Light-activated amino acid transport in Halobacterium halobium envelope vesicles.

Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na-gradient. Sodium-activated amino acid transport for 18 of these amino acids has been shown to occur in direct response to the protonmotive force generated. Glutamate is transported only in response to a sodium gradient. Michaelis constants for the uptake of these amino acids are close or identical whether the amino acids are accumulated in response to a sodium gradient or a protonmotive force (i.e., electrical gradient). On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids, i.e., arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.     

Regulation of the glutamate-glutamine transport system by intracellular pH in Streptococcus lactis.

Various methods of manipulation of the intracellular pH in Streptococcus lactis result in a unique relationship between the rate of glutamate and glutamine transport and the cytoplasmic pH. The initial rate of glutamate uptake by S. lactis cells increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4. A further increase of the cytoplasmic pH to 8.0 was without effect on transport. The different levels of inhibition of glutamate and glutamine transport at various external pH values by uncouplers and ionophores, which dissipate the proton motive force, can be explained by the effects exerted on the intracellular pH. The dependence of glutamate transport on the accumulation of potassium ions in potassium-filled and -depleted cells is caused by the regulation of intracellular pH by potassium movement.     

Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.     

Transport of glutamine by Streptococcus bovis and conversion of glutamine to pyroglutamic acid and ammonia

Streptococcus bovis JB1 cells energized with glucose transported glutamine at a rate of 7 nmol/mg of protein per min at a pH of 5.0 to 7.5; sodium had little effect on the transport rate. Because valinomycin-treated cells loaded with K and diluted into Na (pH 6.5) to create an artificial delta psi took up little glutamine, it appeared that transport was driven by phosphate-bond energy rather than proton motive force. The kinetics of glutamine transport by glucose-energized cells were biphasic, and it appeared that facilitated diffusion was also involved, particularly at high glutamine concentrations. Glucose-depleted cultures took up glutamine and produced ammonia, but the rate of transport per unit of glutamine (V/S) by nonenergized cells was at least 1,000-fold less than the V/S by glucose-energized cells. Glutamine was converted to pyroglutamate and ammonia by a pathway that did not involve a glutaminase reaction or glutamate production. No ammonia production from pyroglutamate was detected. S. bovis was unable to take up glutamate, but intracellular glutamate concentrations were as high as 7 mM. Glutamate was produced from ammonia via a glutamate dehydrogenase reaction. Cells contained high concentrations of 2-oxoglutarate and NADPH that inhibited glutamate deamination and favored glutamate formation. Since the carbon skeleton of glutamine was lost as pyroglutamate, glutamate formation occurred at the expense of glucose. Arginine deamination is often used as a taxonomic tool in classifying streptococci, and it had generally been assumed that other amino acids could not be fermented. To our knowledge, this is the first report of glutamine conversion to pyroglutamate and ammonia in streptococci.     

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.     

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.     

Return of Streptococcus faecalis DNA cloned in Escherichia coli to its original host via transformation of Streptococcus sanguis followed by conjugative mobilization.

It is possible to select transmembrane potential (delta psi)-altered mutants in Streptococcus pneumoniae on the basis of their resistance to the antifolate methotrexate. Comparison of such a mutant strain ( amiA9 ) with its parent was used to evaluate the role of delta psi in the uptake of certain amino acids. The delta psi-dependent uptake of isoleucine, leucine, valine, and asparagine showed a reduced maximum velocity of uptake, and decrease in the transport constant of the energy-dependent, delta psi-independent uptake of lysine, methionine, and glutamine was observed. No reduction of the intracellular pool of ATP or of lactate excretion could be detected in the mutant strain. Moreover, studies on membrane preparations suggest that the phenotype expressed by the amiA mutation is not a consequence of alteration of its ATPase activity or susceptibility to N,N'-dicyclohexylcarbodiimide. Therefore, it is unlikely that the amiA mutation affects the H+ F1F0 ATPase which is involved in the establishment of the proton motive force in anaerobic bacteria. We propose that another function contributes to delta psi in S. pneumoniae. The amiA gene may be the structural gene of that function.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems

