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The Electrochemical Gradient of Protons and Its Relationship to Active Transport in Escherichia Coli Membrane Vesicles

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The Electrochemical Gradient of Protons and Its Relationship to Active Transport in Escherichia Coli Membrane Vesicles Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1892-1896, June 1976 Biochemistry The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles (flow dialysis/membrane potential/energy transduction/lipophilic cations/weak acids) SOFIA RAMOS, SHIMON SCHULDINER*, AND H. RONALD KABACK The Roche Institute of Molecular Biology, Nutley, New Jersey 07110 Communicated by B. L. Horecker, March 17, 1976 ABSTRACT Membrane vesicles isolated from E. coli gen- presence of valinomycin), a respiration-dependent membrane erate a trans-membrane proton gradient of 2 pH units under potential (AI, interior negative) of approximately -75 mV in appropriate conditions when assayed by flow dialysis. Using E. coli membrane vesicles has been documented (6, 13, 14). the distribution of weak acids to measure the proton gradient (ApH) and the distribution of the lipophilic cation triphenyl- Moreover it has been shown that the potential causes the ap- methylphosphonium to measure the electrical potential across pearance of high affinity binding sites for dansyl- and azido- the membrane (AI), the vesicles are shown to generate an phenylgalactosides on the outer surface of the membrane (4, electrochemical proton gradient (AiH+) of approximately -180 15) and that the potential is partially dissipated as a result of mV at pH 5.5 in the presence of ascorbate and phenazine lactose accumulation (6). Although these findings provide ev- methosulfate, the major component of which is a ApH of about idence for the chemiosmotic hypothesis, it has also been dem- -110 mV. As external pH is increased, ApH decreases, reaching o at pH 7.5 and above, while AI remains at about -75 mV and onstrated (6, 16) that vesicles are able to accumulate lactose and internal pH remains at pH 7.5. Moreover, the ability of various other substrates to intravesicular concentrations which are electron donors to drive transport is correlated with their ability 100-fold or greater than those of the external medium. To to generate A4H+. In addition, ApH and Ad can be varied re- sustain concentration gradients of this magnitude, a membrane ciprocally in the presence of valinomycin and nigericin. These potential of at least -120 mV is required. This observation, in data and others (manuscript in preparation) provide convincing addition so numerous negative attempts to establish the exis- support for the role of chemiosmotic phenomena in active tence of a transmembrane pH gradient (1, 6) has left some transport. doubt as to the quantitative relationship between AILH+ and Despite apparently contradictory initial observations (1-3), an solute accumulation. [In the apparent absence of a transmem- increasing accumulation of experimental evidence (4-6) indi- brane pH gradient, it is also not immediately clear how anionic cates that chemiosmotic phenomena, as postulated by Mitchell solutes are accumulated, by the chemiosmotic mechanism. (7-10), play a central role in respiration-linked active transport suggested for this process (10)]. in Escherichia coli membrane vesicles. It now seems clearly Recently, Padan et al. (17) have shown that intact E. coli established that membrane vesicles prepared by the techniques generate a ApH (interior alkaline), and that the magnitude of developed in this laboratory retain the same orientation as the the ApH is very dependent upon external pH, exhibiting a membrane in the intact cell (see ref. 11 for a summary of the maximal value of about 2 pH units at pH 6.0 or below. In ad- evidence), and that oxidation of electron donors which drive dition, Rottenberg (18) has utilized acetate to determine ApH transport in the vesicles results in the generation of a trans- in mitochondria, and suggested that this weak acid might be membrane electrical potential (interior negative) by means of more useful than 5,5'-dimethyloxazolidine-2,4-dione (DMO) electrogenic proton extrusion (6, 12-14). The potential is pos- (19) in certain systems because it might be less permeant (H. tulated to drive solute accumulation via facilitated diffusion Rottenberg; personal communication). of positively charged substrates such as lysine or via coupled The results presented in this paper were obtained by means movements of protons with neutral substrates such as lactose of flow dialysis (20), a technique which allows a rapid, contin- or proline (i.e., "symport"). uous determination of changes in the concentration of solutes According to the chemiosmotic hypothesis, the total driving in the external medium without manipulation of the vesicles. force generated by proton extrusion is the electrochemical Using this technique, it is demonstrated that E. coli membrane potential of protons across the membrane (A,4H+) (7-10). This vesicles generate a large proton gradient under appropriate thermodynamic entity is composed of an electrical and a conditions. In addition, we