Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli

Abstract

Membrane vesicles from E. coli can oxidize D-lactate and other substrates and couple respiration to the active transport of sugars and amino acidl8. The present experiments bear on the nature of the link between respiration and transport.Respiring vesicles were found to accumulate dibenzyldimethylammonium ion, a synthetic lipid-soluble cation that serves as an indicator of an electrical potential. Escherichia coti, like other bacteria, accumulates nutrients from the medium by virtue of specific transport systems ("permeases"). Many nutrients, including amino acids, ,-galactosides, and certain inorganic ions, are transported in the face of large concentration gradients without any apparent chemical modification. Such "active transport" requires the performance of work and implies the coupling of transport systems to the major metabolic pathways of the cell (1-4).A partially resolved system, in which the mechanism of energy coupling may be more amenable to analysis than it is in the intact cell, became available through the work of H. R. Kaback and his associates. In a remarkable research program (5), these investigators demonstrated that isolated membrane vesicles of E. coti and other bacteria can couple active transport of many sugars, amino acids, and certain ions to the oxidation of particular respiratory substrates such as Dlactate. Since these vesicles neither make ATP by oxidative phosphorylation nor utilize exogenous ATP as an energy donor for transport, the coupling of oxidation to transport Abbreviations: DDA+, dibenzyldimethylammonium ion; TPB-, tetraphenylboron; TCS, tetrachlorosalicylanilide; CCCP, carbonylcyanide m-chlorophenylhydrazone; HOQNO, 2-heptyl-4 hydroxyquinoline-N-oxide. * To whom requests for reprints should be addressed, at National Jewish Hospital and Research Center. 1804probably occurs quite directly at the level of the membrane and its constituent proteins (5-7). Kaback and Barnes (8) proposed a tentative mechanism by which the coupling might be effected: the transport carriers are thought to monitor the redox state of the electron-transport chain and themselves undergo cyclic oxidation and reduction of critical sulfhydryl groups; each cycle is accompanied by concurrent changes in the orientation of the carrier center and in its affinity for the substrate, leading to accumulation of the substrate in the lumen of the vesicle. Serious shortcomings of this hypothesis have been pointed out by several investigators (1, 9-11).An alternative mechanism for the coupling of respiration to transport can be envisaged in terms of Mitchell's chemiosmotic hypothesis (12, 13). Briefly, he postulates that the respiratory chain translocates protons outward, thereby generating across the membrane an electrical potential (interior negative) and, under certain conditions, a pH gradient as well (interior alkaline). These gradients, which together constitute a force tending to pull protons back into the vesicle, are held to effect active transport by virtue of carriers that translocate simultaneousl...

Secondary solute transport in bacteria

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The driving force for proton(s) metabolites cotransport in bacterial cells

“…The mechanisms of these cotransport systems (symport) are the subject of this communication. Recently the protonmotive force was determined in various bacterial cells and cell membrane vesicles [5][6][7][8][9][10]. Although the f'mdings differ considerably depending on the species, the type of vesicle and the method of determination, several conclusions can be drawn.…”

Valinomycin‐induced amino acid uptake byStaphylococcus aureus

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