Active Transport Of Proline Research Articles

Role of Asp187 and Gln190 in the Na+/proline symporter (PutP) of Escherichia coli

Asp187 and Gln190 were predicted as conserved and closely located at the Na(+) binding site in a topology and homology model structure of Na(+)/proline symporter (PutP) of Escherichia coli. The replacement of Asp187 with Ala or Leu did not affect proline transport activity; whereas, change to Gln abolished the active transport. The binding affinity for Na(+) or proline of these mutants was similar to that of wild-type (WT) PutP. This result indicates Asp187 to be responsible for active transport of proline without affecting the binding. Replacement of Gln190 with Ala, Asn, Asp, Leu and Glu had no effect on transport or binding, suggesting that it may not have a role in the transport. However, in the negative D187Q mutant, a second mutation, of Gln190 to Glu or Leu, restored 46 or 7% of the transport activity of WT, respectively, while mutation to Ala, Asn or Asp had no effect. Thus, side chain at position 190 has a crucial role in suppressing the functional defect of the D187Q mutant. We conclude that Asp187 is responsible for transport activity instead of coupling-ion binding by constituting the translocation pathway of the ion and Gln190 provides a suppressing mutation site to regain PutP functional activity.

Identification of Mammalian Proline Transporter SIT1 (SLC6A20) with Characteristics of Classical System Imino

Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na+-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the molecular level. We report here the functional characteristics and tissue distribution of Sodium/Imino-acid Transporter 1 (SIT1), which exhibits the properties of classical System IMINO. SIT1, the product of the slc6a20 gene, is a member of the SLC6 Na+- and Cl--dependent neurotransmitter transporter family whose function has remained unknown. When expressed in Xenopus oocytes, rat SIT1 mediated the uptake of imino acids such as proline (K0.5 approximately 0.2 mM) and pipecolate, as well as N-methylated amino acids (e.g. MeAIB, sarcosine). SIT1-mediated proline transport was pH-independent and insensitive to inhibition by alanine or lysine. Proline transport was Na+-dependent, Cl--stimulated, and voltage-dependent. Li+, but not H+, could substitute for Na+. Human SIT1 also functioned as a Na+-dependent proline transporter. Rat SIT1 mRNA was expressed in epithelial cells of duodenum, jejunum, ileum, stomach, cecum, colon, and kidney proximal tubule S 3 segments. SIT1 mRNA was also expressed in the choroid plexus, microglia, and meninges of the brain and in the ovary. Previous reports have documented the marked urinary hyperexcretion of proline in newborn rodents and man. We found that SIT1 was dramatically up-regulated in the kidneys of 3-day-old mice, accounting for the maturation of proline reabsorption in the mouse. The human slc6a20 gene coding SIT1 is an appropriate target for investigation of hereditary forms of iminoaciduria in man.

Effects of epidermal growth factor on placental amino acids uptake in pregnant rats.

To evaluate the effects of epidermal growth factor (EGF) on placental amino acids uptake, transport activities for L-proline, L-leucine, and L-alanine were kinetically examined in placental microvillous vesicles(PMV) obtained from pregnant rats administered with EGF(100 and 200 microg/kg/day) from day 18 to 21 of pregnancy. The Vmax of Na(+)-dependent proline uptake remarkably increased with a dose-dependent manner of EGF, while Km did not change. In contrast, Vmax and Km values of Na(+)-dependent and -independent alanine, and Na(+)-independent leucine uptake were not affected. These results suggested that EGF enhanced proline transport activity in placental microvillous membranes, resulting in an increase of proline concentration in the fetal blood. The selective up-regulation of proline uptake was considered to contribute to fetal growth by EGF.

Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B.

Exogenously provided proline has been shown to serve as an osmoprotectant in Bacillus subtilis. Uptake of proline is under osmotic control and functions independently of the known transport systems for the osmoprotectant glycine betaine. We cloned the structural gene (opuE) for this proline transport system and constructed a chromosomal opuE mutant by marker replacement. The resulting B. subtilis strain was entirely deficient in osmoregulated proline transport activity and was no longer protected by exogenously provided proline, attesting to the central importance of OpuE for proline uptake in high-osmolarity environments. The transport characteristics and growth properties of the opuE mutant revealed the presence of a second proline transport activity in B. subtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane protein. Database searches indicated that OpuE is a member of the sodium/solute symporter family, comprising proteins from both prokaryotes and eukaryotes that obligatorily couple substrate uptake to Na+ symport. The highest similarity was detected to the PutP proline permeases, which are used in Escherichia coli, Salmonella typhimurium and Staphylococcus aureus for the acquisition of proline as a carbon and nitrogen source, but not for osmoprotective purposes. An elevation of the osmolarity of the growth medium by either ionic or non-ionic osmolytes resulted in a strong increase in the OpuE-mediated proline uptake. This osmoregulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Primer extension analysis revealed the presence of two osmoregulated and tightly spaced opuE promoters. The activity of one of these promoters was dependent on sigma A and the second promoter was controlled by the general stress transcription factor sigma B.

