 min and then substrate binding activity was tested as described above (10 nm [³H]TPP⁺ in a 200-μl reaction).