Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids

Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E. coli lipid. Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E. coli galactose transport systems.     

Reconstitution of sugar phosphate transport systems of Escherichia coli

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.     

The association of proton movement with galactose transport into subcellular membrane vesicles of Escherichia coli.

1. Subcellular membrane vesicles were prepared from a strain of Escherichia coli constitutive for the GalP galactose-transport system. 2. The addition of substrates of the GalP transport system to vesicle suspensions promoted alkaline pH changes, which provided direct evidence for the coupling of sugar and proton transport. 3. Respiration-energized galactose transport was progressively inhibited at pH values above 6.0, and was abolished by agents that render the membrane permeable to protons. 4. The combined effects of valinomycin, the nigericin-like compound A217 and pH on galactose transport suggested that both delta pH and delta psi components of the protonmotive force contributed to energization of galactose transport. 5. These results substantiate the conclusion that the GalP transport system operates by a chemiosmotic mechanism.     

Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.     

Reconstitution into liposomes of glucose active transport from the rabbit renal proximal tubule. Characteristics of the system

This paper describes the characteristics of Na⁺-dependent d-glucose transport into liposomes made from soybean phospholipids into which have been reconstituted detergent-solubilized components from the rabbit renal proximal tubular brush border membrane. Conditions for optimal and quantitative reconstitution of glucose carriers are defined. Na⁺-dependent d-glucose uptake occurs via a saturable system with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.125–0.135 mM, is responsive to the volume of the internal liposomal space, and shows ‘overshoot’ as seen in natural membranes. The rate of Na⁺-dependent d-glucose uptake and the magnitude of the ‘overshoot’ are proportional to the concentration of protein used in reconstitution.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Reconstitution of GDP-mannose transport activity with purified Leishmania LPG2 protein in liposomes

Activated nucleotide sugars required for the synthesis of glycoconjugates within the secretory pathway of eukaryotes are provided by the action of nucleotide sugar transporters (NSTs). Typically, NSTs are studied in microsomal preparations from wild-type or mutant lines; however, in this setting it can be difficult to assess NST properties because of the presence of glycosyltransferases and other interfering activities. Here we have engineered Leishmania donovani to express high levels of an active LPG2 Golgi GDP-Man transporter bearing a C-terminal polyhistidine tag. The functional LPG2-HIS was solubilized, purified by metal affinity chromatography, and reconstituted into phosphatidylcholine-containing liposomes using polystyrene SM-2 beads. The proteoliposomes exhibited robust GDP-Man transport activity with an apparent K(m) of 6.6 mum. Transport activity was enhanced by preloading of GMP and showed specificity for multiple substrates (GDP-Ara and GDP-Fuc). In contrast to the activity in crude microsomes, transport was not dependent on the presence of divalent cations. Thus, reconstitution of transport activity using purified LPG2 protein in liposomes provides firm experimental evidence that a single polypeptide is solely required for NST activity and is able to mediate the uptake of multiple substrates. These studies are relevant to the study of NST structure and function in both protozoan parasites as well as their higher eukaryotic hosts.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.     

Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

The Escherichia coli very short patch (VSP) repair pathway corrects thymidine-guanine mismatches that result from spontaneous hydrolytic deamination damage of 5-methyl cytosine. The VSP repair pathway requires the Vsr endonuclease, DNA polymerase I, a DNA ligase, MutS, and MutL to function at peak efficiency. The biochemical roles of most of these proteins in the VSP repair pathway have been studied extensively. However, these proteins have not been studied together in the context of VSP repair in an in vitro system. Using purified components of the VSP repair system in a reconstitution reaction, we have begun to develop an understanding of the role played by each of these proteins in the VSP repair pathway and have gained insights into their interactions. In this report we demonstrate an in vitro reconstitution of the VSP repair pathway using a plasmid DNA substrate. Surprisingly, the repair track length can be modulated by the concentration of DNA ligase. We propose roles for MutL and MutS in coordination of this repair pathway.     

Reconstitution of the melibiose carrier of Escherichia coli

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.     

An essential role of phosphatidylglycerol in the formation of the osmotically stable liposomes of Escherichia coli phospholipids

A temperature sensitive auxotroph of Escherichia coli K-12 requiring unsaturated fatty acids can grow normally at 28 degrees C, but requires an osmotic stabilizer such as a high amount of salt or sugar in the medium for the growth at 42 degrees C. Namely, the apparent osmotic stability of the cells at 28 degrees C and 42 degrees C is quite different. The osmotic properties of liposomes of the phospholipids extracted from these cells were investigated. The osmotically induced volume change of the multilamellar liposomes was examined by the turbidimetric method. The liposomes prepared from cells grown at 28 degrees C can swell and shrink under a wide range of hypo-and hypertonic conditions. However, those from cells grown at 42 degrees C could not swell under hypotonic conditions. These results exhibit a good correlation between the apparent osmotic stability of E. coli cells and the osmotic properties of the liposomes prepared from the extracted total phospholipids. To clarify the role of each phospholipid component, the osmotic properties of the liposomes reconstituted from the purified phospholipid species were further investigated. The results clearly showed that phosphatidylglycerol is the key factor that stabilizes the membranes of E. coli phospholipids against osmotic pressure.     

Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity

Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected. An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations. Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E. coli cells were transformed with plasmids coding for the enzyme and the particular peptides. Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex. It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay.