Effect of lipid acyl chain length on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa

The sodium-dependent leucine transport system of Pseudomonas aeruginosa was reconstituted into liposomes of binary lipid mixtures of dilauroylphosphatidylethanolamine (di(12:0)PE)/phosphatidylcholine (PC) with cis-monounsaturated fatty acid chains (di(n:1)PC) (n = 14-22) or dioleoylphosphatidylethanolamine (di(18:1)PE)/di(n:1)PC (n = 14-22). Leucine carrier proteins can be activated with phosphatidylethanolamine, whereas activation does not occur in PC-reconstituted vesicles (Uratani, Y., and Aiyama, A. (1986) J. Biol. Chem. 261, 5450-5454). Na+-dependent counterflow was measured at 30 degrees C as reconstituted transport activity. Proteoliposomes containing di(12:0)PE exhibited high counterflow activity at the PC acyl carbon number (n) of 18 and 20 but no or low activity at n = 14, 16, and 22. On the other hand, proteoliposomes containing di(18:1)PE exhibited higher transport activity than those vesicles with di(12:0)PE and corresponding di(n:1)PC. A lipid mixture of di(18:1)PE and di(16:1)PC supported maximal activity. These results show that the leucine transport system of P. aeruginosa is dependent on the lipid acyl chain length and suggest that there exists optimal bilayer thickness for maximal carrier activity.     

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.     

Solubilization and reconstitution of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae

The citrate carrier of Klebsiella pneumoniae fermenting this substrate has been solubilized from the bacterial membranes with Triton X-100. The transport function was reconstituted by incorporation of the carrier into proteoliposomes using a freeze-thaw sonication procedure. Citrate uptake into these proteoliposomes required the presence of Na+ ions on the outside; the amount of citrate accumulated increased as the external Na+ concentration increased from 0 to 100 mM. Proteoliposomes preloaded with citrate catalyzed citrate counterflow when added to external [14C] citrate. Sodium ions were required for counterflow activity. The kinetics of citrate uptake, counterflow, or efflux were not influenced by an inside negative membrane potential, and the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone was without effect on citrate uptake. The data therefore suggest an electroneutral Na(+)-citrate symport mechanism for the transport of this tricarboxylic acid into K. pneumoniae.     

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.     

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.     

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.     

Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.     

Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.     

The phospholipid requirement for activity of the lactose carrier of Escherichia coli

The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids. The maximal activity was observed with the natural E. coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine. Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity. The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities. Trinitrophenylation of phosphatidylethanolamine in the E. coli proteoliposomes resulted in a marked reduction in lactose carrier activity. Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E. coli lipid. These results suggest that the amino group of the amino phospholipids (e.g. phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E. coli.     

Effect of different phospholipids on the reconstitution of two functions of the lactose carrier ofEscherichia coli

Summary The lactose carrier was extracted from membranes ofEscherichia coli and transport activity reconstituted in proteoliposomes containing different phospholipids. Two different assays f for carrier activity were utilized: counterflow and membrane potential-driven uptake. Proteoliposomes composed ofE. coli lipid or of 50% phosphatidylethanolamine–50% phosphatidylcholine showed very high transport activity with both assays. On the other hand, proteoliposomes containing asolectin, phosphatilcholine or 25% cholesterol/75% phosphatidylcholine showed good counterflow activity but poor membrane potentialdriven uptake. The discrepancy between the two types of transport activity in the latter group of three lipids is not due to leakiness to protons, size of proteoliposomes, or carrier protein content per proteoliposome. Apparently one function of the carrier molecule shows a broad tolerance for various phospholipids, while a second facet of the membrane protein activity requires very restricted lipid enviroment.     

Acetyl phosphatidylethanolamine in the reconstitution of ion pumps.

Acetyl phosphatidylethanolamine was compared with phosphatidylethanolamine in the reconstitution of several biological membrane activities with the following results. 1. The proton pump reconstituted with the purple membrane of Halobacterium halobium and acetyl phosphatidylethanolamine was quite active. However, some differences in the kinetic properties, particularly in the decay rate, were noted between vesicles reconsituted with phosphatidylethanolamine and acetyl phosphatidylethanolamine. 2. Acetyl phosphatidylethanolamine could not replace phosphatidylethanolamine in the reconstitution of a Ca-2 plus pump with ATPase isolated from sacoplasmic reticulum. However, inclusion of suitable amounts of stearylamine or oleylamine during reconstitution yielded acetyl phosphatidylethanolamine vesicles with Ca-2 plus translocation activity comparable to that of phosphatidylethanolamine vesicles. 3. A mixture of acetyl phosphatidylethanolamine and stearylamine or oleylamine substituted for phosphatidylethanolamine in the reconstitution of mitochondrial hydrophobic proteins to form vesicles that catalyze 32-Pi-ATP exchange. Since phosphatidylcholine is also required in this system, these findings point to two functions of phosphatidylethanolamine, one related to the specific properties of its amino group, the other to a structural role of its small polar head group. A hydrophobic alkylamine can fullfill the first function, acetyl phosphatidylethanolamine the second. 4. The importance of the charge was also observed in experiments with the reconstituted rutamycin-sensitive ATPase of mitochondria. After depletion of phospholipids from the hydrophobic proteins, ATPase activity and rutamycin sensitivity were restored only if a phospholipid as well as the appropriate charge were present.     

