Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: XIII. VALINOMYCIN-INDUCED RUBIDIUM TRANSPORT

Abstract

Valinomycin-induced uptake of rubidium by membrane vesicles prepared from Escherichia coli, Staphylococcus aureus, and Micrococcus denitrificans is analogous in nearly all respects to the transport of sugars or amino acids, or both, by these membrane vesicles. In E. coli membrane vesicles, concentrative rubidium uptake is stimulated maximally by d -lactate and by the artificial electron donor ascorbate-phenazine methosulfate, to a lesser extent by l -lactate, dl -α-hydroxybutyrate, succinate, and reduced diphosphopyridine nucleotide, and not at all by ATP, phosphoenolpyruvate, or a number of other metabolites. There is no direct relationship between the ability of various electron donors to stimulate rubidium uptake (ascorbate-phenazine methosulfate >> d -lactate >> l -lactate g dl -α-hydroxybutyrate g succinate g reduced diphosphopyridine nucleotide) and their rates of oxidation by membrane vesicles (ascorbate-phenazine methosulfate >> reduced diphosphopyridine nucleotide g succinate g d -lactate g l -lactate g dl -α-hydroxybutyrate). In the presence of d -lactate and valinomycin, the initial rate of rubidium uptake versus the rate of d -lactate oxidation yields a ratio of nearly 2:1. Initial rates of d -lactate-dependent rubidium uptake are saturable with respect to rubidium and valinomycin concentrations and exhibit the same temperature optimum at 50° as that observed for d -lactate oxidation. Steady state levels of rubidium accumulation vary with temperature and can be shifted readily from one steady state level to another by raising or lowering the temperature. Competitive uptake and displacement experiments indicate that rubidium and potassium are equivalent substrates for valinomycin-induced uptake. Rubidium uptake in the presence of valinomycin is markedly inhibited by anaerobiosis, by the electron transfer inhibitors oxamate, amytal, 2-heptyl-4-hydroxyquinoline-N-oxide and cyanide, and by p-chloromercuribenzenesulfonate, but not by arsenate or dicyclohexylcarbodiimide; inhibition by p-chloromercuribenzenesulfonate is reversed by dithiothreitol. Moreover, anaerobiosis, amytal, 2-heptyl-4-hydroxyquinoline-N-oxide, and cyanide produce rapid efflux of rubidium from the vesicles. Oxamate and p-chloromercuribenzenesulfonate, however, cause little or no efflux of rubidium accumulated in the presence of valinomycin, even though p-chloromercuribenzenesulfonate-treated vesicles catalyze exchange of intravesicular rubidium with external potassium at normal rates. These findings indicate that valinomycin does not simply catalyze the passive transfer of rubidium across the vesicle membrane. Valinomycin-induced active rubidium uptake is accompanied by the rapid efflux of intravesicular sodium against its concentration gradient. The vesicles are passively permeable to the lipophilic dibenzyldimethylammonium ion; however, this cation does not inhibit rubidium uptake or rubidium-induced sodium efflux and does not cause sodium efflux. Moreover, radioactive dibenzyldimethylammonium is not accumulated by membrane vesicles under conditions in which rubidium is actively transported. Valinomycin, in the presence of potassium, has no significant effect on the transient acidification of the medium observed upon addition of d -lactate to the vesicles. In addition, vesicles treated with Tween 40 such that they are rendered devoid of a diffusion barrier exhibit pH changes similar to those of untreated vesicles. These findings indicate that active rubidium uptake is an electrogenic process and that proton or potential gradients are not the primary driving force for respiration-linked active transport in isolated bacterial membrane vesicles.