Figure 4.

Lactose efflux and equilibrium exchange by RSO membrane vesicles containing wild-type permease, E325A, R302A, and R302S permease mutants. RSO vesicles were equilibrated with 10 mM [1-¹⁴C]lactose (pH 7.5) and assayed by dilution into equilibration buffer without lactose (efflux; open circles) or with 10 mM lactose (exchange; solid circles) as described in Experimental Procedures. Nonspecific, passive lactose efflux was assayed after NEM treatment and is shown only for wild-type permease (solid triangles). Although not shown, the loss of radioactivity from NEM-treated vesicles containing E325A, R302A, or R302S permease was identical to the permease-mediated efflux activity of these mutants (i.e., coincides with the empty circles).