Fig. 2.

Functional properties of LacY mutants with Gly→Trp replacements. (A) Lactose transport in E. coli cells harboring WT LacY (●), mutants with single or double Gly→Trp replacements: G46W (▲), G159W (), G262W (■), G370W (◆), G46W/G262W (▼), G46W/G370W (★), or pT7-5 vector only with no LacY insert (○). Active sugar accumulation was measured at 0.4 mM [¹⁴C]-lactose, as described in Materials and Methods. One hundred percent transport corresponds to 160 nmol/mg protein. (B). Equilibrium exchange of lactose by RSO vesicles containing WT LacY (●), NEM-treated WT LacY (□), mutants with double Gly→Trp replacements G46W/G262W (▲), and G46W/G370W (▼), or no permease (○). RSO vesicles equilibrated with 10 mM [¹⁴C]lactose were diluted (1:200) into buffer containing 10 mM nonradioactive lactose and, at given times, radioactive lactose retained inside of vesicles was measured by rapid filtration as described in Materials and Methods. Nonspecific exchange of lactose in RSO vesicles containing WT LacY was assayed with an NEM-treated sample (see Materials and Methods for details).