The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.     

Energy transduction in Escherichia coli. The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures.

1. The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium. 2. Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake. 3. The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide. 4. Chaotrope-treated membranes were found to lack Mg2+-ATPase activity. Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions. 5. Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents. 6. Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate. 7. It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase.     

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.     

Immunochemical analysis of membrane vesicles from Escherichia coli.

Membrane vesicles isolated from Escherichia coli ML 308--225 have been analyzed by crossed immunoelectrophoresis, and immunoprecipitates corresponding to the following cellular components have been identified: ATPase (EC 3.6.1,3), two or three NADH dehydrogenases (EC 1.6.99.3), D-lactate dehydrogenase (EC 1.1.1.27), glutamate dehydrogenase (EC 1.4.1.4), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), beta-galactosidase (EC 3.2.1.23), lipopolysaccharide, and Braun's lipoprotein. The cellular origin of many of the vesicle immunogens is determined, and Braun's lipoprotein is used as a marker to quantitate the extent of outer membrane contamination (less than 3%). Membrane antigens are also characterized with regard to their amphiphilic or hydrophilic properties by charge-shift crossed immunoelectrophoresis. Furthermore, the following immunogens cross-react with components in membrane vesicles prepared from Salmonella typhimurium: one of the three NADH dehydrogenases, ATPase, polynucleotide phosphorylase, 6-phosphogluconate dehydrogenase, Braun's lipoprotein, and three unidentified antigens. In the accompanying paper [Owen, P., & Kaback, H. R. (1979) Biochemistry 18 (following paper in this issue)] quantitative immunoadsorption is utilized to establish the topology of the vesicles with respect to the distribution of antigens on the inner and outer faces of the membrane.     

Adenosine 5''-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.     

Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli.

Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated. Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide. Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation. ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase. Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium. Valinomycin plus nigericin completely inhibited ATP synthesis. The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined. NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect. These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Uncoupling action of amytal in membrane vesicles from Escherichia coli.

The barbiturate amytal (5-ethyl-5-isopentylbarbituric acid) has been shown to inhibit amino acid transport in membrane vesicles from anaerobically grown Escherichia coli. Amytal has no effect on the activity of the enzymes of the nitrate respiration system, nor on electron transfer in this system. However, addition of amytal to the membrane vesicles results in a decrease of the membrane potential from -90 mV to -72 mV, and to a decrease of the pH-gradient of -61 mV to undetectable values. Furthermore, amytal causes an increase in the rate of ferricyanide reduction in liposomes, indicating that amytal increases the proton permeability of phospholipid membranes. These results demonstrate that amytal acts as an uncoupler in membrane vesicles from anaerobically grown E. coli.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

A chemically cross-linked nonlinear proOmpA molecule can be translocated into everted membrane vesicles of Escherichia coli in the presence of the proton motive force.

The chemical cross-linking between the two cysteine residues at positions + 290 and + 302 of proOmpA was performed with N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine. In the absence of the proton motive force (delta muH+), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates. In the presence of delta mu H+, the cross-linked proOmpA was completely translocated. The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker. It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated.     

Interaction of the maltose-binding protein with membrane vesicles of Escherichia coli.

The interaction of the radioactively labeled purified maltose-binding protein of Escherichia coli with membrane vesicles was studied. The maltose-binding protein bound specifically to the vesicles, in the presence of maltose, on few sites. Under conditions in which a potential was imposed across the membrane, the specific binding was (i) increased, (ii) dependent on maltose, and (iii) abolished in a mutant defective in the tar gene product, one of the methyl-accepting chemotaxis proteins. At least 1,300 binding sites were present in the membrane fraction of logarithmically growing cells.     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities.

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0 degrees C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.