have shown that ApH and AI can chemical parameter according to the following relationship: be manipulated reciprocally by the ionophores valinomycin and nigericin. The results to be presented, and others (S. Ramos 2.3RTA and H. R. Kaback, manuscript in preparation) which will be A =H+= A - F ApH [1] discussed, leave little doubt as to the primary role of chemios- motic phenomena in respiration-dependent active transport. where Ai represents the electrical potential across the mem- brane, and ApH is the chemical difference in proton concen- METHODS tration across the membrane (2.3RT/F is equal to 58.8 mV at Growth of Cells and Preparation of Membrane Vesicles. room temperature). E. coli ML 308-225 (i-z-y+a+) was grown on minimal me- Through the use of lipophilic cations and rubidium (in the dium A with 1.0% sodium succinate (hexahydrate), and membrane vesicles were prepared as described (21, 22). Vesicles Abbreviations: DMO, 5,.5'-dimethyloxazolidine-2,4-dione; TPMP', M and triphenylmethylphosphonium (bromide salt); PMS, phenazine were suspended in 0.1 potassium phosphate (pH 6.6) methosulfate; CCCP, carbonylcyanide m-chlorophenylhydrazone. stored in liquid nitrogen. * Present address: Department of Molecular Biology, Hadassah For studies at various pH's, membrane suspensions containing Medical School, Hebrew University, Jerusalem, Israel. about 4 mg of protein per ml were removed from storage, 1892 Downloaded by guest on October 2, 2021 Biochemistry: Ramos et al. Proc. Natl. Acad. Sci. USA 73 (1976) 1893 thawed rapidly at 460, diluted at least 10-fold with 0.1 M po- tassium phosphate buffer at the desired pH, and incubated for 10 min at 25°. The suspension was centrifuged at 40,000 X g for 30 min, and the pellet resuspended and washed once in a similar volume of the same buffer. The final pellet was then resuspended to an appropriate protein concentration in 0.1 M potassium phosphate at the same pH. 0 Transport Assays. Filtration assays (6, 23) were carried out x on Millipore Cellotate filters (0.5 ,um pore size). Electron donors ., and isotopically labeled solutes were used as described. Flow 0. 15 dialysis was performed as described (24) except that the ap- paratus was modified so that the upper chamber was com- pletely open to the atmosphere, and the reaction mixture was gassed with oxygen. The upper and lower chambers were separated by Spectrapor 1 dialysis tubing (600-8000 molecular weight cut-off; Fisher Scientific), and both chambers were stirred by means of magnetic bars. Membrane vesicles sus- FRACTION NUMBER pended in 0.05 M potassium phosphate at a given pH containing FIG. 1. Ascorbate-PMS-dependent acetate uptake by E. coli ML 0.'01 M magnesium sulfate were added to the upper chamber 308-225 membrane vesicles as determined by flow dialysis. The assay (total volume 0.8 ml), and electron donors, isotopically labeled shown was carried out at pH 5.5 as described in Methods with sodium solutes, and ionophores were added as indicated. Potassium [3H]acetate (685 mCi/mmol) at a final concentration of 18 gM and phosphate (0.05 M at the same pH as the buffer in the upper E. coli ML 308-225 membrane vesicles at a final concentration of 2.5 mg ofprotein per ml in the upper chamber. As indicated by the arrows, chamber) was pumped from the lower chamber at a rate of 6.0 ascorbate and phenazine methosulfate (ASC/PMS), valinomycin ml/min with a Pharmacia pump (model P3). Fractions of about (VAL), and nigericin (NIG) were added to the upper chamber at final 1.7 ml were collected and assayed for radioactivity by liquid concentrations of 20 mM and 0.1 mM, 1 ,uM and 1 gM, respectively scintillation spectrometry. Since phenazine methosulfate (PMS) (closed symbols). Open symbols were obtained from an identical ex- causes about 5.5% quenching of tritium under the conditions periment carried out in the absence of ascorbate and PMS. The data described, control experiments were carried out in the absence have been corrected for a control performed in the absence of mem- of membrane vesicles, and the data were corrected appro- brane vesicles as described in Methods. priately. appears in the dialysate, increasing linearly for about 2 min, and Determination of ApH. ApH.was determined by measuring reaching a maximum which then decreases at a slow and con- the accumulation of acetate, propionate, butyrate or DMO by stant rate (open symbols). When ascorbate and PMS are added flow dialysis unless otherwise stated. Data were quantitated to the upper chamber (closed symbols), the vesicles accumulate assuming that dialysis rates obtained after addition of nigericin acetate, and its concentration in the dialysate decreases mark- (Fig. 1) represent 0 ApH. edly to about 60% of the level observed in the absence of elec- Determination of AI. The electrical potential across the tron donor. Addition of valinomycin, an ionophore which membrane (Au') was determined by measuring the accumu- specifically increases the potassium permeability of the mem- lation of [3H]triphenylmethylphosphonium (TPMP+) by either brane (27), causes the vesicles to accumulate more acetate, and filtration (6) or flow dialysis.
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