Mechanisms Potentail and Active Transport in Membrane Vesicles from Vesicles from Escherichia coli

Membrane vesicles isolated from Escherichia coli ML 308-225 accumulate triphenylmethylphosphonium and safranine O in the presence of appropriate electron donors. Moreover, these cations are accumulated when a potassium diffusion gradient is imposed across the vesicle membrane ([K⁺]_in > [K⁺]_out), and the vesicles exhibit the same steady-state levels of accumulation for triphenylmethylphosphonium, dimethyldibenzylammonium (in the presence of tetraphenylboron), and rubidium (in the presence of valinomycin). Triphenylmethylphosphonium accumulation by the vesicles is not dependent on the presence of ionophores or lipophilic anions, occurs with vesicles prepared and assayed in either potassium- or sodium-containing media, and does not exhibit certain properties associated with carrier-mediated transport systems. These results provide strong evidence in support of the hypothesis that oxidation of D-lactate or reduced phenazine methosulfate by the vesicles generates an electrical potential, interior negative, across the vesicles membrane. Accumulation of triphenylmethylphosphonium by the vesicles is relatively specific for d-lactate or reduced phenazine methosulfate as electron donors. l-Lactate, succinate, and NADH are oxidized more rapidly than d-lactate, but l-lactate and succinate do not support triphenylmethylphosphonium uptake as well as d-lactate, and NADH is ineffective. These and other observations suggest that there is an energy-coupling site located primarily between d-lactate dehydrogenase and cytochrome b₁ which is responsible for the generation of the membrane potential. Anoxia, various electron transfer inhibitors, and proton conductors block d-lactate dependent triphenylmethylphosphonium accumulation and proton extrusion. However, only proton conductors and electron transfer inhibitors which block electron flow after the energy-coupling site produce efflux of previously accumulated triphenylmethylphosphonium or collapse the proton gradient established as a result of d-lactate oxidation. The observations suggest that the membrane potential may be in equilibrium with the redox state of the respiratory chain at the site of energy coupling. Evidence is also presented which demonstrates that a membrane potential, interior negative, is intimately associated with the ability of the vesicles to catalyze active transport. Steady-state levels of lactose, proline, tyrosine, glutamic acid, and glycine accumulation are directly related to the steady-state level of triphenylmethylphosphonium accumulation. Moreover, addition of lactose to vesicles containing the β-galactoside transport system partially inhibits the uptake of proline and triphenylmethylphosphonium. The effects are not observed in vesicles devoid of the β-galactoside transport system. Although most of the data support a chemiosmotic mechanism for active transport, evidence is presented which indicates that the membrane potential in itself may not be sufficient to account for the totality of active transport. Possible explanations for these inconsistencies are discussed.