Solubilization and reconstitution characteristics of hepatic system A-mediated amino acid transport

In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 degrees C or for 2 days at 4 degrees C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteoliposomes at specific activities greater than those in the native vesicles.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Evidence for inherent differences in the system A carrier from normal and transformed liver tissue. Differential inactivation and substrate protection in membrane vesicles and reconstituted proteoliposomes

Plasma membrane vesicles isolated from intact rat liver (normal hepatocyte) or cultured rat H4 hepatoma cells retain Na+-dependent uptake of 2-aminoisobutyric acid mediated by System A. The carrier was inactivated in normal liver membrane vesicles by either N-ethylmaleimide (NEM) or p-chloromercuribenzene sulfonate (PCMBS). The concentrations required to produce half-maximal inhibition were approximately 370 and 110 microM for NEM and PCMBS, respectively. In contrast, transport of System A in H4 hepatoma membrane vesicles was sensitive to PCMBS (K 1/2 = 180 microM), yet totally unaffected by NEM at concentrations up to 5 mM. Substrate-dependent protection from PCMBS activation was observed for the System A activity in H4 hepatoma membranes, but not in vesicles from normal hepatocytes. Subsequent inactivation of the substrate-protected carrier by sulfhydryl-specific reagents, added following the removal of the protective amino acid, suggests that one or more cysteine residues become less reactive in the presence of System A substrates. Treatment of solubilized membrane proteins with NEM prior to reconstitution into artificial proteoliposomes showed that the selective inactivation by NEM of the carrier in normal liver membranes is not dependent on the lipid environment or on the integrity of the plasma membrane. The results support the hypothesis that there are inherent differences in the System A carriers that are present in normal and transformed liver tissue.     

Expressing and purifying membrane transport proteins in high yield

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.     

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Solubilization and functional reconstitution of the proline transport system of Escherichia coli

The membrane carrier for L-proline (product of the putP gene) of Escherichia coli K12 was solubilized and functionally reconstituted with E. coli phospholipid by the cholate dilution method. The counterflow activity of the reconstituted system was studied by preloading the proteoliposomes with either L-proline or the proline analogues: L-azetidine-2-carboxylate or 3,4-dehydro-L-proline. The dilution of such preloaded proteoliposomes into a buffer containing [3H]proline resulted in the accumulation of this amino acid against a considerable concentration gradient. A second driving force for proline accumulation was an electrochemical potential difference for Na+ across the membrane. More than a 10-fold accumulation was seen with a sodium electrochemical gradient while no accumulation was found with proton motive force alone. The optimal pH for the L-proline carrier activities for both counterflow and sodium gradient-driven uptake was between pH 6.0 and 7.0. The stoichiometry of the co-transport system was approximately one Na+ for one proline. The effect of different phospholipids on the proline transport activity of the reconstituted carrier was also studied. Both phosphatidylethanolamine and phosphatidylglycerol stimulate the carrier activity while phosphatidylcholine and cardiolipin were almost inactive.     

Reconstitution of a partially purified Na+-independent D-glucose transport system from rat jejunal basolateral membranes

1. Basolateral membranes of rat small intestine were first solubilized in a 0.6% cholate buffer and then the insoluble fraction was reextracted with a 1.2 or 1.6% cholate buffer. 2. Proteoliposomes reconstituted from the 1.2 or 1.6% cholate-extracted membrane fraction demonstrated characteristic Na+-independent D-glucose transport of the native basolateral membrane vesicles: inhibitable by mercuric chloride and D-galactose. 3. To further purify this D-glucose transport system, the 1.6% cholate-extracted membrane fraction was chromatographed on either hydroxylapatite, concanavalin A, wheat-germ lectin or castor bean lectin-120 affinity gels. 4. Proteoliposomes reconstituted from the membrane proteins adsorbed on hydroxylapatite and subsequently passed through agarose-castor bean lectin-120 showed a 12-fold enrichment of Na+-independent D-glucose transport activity over that of the native membrane vesicles. 5. SDS-electrophoretic analysis showed that the protein composition of the hydroxylapatite-castor bean lectin-120 treated fraction was much simpler than that of both 1.6% cholate-extracted fraction and the native membrane vesicles.     

