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.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Solubilization of a functionally active proline carrier from membranes of Escherichia coli with an organic solvent.

A proline transport carrier was extracted from the membranes of Escherichia coli with acidic n-butanol. Vesicles reconstituted from the butanol extract and E. coli phospholipids and preloaded with K⁺ showed rapid uphill uptake of proline when energy was supplied as a membrane potential introduced by K⁺-diffusion via valinomycin. Proline uptake by the reconstituted vesicles, like that of intact cells and isolated membrane vesicles, was inhibited by 3,4-dehydroproline, SH reagents, and a proton conducting uncoupler. Reconstituted vesicles of mutants defective in proline transport showed little or no proline uptake. The proline carrier was partially purified from the extract and separated from the bulk of phospholipids on Sephadex LH-20.     

Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein

We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles.

The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.     

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.     

Electrogenic sucrose transport in developing soybean cotyledons

Addition of sucrose to a solution bathing an excised developing soybean cotyledon causes a transient depolarization of the membrane potential, as measured using standard electrophysiological techniques. The magnitude of the depolarization is dependent on the concentration of both sucrose and protons in a manner which suggests carrier mediation; this process has an apparent K_m for sucrose of about 10 millimolar. Agents interfering with the generation or maintenance of a proton electrochemical gradient eliminate these depolarizations. Electrogenic sugar transport is sensitive to sulfhydryl-modifying reagents; their effect appears to be through a direct interaction with the carrier protein and/or with the process establishing the proton electrochemical gradient across the plasma membrane. p-Chloromercuribenzene sulfonate appears to be a selective inhibitor of the carrier-mediated process itself.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF₁) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.     

A novel type of coupling between proline and galactoside transport in Escherichia coli.

Exit of thiomethylgalactoside (TMG) from preloaded cells induced the accumulation of proline. Likewise, proline exit stimulated TMG accumulation. Since a proton ionophore (carbonylcyanide-m-chlorophenylhydrazone) abolished these effects, a protonmotive force was implicated as the "intermediate" in the coupling reaction. The evidence suggests that the exit of TMG resulted in proton exit, which produced either a membrane potential (inside negative or a pH gradient (outside acid) or both. This inwardly directed protonmotive force provided the energy for proline entry and accumulation. Thus the energy coupling was not via a common transport protein but by proton movements which coupled the two separate H+-dependent transport processes.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.     

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).     

Chloride-dependence of amino acid transport in rabbit ileum

Chloride-dependence of influx across the brush-border membrane of distal rabbit ileum was examined for beta-alanine, 2-methylaminoisobutyric acid (MeAIB), leucine, lysine, proline and D-glucose. Influx of leucine at 2 mM and of D-glucose at 0.5 mM was chloride-independent indicating that substitution of isethionate for chloride has no unspecific effect on sodium gradient driven transport processes. In contrast influx of beta-alanine and MeAIB was totally dependent on the presence of chloride ions. In the absence of chloride, proline transport was reduced to 20% of its control level. This remaining transport can be accounted for by the function of the carrier of alpha-amino-monocarboxylic acids. Transport of leucine at 0.1 mM was reduced by absence of chloride. This is in accordance with the observation of leucine transport by the beta-alanine carrier. The kinetics of chloride and sodium activation of transport of MeAIB were examined at 1 mM MeAIB. Chloride activation was characterized by a Hill coefficient of 1 and a K1/2 of 23.5 mM, and sodium activation by a Hill coefficient of 2 and a K1/2 of 51 mM. Thus cotransport of chloride with an imino acid would be compatible with the known rheogenic nature of this transport. This study adds the imino acid carrier and the beta-alanine carrier to the group of chloride-dependent, epithelial amino acid transport systems.     

A new method for the preparation of delta 1-pyrroline 5-carboxylic acid and proline.

Hydroxylysine was oxidized with sodium metaperiodate and the major product characterized as Δ¹-pyrroline 5-carboxylic acid by the visible spectrum of its o-aminobenzaldehyde complex and by its reduction to proline by subsequent treatment with sodium borohydride. The reduction product was positively identified as proline by proton nuclear magnetic resonance spectroscopy and mass spectral analysis. The pH dependence for the preparation of Δ¹-pyrroline 5-carboxylic acid was determined by a study of the yields of proline obtained by variation of the pH values of the oxidative step. These observations support the hypothesis of intramolecular cyclization of α-aminoglutaric γ-semialdehyde as the second step in the reaction mechanism.     

Protein thermostabilization by proline substitutions

Many recent approaches involving site-directed mutants have succeeded in increasing the thermostability of proteins. It is well known that replacements with proline residues reduce the conformational degrees of freedom in the main polypeptide chain and thus can increase protein thermostabilization. We have studied protein thermostabilization by introducing proline substitutions in the homologous oligo-1,6-glucosidases from various Bacillus strains which grow within different temperature ranges. As a consequence, the `proline rule' was proposed for protein thermostabilization. The principle of this rule is that an increase in the frequency of proline occurrence at β-turns and/or an increase in the total number of hydrophobic residues can enhance protein thermostability. We have generated several lines of evidence supporting the theory from the comparative analysis of oligo-1,6-glucosidases in their primary and secondary structures and molecular properties, the X-ray crystal structure analysis of the Bacillus cereus oligo-1,6-glucosidase, and the enhancement in thermostability of the oligo-1,6-glucosidase by cumulative replacements with prolines. As a new finding from the studies, two specific sites (second positions at β-turns and N1 positions of α-helices) were found to be the most critical to protein thermostabilization dependent on several structural prerequisites for proline substitution.     

The action of tributyltin chloride on the uptake of proline and glutamine by intact cells of Escherichia coli.

Tributyltin chloride inhibits growth and uptake of glutamine and proline into intact cells of Escherichia coli. It causes efflux of the accumulated amino acids. A pH gradient generated in intact cells and everted membrane vesicles is dissipated by this compound. These effects do not require lipoic acid but are dependent on the presence of chloride, bromide, or iodide ions. We conclude that tributyltin chloride can catalyse a transmembrane OH- -anion exchange exchange reaction and that this is its mode of inhibition of the uptake of these amino acids. The response of proline and glutamine uptake to the inhibitor is similar and is consistent with the transport of both amino acids requiring an electrochemical gradient of protons.