Fig. 5. Direct determination of the vinblastine/LmrA transporter stoichiometry. The average number of vinblastine molecules bound per LmrA homodimer is plotted as a function of the total number of vinblastine molecules available per transporter. Specific binding of vinblastine to inside-out membrane vesicles containing LmrA was measured in equilibrium binding experiments in which the total amount of membrane protein was varied, at a fixed concentration of 160 nM vinblastine. The number of LmrA molecules per milligram of membrane protein in inside-out membrane vesicles was determined by quantitative immunoblotting and densitometry. The data show that each homodimer of LmrA binds two vinblastine molecules under conditions that saturate the vinblastine-binding sites in the protein.