N- and C-terminal domains are shown in green and blue, respectively, and hydrophobic patches are in yellow. Arg141-Arg149 stretch is colored as black. “L” denotes positions of ionic locks. a, Left, outward partially occluded conformation (Mol-A); right, threading model for inward conformation. b, Residues forming ionic locks are shown in sticks connected by broken lines. Gray spheres represent the residues involved in sugar binding. c, Cys mutant of Arg residue at position 295, 141, or 363. Intact E. coli DW2 cells containing WT MelBSt or a given mutant were assayed for [3H]melibiose transport (0.4 mM, 10 mCi/mmol) in the absence or presence of 20 mM NaCl or LiCl. Intracellular melibiose expressed as nmol/mg of total proteins is plotted as a function of time. Each single-site mutation is in the WT background. Error bar, s.e.m., n = 2 for the mutants and n = 3 for the WT. d, Western blotting. 25 μg of RSO vesicles was loaded on each well and detected by anti-His-tag antibody.