PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.     

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na⁺ dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K_M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K_M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na⁺- free media or after treatment with ouabain suggested that system 1 is Na⁺ independent and that system 2 is Na⁺ dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

Uptake of 14C-α-aminoisobutyric acid by Phaseolus vulgaris

Specific uptake (S.U.) of α-aminoisobutyric acid ([1-¹⁴C]AIB), a non-metabolizable neutral amino acid analog, by dwarf bush bean plants (Phaseolus vulgaris cv Top Crop) demonstrated wide differences in active transport between various plant organs. The kinetic and timed uptake data reported were expressed as S.U. because this corrects for the diffusion of AIB which is part of the total AIB uptake process. Roots accumulated AIB to concentrations up to 18 times and leaf disks to twice those of the incubation medium. Stem tissue showed very little uptake, if any, that could not be accounted for by simple diffusion or water free space. Although initial rate kinetic studies demonstrated the presence of a normal transport system, timed uptake studies revealed greatly decreased transport by etiolated plants, suggesting a relationship between active transport and the lack of photosynthate. The reproducibility of the AIB uptake pattern by mature roots strongly supports the concept that the transport of neutral amino acids is biphasic and suggested one or more carrier systems are inducible by either low intracellular concentrations or repressed by high intracellular concentrations of the amino acid.     

Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.     

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves: II. Evidence for Multiple Aliphatic, Neutral Amino Acid Symports

Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K_m and K_i values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the β-carbon position that places a dense electron cloud close to the α-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity.     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-β-lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. ΔpH-Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N′-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate-sensitive K⁺-Mg²⁺-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Incorporation of 14C-amino acids by malarial plasmodia (Plasmodium iophurae). VI. Changes in the kinetic constants of amino acid transport during infection

The majority (10 of 17) of amino acids tested entered the mature duck erythrocyte by a saturable, non-uphill transport system, whereas for the erythrocyte-free malarial parasite, Plasmodium lophurae, the converse was true: most amino acids entered the parasite by simple diffusion. Only five amino acids (glutamic and aspartic acids, cysteine, lysine, arginine) showed mediated entry into P. lophurae. The pattern of mediated amino acid transport into the duck erythrocyte was altered upon infection, e.g., either entry was by diffusion or there was a reduced affinity for the amino acid. Transport characteristics similar to those found in the malaria-infected erythrocyte were produced by treating normal duck red cells with a cell-free extract of malaria-infected erythrocytes and quinine (a depressor of red cell ATP). It is suggested that depletion of host cell ATP as well as elaboration of as yet unidentified substances by the parasite promote the changes in permeability seen in the malaria-infected cell.     

Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

Transport and metabolism of N-δ-chloroacetyl-l-ornithine by Saccharomyces cerevisiae

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids (M. Grenson, C. Hou, and M. Crabeel, 1970,J. Bacteriol. 103, 770–777; J. Rytka, 1975,J. Bacteriol.121, 562–570; C. Darte and M. Grenson, 1975,Biochem. Biophys. Res. Commun.67, 1028–1033). We have previously demonstrated that this transport system can be inhibited by the amino acid, N-δ-chloroacetyl-l-ornithine (NCAO) (F. S., Larimore and R.J. Roon, 1978,Biochemistry17, 431–436). In the present study radiolabeled NCAO was synthesized and its transport and metabolism studied. Under initial rate conditions: (a) NCAO was transported by the general amino acid transport system with a K_m of 52 μm, a V of 32 nmol/min/mg cells, and a pH optimum of 5.0; (b) the V for NCAO transport in gap mutants, which lack the general amino acid transport system, was approximately 1% of that observed with wild-type cells; (c) the V for NCAO in cells deprived of glucose was less than 5% of that observed when glucose was present. NCAO was transiently concentrated more than 1000-fold by yeast cells when glucose served as an energy source. The internal pool of NCAO was metabolized by the yeast cells and the products were excreted. When 100 μm [¹⁴C]NCAO was incubated with a yeast cell suspension for 8 h, more than 95% of the compound was converted into two ninhydrin-negative excretory products. The effect of NCAO on the growth of yeast cells was determined. Wild-type strains did not grow when 1 mm NCAO was present in the medium. The growth of gap mutants was not inhibited by 1 mm NCAO.     