Transport systems for branched-chain amino acids in Pseudomonas aeruginosa.

The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.     

Effect of cholesterol on the branched-chain amino acid transport system of Streptococcus cremoris.

The effect of cholesterol on the activity of the branched-chain amino acid transport system of Streptococcus cremoris was studied in membrane vesicles of S. cremoris fused with liposomes made of egg yolk phosphatidylcholine, soybean phosphatidylethanolamine, and various amounts of cholesterol. Cholesterol reduced both counterflow and proton motive force-driven leucine transport. Kinetic analysis of proton motive force-driven leucine uptake revealed that the Vmax decreased with an increasing cholesterol/phospholipid ratio while the Kt remained unchanged. The leucine transport activity decreased with the membrane fluidity, as determined by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the fused membranes, suggesting that the membrane fluidity controls the activity of the branched-chain amino acid carrier.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Effect of internally loaded iodide, thiocyanate, and perchlorate on sodium-dependent iodide uptake by phospholipid vesicles reconstituted with thyroid plasma membranes: iodide counterflow mediated by the iodide transport carrier

Na+-dependent I- transport and I- counterflow were studied using phospholipid vesicles (P-vesicles) made of porcine thyroid plasma membranes and soybean phospholipid by sonication. 1) I- uptake by P-vesicles incubated in the presence of external Na+ was higher than that by P-vesicles incubated in choline+ instead of Na+. The vesicles exhibited Na+-dependent I- uptake. When P-vesicles were internally loaded with I- prior to incubation in Na+, a further increase in Na+-dependent I- uptake was observed, although the concentration of internal I- was very much higher than that outside. In the absence of external Na+, I- uptake by P-vesicles preloaded with I- was comparable to baseline uptake. 2) Na+-dependent I- uptake by P-vesicles not loaded with I- and enhanced Na+-dependent I- uptake by P-vesicles preloaded with I- were both inhibited by either of SCN- and ClO4- added outside the vesicles. 3) When P-vesicles were loaded with SCN- instead of I- and incubated in Na+, I- uptake by these vesicles was also higher than baseline Na+-dependent I- uptake. However, a ClO4- load did not result in an increase in I- uptake. These results indicate that Na+-dependent I- transport including Na+-dependent I- counterflow is specifically mediated by the thyroid I- carrier. SCN- - I- counterflow in addition to I- - I- counterflow occurs dependently on Na+, but ClO4- - I- counterflow does not.     

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla studies on (Na+ + K+)-activated ATPase, XXXIX.

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.     

Lipid requirement of the branched-chain amino acid transport system of Streptococcus cremoris

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.     

A cytochrome that can pump sodium ion

Previous studies have shown that the bacterium, Vitreoscilla, generates a respiratory-driven delta psi Na+. Two major respiratory electron transport proteins, NADH dehydrogenase (NADH:Quinone oxidoreductase), and cytochrome o terminal oxidase are candidates for the electrogenic Na+ pumping that mediates the delta psi Na+ formation. The NADH oxidase activity of the membranes was enhanced more by Na+ than by Li+. The NADH:Quinone oxidoreductase activity in the respiratory chain was enhanced by Na+ and Li+, whereas the quinol oxidase activity of cytochrome o was enhanced specifically by Na+, and not by Li+, K+, or choline. Purified cytochrome o, reconstituted into Na(+)-loaded liposomes in the right-side-out orientation, catalyzed a net Na+ extrusion when energized with Q1H2(1). In nonloaded inside-out proteoliposomes, this cytochrome catalyzed a net uptake of 22Na+ when energized with ascorbate/TMPD. Both Na(+)-pumping activities were inhibited by CN-. These results are consistent with the Vitreoscilla cytochrome o being a redox-driven Na+ pump.     

Modulatory effect of two cardioglycosides on reconstituted Na+/K(+)-ATPase in proteoliposomes.

1. Na,K-ATPase was extracted from Cavia cobaya kidneys, solubilized with nonionic detergent C12E8 (octaethyleneglycol dodecyl monoether) in mixed lipid-detergent-protein micelles. The Na,K-ATPase specific activity was 30-35 IU/mg protein. 2. The enzyme was reconstituted in vesicles, made of phosphatidylethanolamine and cholesterol: an enhancement of +60% in specific activity was obtained. 3. Two different vesicle-types were carried out: open liposomes (partially organized membranes) and closed liposomes. 4. Proteoliposomes were employed for measuring the modulatory effect of two cardioglycosides: ouabain and digoxin. 5. Inhibition of the Na,K-ATPase activity revealed apparent Ki of 1.25 microM for ouabain and 0.25 microM for digoxin in open liposomes, and apparent Ki of 0.75 microM for ouabain and of 1.75 microM for digoxin in closed liposomes. 6. Maximum enhancement of enzymatic activity was found at concentrations of 5-0.5 nM for ouabain and 5-1 nM for digoxin in open liposomes, and 25-1 nM for both digoxin and ouabain in closed liposomes.