MECHANISM OF LACTOSE TRANSLOCATION IN MEMBRANE-VESICLES FROM ESCHERICHIA-COLI .1. EFFECT OF PH ON EFFLUX, EXCHANGE, AND COUNTERFLOW

Carrier-mediated lactose efflux down a concentration gradient was used to probe the mechanism of β-galactoside translocation in Escherichia coli membrane vesicles, with particular emphasis on proton/lactose symport. The maximal rate of efflux is dependent on pH, increasing about threefold from pH 5.5 to 7.5 (t1/2 = 45, 27, and 15 s at pH 5.5, 6.6, and 7.5, respectively). In contrast, experiments performed under identical conditions with equimolar lactose in the external medium (i.e., under exchange conditions) demonstrate that the exchange reaction is insensitive to pH and very fast relative to efflux (t1/2 < 2 s). Proton symport occurs during lactose efflux, resulting in the transient formation of a membrane potential (ΔΨ, interior negative) as demonstrated by efflux-dependent accumulation of rubidium (in the presence of valinomycin) and active transport of proline, both of which are abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone. Moreover, the magnitude of the ΔΨ generated increases with pH in much the same manner as the rate of lactose efflux, suggesting tight coupling between the processes. Comparison of the efflux and exchange re-actions suggests that the rate-determining step for efflux involves the return of the unloaded carrier to the inner surface of the membrane and that either loss of the symported proton from the carrier or translocation of the unloaded carrier may be limiting. Counterflow experiments conducted at various pH values reveal that external lactose affects proton loss from the carrier. When external lactose is present at concentrations below the apparent Km of the carrier, counterflow is pH dependent and decreases from pH 5.5 to 7.5, indicating that deprotonation of the carrier occurs frequently under these conditions to limit the counterflow process. When the external lactose concentration is saturating, however, counterflow is unaffected by pH. Moreover, the transient formation of ΔΨ observed during lactose efflux is abolished under these conditions. The observations are consistent with an ordered mechanism for efflux whereby lactose is released first, followed by loss of a proton. In addition, it is postulated that the loaded carrier recycles in the protonated form during counterflow and exchange. © 1979, American Chemical Society. All rights reserved.