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.

Parathyroid hormone regulation of proline uptake by cultured neonatal mouse osteoblastlike cells

Regulation of proline uptake by the synthetic amino-terminal fragment of bovine parathyroid hormone [bPTH-(1-34)] has been studied in confluent primary cultures of osteoblastlike cells isolated from neonatal mouse calvaria. The initial velocity of proline transport was increased by 85% in cultures treated with 24 nM bPTH-(1-34) for 6 h. Cycloheximide, at a concentration that inhibited protein synthesis by 97%, did not prevent this effect. However, adding the inhibitor during the first 1-2 h of hormone treatment did significantly reduce its magnitude. Exposure of cells to the inhibitor alone caused a time-dependent decrease in the basal rate of proline uptake. In the absence of protein synthesis, the maximal velocity (Vmax) of transport was 60% greater in cultures treated with 24 nM bPTH-(1-34) than in controls. The concentration of proline at which half-maximal transport occurred (Km) was unchanged. In cultures treated with cycloheximide alone, proline transport decreased as a first-order exponential with a half-life of 250-280 min. Parathyroid hormone significantly reduced this decline, increasing the half-life of proline transport activity about fourfold. These effects were duplicated by 1 mM DBcAMP. It is concluded that bPTH-(1-34) increases proline transport in osteoblastlike cells by decreasing the degradation of amino acid transport system A proteins. The hormone may also affect the synthesis of these molecules. These effects appear to be mediated by cAMP.

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase.

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO 3 - as the terminal acceptor, readily incorporated [14C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO 3 - , NO 2 - , N2O or O2. Light stimulated the accumulation of proline in these cells. The addition of NO 3 - to washed cells in light decreased the K m for proline from 40 μM to 5.7 μM. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H+/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions Δμpro increased but Δp decreased markedly above pH 7.0.

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.

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12.

Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

Active transport of proline by Coxiella burnetii.

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3.0 to 4.5, a pH range approximately that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent Km for proline transport from 45 microM to 15 microM, with the Vmax increasing from 3.6 pmol s-1 (mg dry wt)-1 to 4.8 pmol s-1 (mg dry wt)-1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. D-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.

Colicin V-treated Escherichia coli does not generate membrane potential.

Colicin V-treated Escherichia coli was inhibited in its capacity to carry out active transport of proline and was unable to generate a membrane potential. Colicin V also prevented membrane potential formation by isolated cytoplasmic membrane vesicles. We conclude that a primary effect of this colicin involves the cytoplasmic membrane as a target.

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli.

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.

Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei.

Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K(1), menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K(1). Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca(2+). These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.

Amino acid and sugar transport in Escherichia coli (ColIb) during abortive infection by bacteriophage T5

T5 bacteriophage cannot replicate in Escherichia coli containing the colicinogenic factor ColIb. We show that active transport of proline and glutamine begins to decline at about 10 min after infection, the same time at which macromolecular synthesis stops during abortive infection. Uptake of alpha-methylglucoside is stimulated, however, and this change is evident even by 5 min after infection. These changes in membrane function do not occur during infections that are productive because of mutations on the plasmid or phage. The results suggest that the abortive infection is caused by membrane depolarization.

Amino acid transport in chick embryo fibroblasts: Evidence for transcriptional regulation of transport following serum addition

AbstractChanges in amino acid transport activity by system A (a Na+‐dependent agency with affinity for a discrete group of neutral amino acids) caused by the addition of serum to serum‐deprived cultured chick embryo fibroblasts have been evaluated by measurements of 14C‐labeled L‐proline and α‐methylaminoisobutyric acid uptake under conditions approaching initial entry rates. Dialysed serum was as effective as undialysed serum in stimulating amino acid transport. This effect was inhibited by 7 μM cycloheximide, by 80 nM actinomycin D and by 40 μM cordycepin, but not by 0.3 mM cytosine arabinoside. Cultured avian fibroblasts previously incubated in a cycloheximide‐containing medium (phase of inhibited translation) in the presence of serum, subsequently exhibited a net increase of proline transport activity when transferred to a medium containing actinomycin D (phase of inhibited transcription). Omission of serum in the cycloheximide‐phase prevented the increase of transport activity during subsequent incubation in the actinomycin D‐phase; omission of serum in the actinomycin D‐phase allowed a shorter and less pronounced increase of transport activity than in the presence of serum. Additions of actinomycin D or cycloheximide slightly increased the rate of decay of amino acid transport caused by serum withdrawal. These observations suggest that in cultured avian fibroblasts, serum modulates the activity of transport system A by a mechanism acting at the transcription level.

Removal by Bovine Serum Albumin of Fatty Acids from Membrane Vesicles and Its Effect on Proline Transport Activity in Escherichia coli1

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP + by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of E. coli , W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain et al. , Biochem. J. (1977) 166 , 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis et al. (FEBS Lett. (1977) 75 , 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of E. coli .

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

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

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.