The substrate specificities of the amino acid transport systems of vacuoles of the yeast, Saccharomyces cerevisiae, were investigated using purified vacuolar-membrane vesicles (Ohsumi, Y., and Anraku, Y. (1981) J. Biol. Chem. 256, 2079-2082). Ten amino acids: arginine, lysine, histidine, phenylalanine, tryptophan, tyrosine, glutamine, asparagine, isoleucine, and leucine, were taken up actively into the vesicles. Kinetic studies indicated the presence of seven independent H+/amino acid antiport systems with narrow substrate specificity, which were all driven by a proton motive force established by ATP hydrolysis. The Kt and Vmax values, and the specific inhibitors for the arginine, arginine-lysine, histidine, phenylalanine-tryptophan, tyrosine, glutamine-asparagine, and isoleucine-leucine transport systems were determined.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport.

The maximum specific growth rate of Streptococcus lactis and Streptococcus cremoris on synthetic medium containing glutamate but no glutamine decreases rapidly above pH 7. Growth of these organisms is extended to pH values in excess of 8 in the presence of glutamine. These results can be explained by the kinetic properties of glutamate and glutamine transport (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:2755-2761, 1987). At alkaline pH the rate of growth in the absence of glutamine is limited by the capacity to accumulate glutamate due to the decreased availability of glutamic acid, the transported species of the glutamate-glutamine transport system. Kinetic analysis of leucine and valine transport shows that the maximal rate of uptake of these amino acids by the branched-chain amino acid transport system is 10 times higher in S. lactis cells grown on synthetic medium containing amino acids than in cells grown in complex broth. For cells grown on synthetic medium, the maximal rate of transport exceeds by about 5 times the requirements at maximum specific growth rates for leucine, isoleucine, and valine (on the basis of the amino acid composition of the cell). The maximal rate of phenylalanine uptake by the aromatic amino acid transport system is in small excess of the requirement for this amino acid at maximum specific growth rates. Analysis of the internal amino acid pools of chemostat-grown cells indicates that passive influx of (some) aromatic amino acids may contribute to the net uptake at high dilution rates.     

Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by beta-hydroxyaspartate and cysteic acid but not by alpha-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. gltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.     

Uterine uptake of amino acids and placental glutamine--glutamate balance in the pregnant ewe

The uterine uptake of amino acids was studied in 10 pregnant sheep with gestational ages of 114-146 days. After recovery from surgery, arterial and uterine venous samples were drawn simultaneously via indwelling catheters and analysed for amino acid and oxygen content. In seven ewes, amino acid concentrations were measured by a chromatographic technique. In four ewes, glutamate and glutamine arterio-venous differences across the uterine and umbilical circulations were measured by an enzymatic method. The uptake of neutral and basic amino acids was 66 mumol/mmol O2 and 17.3 mumol/mmol O2, respectively. Comparison of uterine and umbilical uptake shows that the bulk of the neutral and basic amino acids taken up by the pregnant uterus are transferred to the fetus. there was no significant uptake of acidic amino acids (i.e. glutamate, aspartate and taurine). glutamate was delivered from the fetus to the placenta but excretion of glutamate into the uterine circulation was negligible. Glutamine and asparagine were delivered to the fetus in amount which were two to three times larger than the placental uptake of glutamate and aspartate. Therefore placental conversion of exogenous glutamate and aspartate to glutamine and asparagine cannot account entirely for the fetal uptake of these amino acids.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].     

