Energy Coupling in Membrane Vesicles of Escherichia coli: I. ACCUMULATION OF METABOLITES IN RESPONSE TO AN ELECTRICAL POTENTIAL

Abstract

Abstract Membrane vesicles from Escherichia coli have been shown to accumulate sugars and amino acids in response to an artificially imposed membrane potential, in the absence of any metabolizable substrate. Membrane vesicles were first loaded with K+ by incubation at 40° in 0.5 m potassium phosphate and then transferred to K+-free medium. Addition of the potassium ionophores valinomycin, monactin, or enniatin elicited rapid efflux of K+ ions, down the concentration gradient, with the generation of a membrane potential, interior negative. These potassium ionophores also induced concentrative uptake of amino acids and sugars by the vesicles. By contrast, nigericin and monensin, which catalyze electrically neutral exchange of K+ or Na+ for H+, did not induce metabolite accumulation. Accumulation of proline in response to valinomycin was a function of the gradient of K+ concentration across the membrane. Accumulation was not affected by inhibitors of the respiratory chain nor by N, N'-dicyclohexylcarbodiimide, but was completely abolished by reagents which render the membrane permeable to protons or which inhibit the transport carrier itself. The optimal external pH for valinomycin-induced proline uptake lay between 7 and 8; in addition, membrane vesicles prepared so as to create an alkaline lumen accumulated proline better than did those whose lumen pH was acid. The results suggest that the driving force for proline accumulation is the electrical potential; a pH gradient is not required. Potassium-loaded vesicles, prepared from a mutant defective in proline transport, failed to accumulate proline in response to valinomycin, but did accumulate glycine and thiomethyl-β-d-galactoside. This and other experiments with mutants and inhibitors strongly suggest that the accumulation driven by an artificial membrane potential is mediated by the physiological transport systems. The results are consistent with the chemiosmotic interpretation of metabolite transport by membrane vesicles.