Hormone-induced system A amino acid transport activity in rat liver plasma membrane and Golgi vesicles. Evidence for a differential sensitivity to inactivation by N-ethylmaleimide during carrier maturation

We have investigated the biogenesis and processing of the rat hepatic System A amino acid carrier following induction of its de novo synthesis by the combined action of glucagon and dexamethasone. Golgi subfractions isolated from hormone-treated rat liver form transport competent vesicles and possess characteristic System A activity based on pH sensitivity and 2-(methylamino)isobutyric acid inhibition of Na(+)-dependent 2-aminoisobutyric acid uptake. We have monitored the time course for appearance of the newly synthesized carrier in the Golgi and plasma membrane fractions after the administration of hormones. Our data suggest that it may also be possible to detect processing intermediates of the System A carrier in the Golgi. Perfusion of whole rat liver with 5 mM N-ethylmaleimide followed by isolation of Golgi subfractions and plasma membrane revealed a differential sensitivity such that the plasma membrane or trans Golgi activities were inactivated to a much greater extent than those of the cis or medial Golgi. In vitro N-ethylmaleimide treatment of membrane fractions isolated from an intact rat results in an inactivation of the trans Golgi and plasma membrane System A carrier protein, whereas the cis and medial Golgi fractions retained their transport activity.     

Partial purification and reconstitution of the aspartate transport system from Halobacterium halobium

Membrane vesicles of Halobacterium halobium R1Wrm bind to an aspartic acid-agarose affinity column. After disruption of the bound vesicles by low ionic strength, a protein fraction is eluted from the column with 2.5% cholate in 3 M NaCl. When this fraction is reconstituted with soybean lipids to form proteoliposomes, the proteoliposomes exhibit active aspartate accumulation. Aspartate transport in the reconstituted system is driven by a chemical sodium gradient (out greater than in), exhibits sensitivity to an electrical potential, and is specific for L-aspartate. These characteristics are consistent with observations on aspartate transport in intact membrane vesicles of H. halobium. Initial aspartate transport rates in the reconstituted system are about ninefold enhanced over the native system. The system developed should be useful in future purification schemes and studies of the molecular details of membrane transport.     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.     

Resolution and reconstitution of active transport of calcium by a protein(s) from Mycobacterium phlei.

Membrane protein(s) responsible for the active transport of calcium in membrane vesicles from Mycobacterium phlei have been solubilized from membranes by sodium cholate treatment and partially purified using a hydrophobic resin. Reconstitution of calcium transport was demonstrated by reconstitution of detergent extracted membranes with the partially purified protein. The uptake of calcium in the reconstituted system was sensitive to proton-conducting uncouplers. Liposomes prepared with partially purified calcium translocating protein were capable of accumulating calcium. The uptake of calcium in this system occurred as a result of an artificial proton gradient generated by the reduction of entrapped ferricyanide with ascorbate-benzoquinone serving as a hydrogen carrier. The addition of the ionophore A23187 caused efflux of accumulated calcium in both native and proteoliposomal-reconstituted system.     

Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.     

Reconstitution of Neutral Amino Acid Transport System from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Reconstitution of Neutral Amino Acid Transport Systems from Ehrlich Ascites Tumor Cells

Amino acid transport systems for alanine and leucine were reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1mM dithiothreitol, and 0.5 mM EDTA a mixture that solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue-staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valino-mycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Isolation, purification, and reconstitution of a proline carrier protein from Mycobacterium phlei.

Membrane vesicles from Mycobacterium phlei contain carrier proteins for proline, glutamine, and glutamic acid. The transport of proline is Na+ dependent and required substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. Electron microscopic observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by means of sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a molecular weight of 20,000 +/- 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by sulfhydryl reagents.     

Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.     

The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at ?70°C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high d-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific d-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extrac decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.     

Proton-motive force-driven D-galactose transport in plasma membrane vesicles from the yeast Kluyveromyces marxianus.

Galactose transport was studied in membrane vesicles, prepared by fusion of plasma membranes from the yeast Kluyveromyces marxianus with proteoliposomes containing beef heart cytochrome c oxidase as a proton-motive force-generating system. Sugar transport studies performed under nonenergized conditions revealed that, even at high protein to phospholipid ratios, not all vesicles contained a D-galactose-specific transporter. The amount of vesicles containing an active carrier proved to be proportional to the amount of plasma membrane protein present in the fusion mixture. By addition of a suitable electron donor system a proton-motive force of -160 mV could be generated, inside alkaline and negative. Moreover, D-galactose accumulation was observed. It was found that D-galactose accumulation was highly dependent on the phospholipid composition of the vesicles, whereas generation of a proton-motive force was not. Best results were obtained with vesicles prepared with Escherichia coli phospholipid, giving a galactose accumulation of 14 times. Uphill transport could be established under conditions where only the pH gradient or the electrical gradient was present. Moreover, kinetic analysis of the galactose transport activity in energized vesicles revealed influx with a Km value of 540 microM, which is in good agreement with the apparent affinity constant obtained with whole cells. These results establish that galactose transport of K. marxianus is a proton-motive force-driven process. Moreover it demonstrates that plasma membrane vesicles co-reconstituted with cytochrome c oxidase are a valuable resource for the analysis of proton-motive force-driven sugar transport systems of yeast.