Lactate and Amino Acid Catabolism in the Cheese-Ripening Yeast Yarrowia lipolytica

The consumption of lactate and amino acids is very important for microbial development and/or aroma production during cheese ripening. A strain of Yarrowia lipolytica isolated from cheese was grown in a liquid medium containing lactate in the presence of a low (0.1×) or high (2×) concentration of amino acids. Our results show that there was a dramatic increase in the growth of Y. lipolytica in the medium containing a high amino acid concentration, but there was limited lactate consumption. Conversely, lactate was efficiently consumed in the medium containing a low concentration of amino acids after amino acid depletion was complete. These data suggest that the amino acids are used by Y. lipolytica as a main energy source, whereas lactate is consumed following amino acid depletion. Amino acid degradation was accompanied by ammonia production corresponding to a dramatic increase in the pH. The effect of adding amino acids to a Y. lipolytica culture grown on lactate was also investigated. Real-time quantitative PCR analyses were performed with specific primers for five genes involved in amino acid transport and catabolism, including an amino acid transporter gene (GAP1) and four aminotransferase genes (ARO8, ARO9, BAT1, and BAT2). The expression of three genes involved in lactate transport and catabolism was also studied. These genes included a lactate transporter gene (JEN1) and two lactate dehydrogenase genes (CYB2-1 and CYB2-2). Our data showed that GAP1, BAT2, BAT1, and ARO8 were maximally expressed after 15 to 30 min following addition of amino acids (BAT2 was the most highly expressed gene), while the maximum expression of JEN1, CYB2-1, and CYB2-2 was delayed (≥60 min).     

α-Methyl-l-glutamic acid uptake by high affinity dicarboxylic amino acid transport system in Streptococcus faecalis

The transport of α-methyl-l-glutamic acid was studied in Streptococcus faecalis. Energy-dependent uptake against substantial concentration gradients was observed. Kinetic experiments indicated that, in contrast to l-glutamic acid, only a single catalytic component (high affinity) and a diffusion controlled process participated in α-methyl-l-glutamic acid uptake. At concentrations up to 10 mM, α-methylglutamate transport was almost completely abolished in a mutant strain lacking a high affinity dicarboxylic amino acid transport system. In competition experiments, α-methylglutamic acid antagonized glutamate uptake via the high affinity system, and only slightly via the low affinity system. Column chromatography of cell extracts showed that very little (approx. 5%) of the accumulated amino acid was converted to metabolites during short term incubations. These studies indicate that, at concentrations up to 3–5 mM, α-methyl-l-glutamic acid can be used as a specific, relatively metabolically inert substrate of the high affinity dicarboxylic amino acid transport system in S. faecalis.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Analysis of Cationic Amino Acid Transport Activity in Canine Lens Epithelial Cells

Cationic amino acid transport activity in a canine lens epithelial cells (LEC) line was investigated. The transporter activity of arginine was 0.424 ± 0.047 nmol/mg protein min, while the presence of N-ethylmaleimide, an inhibitor of the canine cationic amino acid transporter (CAT), reduced transport activity by 30%. A full-length cDNA sequence of canine CAT1 was 2558 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence of canine CAT1 showed similarities of 92.1% and 88.6% to those of the human and mouse, respectively. Western blot analysis detected a band at 70 kDa in a membrane protein sample of LEC. RT-PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined.     

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves: I. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems

Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. ΔpH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent K_m values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. ΔpH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (ΔΨ) on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When ΔΨ was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative ΔΨ, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing ΔΨ on the kinetics of alanine transport altered V_max with no apparent change in K_m. Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N′-dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline. Although each symport was relatively specific for a given group of amino acids, each system exhibited some crossover specificity for amino acids in other groups.