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Role of an Electrical Potential in the Coupling of Metabolic Energy To Proc. Nat. Acad. Sci. USA Vol. 70, No. 6, pp. 1804-1808, June 1973 Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli (chemiosmotic hypothesis/lipid-soluble ions/valinomycin/ionophores/amino-acid transport) HAJIME HIRATA, KARLHEINZ ALTENDORF, AND FRANKLIN M. HAROLD* Division of Research, National Jewish Hospital and Research Center, Denver, Colorado 80206; and Department of Microbiology, University of Colorado Medical Center, Denver, Colorado 80220 Communicated by Saul Roseman, April 12, 1973 ABSTRACT Membrane vesicles from E. coli can oxidize probably occurs quite directly at the level of the membrane D-lactate and other substrates and couple respiration to its the active transport of sugars and amino acidl8. The pres- and constituent proteins (5-7). Kaback and Barnes (8) ent experiments bear on the nature of the link between proposed a tentative mechanism by which the coupling might respiration and transport. be effected: the transport carriers are thought to monitor Respiring vesicles were found to accumulate dibenzyl- the redox state of the electron-transport chain and themselves dimethylammonium ion, a synthetic lipid-soluble cation undergo cyclic oxidation and reduction of critical sulfhydryl that serves as an indicator of an electrical potential. The results suggest that oxidation of it-lactate generates groups; each cycle is accompanied by concurrent changes in a membrane potential, vesicle interior negative, of the the orientation of the carrier center and in its affinity for the order of -100 mV. In vesicles lacking substrate, an electri- substrate, leading to accumulation of the substrate in the cal potential was created by induction of electrogenic lumen of the vesicle. Serious shortcomings of this hypothesis efflux of K+ with the aid of the K+ ionophores, valino- out mycin and monactin. These conditions induced transient have been pointed by several investigators (1, 9-11). accumulation by the vesicles of [14Cjproline and other An alternative mechanism for the coupling of respiration to metabolites. Experiments with inhibitors and ionophores transport can be envisaged in terms of Mitchell's chemiosmo- indicate that neither ATP nor the respiratory chain is tic hypothesis (12, 13). Briefly, he postulates that the involved; the electrical potential generated by K+ efflux respiratory chain translocates protons outward, thereby is coupled directly to the transport systems. The results verify two predictions derived from Mitchell's chemios- generating across the membrane an electrical potential (in- motic hypothesis: respiring vesicles generate an electrical terior negative) and, under certain conditions, a pH gradient as potential of the proper polarity and magnitude; and a well (interior alkaline). These gradients, which together membrane potential is in itself sufficient to drive the constitute a force tending to pull protons back into the vesicle, active transport of amino acids and other metabolites. are held to effect active transport by virtue of carriers that Escherichia coti, like other bacteria, accumulates nutrients translocate simultaneously both protons and the particular from the medium by virtue of specific transport systems substrate (symport). The application of chemiosmotic prin- ("permeases"). Many nutrients, including amino acids, ,- ciples to transport in bacteria and mitochondria has been galactosides, and certain inorganic ions, are transported in the considered more fully in several recent reviews (1, 14, 15). face of large concentration gradients without any apparent Kaback and his associates reject the chemiosmotic inter- chemical modification. Such "active transport" requires the pretation of transport by vesicles for several reasons (5, 16), performance of work and implies the coupling of transport one of their chief arguments being that vesicles do not gen- systems to the major metabolic pathways of the cell (1-4). erate the predicted electrical potential. We believe this con- A partially resolved system, in which the mechanism of clusion to be erroneous (9) and have begun to systematically energy coupling may be more amenable to analysis than it is examine active transport in E. coli from the chemiosmotic in the intact cell, became available through the work of H. R. perspective. Our approach relies on the use of ionophores to Kaback and his associates. In a remarkable research program monitor and manipulate ion gradients across the membrane; (5), these investigators demonstrated that isolated membrane the modes of action of these reagents are now well established vesicles of E. coti and other bacteria can couple active trans- and have been reviewed repeatedly (1, 15, 17, 18). port of many