Branched-Chain Amino Acid Transport in Cytoplasmic Membranes of Leuconostoc mesenteroides subsp. dextranicum CNRZ 1273

Membrane vesicles of Leuconostoc mesenteroides subsp. dextranicum fused with proteoliposomes prepared from Escherichia coli phospholipids containing beef heart cytochrome c oxidase were used to study the transport of branched-chain amino acids in a strain isolated from a raw milk cheese. At a medium pH of 6.0, oxidation of an electron donor system comprising ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and horse heart cytochrome c resulted in a membrane potential (Deltapsi) of -60 mV, a pH gradient of -36 mV, and an l-leucine accumulation of 76-fold (Deltamu(Leu)/F = 108 mV). Leucine uptake in hybrid membranes in which a Deltapsi, DeltapH, sodium ion gradient, or a combination of these was imposed artificially revealed that both components of the proton motive force (Deltap) could drive leucine uptake but that a chemical sodium gradient could not. Kinetic analysis of leucine (valine) transport indicated three secondary transport systems with K(t) values of 1.7 (0.8) mM, 4.3 (5.9) muM, and 65 (29) nM, respectively. l-Leucine transport via the high-affinity leucine transport system (K(t) = 4.3 muM) was competitively inhibited by l-valine and l-isoleucine (K(i) and K(t) values were similar), demonstrating that the transport system translocates branched-chain amino acids. Similar studies with these hybrid membranes indicated the presence of high-affinity secondary transport systems for 10 other amino acids.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

凤眼莲根分泌物氨基酸组分对根际肠杆菌属F2细菌的趋化作用

凤眼莲(Eichhornia crassipes)的根分泌物中含有Met等多种氨基酸,其中Met、GABA、Gly、Ala、Asp、Ser、Val和Leu(10^-7～10^-2mol·L^-1)均对凤眼莲的根际肠杆菌属F₂(Enterobacter sp.F₂)细菌有强烈的正趋化作用;Glu、Thr和His(10^-7～10^-3mol·L^-1)也对该菌有一定的正趋化作用;而Lys、Cys、Arg、Tyr、Pro、Asn、Gln、Ile、Phe和Typ则对该菌表现出一定的负趋化作用.对细菌的正趋化作用存在一个趋化物的最适浓度范围.具有正趋化作用的氨基酸在凤眼莲根际的浓度都较高,而具有负趋化作用的浓度则较低,这正是凤眼莲与该根际细菌结合为根际微生态系统的原因之一.     

Neutral amino acid transport by membrane vesicles of Streptococcus cremoris is subject to regulation by internal pH.

The pH dependence of transport of the neutral amino acids L-serine and L-alanine by membrane vesicles of Streptococcus cremoris have been studied in detail. The rates of four modes of facilitated diffusion (e.g., influx, efflux, exchange, and counterflow) of L-serine and L-alanine increase with increasing H+ concentration. Rates of artificially imposed electrical potential across the membrane (delta psi)-driven transport of L-serine and L-alanine show an optimum at pH 6 to 6.5. Under similar conditions, delta psi- and pH gradient across the membrane (delta pH)-driven transport of L-leucine is observed within the pH range studied (pH 5.5 to 7.5). The effect of ionophores on the uptake of L-alanine and L-serine has been studied in membrane vesicles of S. cremoris fused with proteoliposomes containing beef heart mitochondrial cytochrome c oxidase as a proton motive force (delta p)-generating system (Driessen et al., Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985). An increase in the initial rates of L-serine and L-alanine uptake is observed with decreasing pH, which is not consistent with the pH dependency of delta p. Nigericin, an ionophore that induced a nearly complete interconversion of delta pH into delta psi, stimulated both the rate and the final level of L-alanine and L-serine uptake. Valinomycin, an ionophore that induced a collapse of delta psi with a noncompensating increase in delta pH, inhibited L-alanine and L-serine uptake above pH 6.0 more efficiently than it decreased delta p. Experiments which discriminate between the effects of the internal pH and the driving force (delta pH) on solute transport indicate that at high internal pH the transport systems for L-alanine and L-serine are inactivated. A unique relation exists between the internal pH and the initial rate of uptake of L-serine and L-alanine with an apparent pK of 7.0. The rate of L-alanine and L-serine uptake decreases with increasing internal pH. The apparent complex relation between the delta p and transport of L-alanine and L-serine can be explained by a regulatory effect of the internal pH on the activity of the L-serine and L-alanine carriers.     

A family of yeast proteins mediating bidirectional vacuolar amino acid transport

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.