sugars, amino acids, and certain ions to the In this communication, we report that -E. coli membrane oxidation of particular respiratory substrates such as D- vesicles can, in fact, generate a membrane potential, interior lactate. Since these vesicles neither make ATP by oxidative negative, of an order of magnitude sufficient to account for phosphorylation nor utilize exogenous ATP as an energy the active transport of amino acids by symport with protons. donor for transport, the coupling of oxidation to transport Moreover, vesicles lacking oxidizable substrate can accumu- late proline in response to an artificially imposed membrane Abbreviations: DDA+, dibenzyldimethylammonium ion; TPB-, potential. tetraphenylboron; TCS, tetrachlorosalicylanilide; CCCP, car- bonylcyanide m-chlorophenylhydrazone; HOQNO, 2-heptyl-4 METHODS hydroxyquinoline-N-oxide. * To whom requests for reprints should be addressed, at National Organisms. E. coli ML-3088 (i-z-y+) was used, unless Jewish Hospital and Research Center. otherwise stated. In a few experiments, we used E. coli W-157, 1804 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) Chemiosmotic Coupling of Active Transport 1805 a mutant auxotrophic for proline and defective in proline transport, generously provided by Prof. Martin Lubin. 'S:S Preparation of Na+-Vesicles. The organisms were grown 2010 under aeration in the minimal medium of Davis and Mingioli 0I (19), with 1% succinate as the energy source. Membrane E vesicles were prepared exactly as described by Kaback (20), except for two modifications: the amount of DNase I in the 0. final lysate was reduced to 3 gug/ml and sodium replaced E potassium throughout the procedure. Thus, spheroplasts FIG.~~01.Utk0fDAod 4 yK-oddvsce nrsos were lysed in 0.05 M sodium phosphate (pH 6.6). The vesicles were then layered upon 60% sucrose buffered with sodium phosphate. Intact cells were removed by centrifugation at 0 5 10 64,000 X g. The vesicles were collected by centrifugation at Minutes 30,000 X g, washed with sodium phosphate containing eth- FIG. 1. Uptake of. DDA + by K +loaded vesicles in response ylene diamine tetraacetic acid, resuspended in 0.1 M sodium to a membrane Potential. K+4loaded membrane vesicles were phosphate (pH 6.6), and stored at -80°. Since our vesicles prepared as described in Methode. Reaction mixtures contained contain sodium phosphate they will be referred to as Na+- the following (final concentrations) in a volume of 1 ml: 3.5 mg vesicles. Vesicles prepared by Kaback's procedure, K+- of protein, 280 mM sucrose, 13 mM MgSO4, 100 mM Tris. vesicles, were used in a few experiments. maleate (pH 7), 0.01 mM TPB-, and 0.5 mM [3H]DDA+ (2.5 uCi/ml). The mixture was incubated for 10 min at 250, and Potassium-Loaded Vesicles. For experiments in which an the following additions were made (arro'w): *--, valinomycin artificial membrane potential was generated by K+ efflux, (2 jug), TPB- present; A- A, valinomycin (2 ,ug), TPB- vesicles containing a higher internal K+ concentration were absent; o-O, ethanol only (2 Ml), TPB- present; A-A, required. In preliminary experiments this was accomplished ethanol only, TPB- absent. At intervals, 0.1-ml aliquots of the by brief sonication in the appropriate buffer, but for the suspension were filtered and washed with 5 ml of 0.4 M sucrose experiments described here a gentler procedure was used: containing 5 mM MgSO4. sodium vesicles, as prepared above, were resuspended in 0.5 M potassium phosphate (pH 8) containing 10 mM MgSO4. and monensin; and Dr. Keller-Schierlein (Eidgen6ssische The suspension was kept at 480 for 10 min, then chilled in Technische Hochschule, Zurich) for monactin. Other in- ice; the vesicles were collected by centrifugation at 30,000 X g hibitors were purchased commercially; DDA+ was purchased for 10 min, washed once with 0.4 M sucrose containing 5 mM frQm K and K Laboratories. Ionophores were added in MgSO4, and resuspended in sucrose-magnesium. Such vesicles ethanol; control suspensions received ethanol alone. are stable for several hours. RESULTS Chloride (DDA+). Tri- [3H]Dibenzyldimethylammonium Uptake of ['H] DDA+ as an index of the tiated DDA+ was prepared to order by New England Nuclear membrane potential Corp.; the final product has a specific activity of 12.5 Ci/ mol, and was warranted to have a radiochemical purity of Skulachev, Liberman, and their associates (21, 22) demon- 98% or better by thin-layer chromatography. Purity was strated that DDA+ behaves as a permeant cation for artificial confirmed in this laboratory by infrared and ultraviolet lipid-bilayer membranes, especially in the presence of a trace spectroscopy and by crystallization to constant specific of the anion TPB- (tetraphenylboron), and pioneered the use activity after
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