Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
List of types of proteins wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Cell membrane wikipedia , lookup
Protein structure prediction wikipedia , lookup
Endomembrane system wikipedia , lookup
Magnesium transporter wikipedia , lookup
Genetic code wikipedia , lookup
Plant Physiol. (1990) 94, 268-277 0032-0889/90/94/0268/1 0/$01 .00/0 Received for publication April 6, 1990 Accepted June 11, 1990 ApH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves 1. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems Zhen-Chang Li and Daniel R. Bush* Photosynthesis Research Unit, U.S. Department of Agriculture, Agricultural Research Service (D.R.B.), and Department of Plant Biology (D.R.B., Z. -C.L.), University of Illinois, Urbana, Illinois 61801 ABSTRACT port those amino acids to the heterotrophic tissues that are dependent upon imported carbon and nitrogen for normal growth and development. Amino acid transport in plants is frequently associated with accumulation against a significant free energy gradient across the plasma membrane. Several lines of evidence have linked this active transport process to the proton electrochemical difference generated by the plasma membrane proton-pumping ATPase (10, 13, 15, 20, 23-25) and recently, Bush and Langston-Unkefer (6) provided the first in vitro evidence demonstrating proton-amino acid symport activity in isolated plasma membrane vesicles. Most amino acid transport experiments have been conducted with intact tissues or suspension culture cells (2, 11, 13, 15, 17, 18, 20, 25). Although these systems have provided important insight into the nature of amino acid transport, this experimental approach is limited by a number of problems which can compromise investigations of membrane bound transport systems. For example, rapid metabolism of the transported amino acid can lead to an overestimate of transport stoichiometries and metabolically linked feedback control systems may significantly influence transport activity (3, 20, 24). Furthermore, differential rates of accumulation by the various cell types present in an intact tissue and the existence of ill-defined diffusion barriers may further complicate interpretation. Nevertheless, results from studies with intact tissues and cells have lead to the general conclusion that amino acid transport is mediated by specific carrier proteins (2, 7, 11, 13, 14, 20, 21, 25), although progress in characterizing these transport systems has been slow. Currently, our knowledge of amino acid transport at the membrane level is quite limited. This is due not only to the difficulty encountered in the solubilization and reconstitution of membrane bound transport proteins, but also to the fact that the amino acid transport systems in plants are not well understood (20). For example, an amino acid transport protein has yet to be identified and, furthermore, even the number of independent transport systems is still unknown (2, 7, 14, 18, 20). These observations suggest a simpler experimental system may be more suitable when examining amino acid transport at the membrane level. In the work reported here, we used highly purified plasma membrane vesicles from sugar beet leaves to study amino acid transport. Isolated membrane vesicles eliminate many of Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Westem) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. ApH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent Km values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. ApH-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 (A*) on ApHdependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When A' was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative A+, 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 AI on the kinetics of alanine transport altered Vmax with no apparent change in Km. 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. The concentration of amino acid nitrogen in the sieve tube sap of castor bean can be as high as 230 mm. In contrast, the concentration of inorganic nitrogen in the same sap is less than 2 mM (22). In this system, as in many other higher plants, amino acids are the primary form of transported nitrogen. This is especially true for those plants that incorporate reduced nitrogen into amino acids in the leaf and subsequently trans268 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. PROTON-AMINO ACID TRANSPORT the experimental problems associated with intact tissues and allow us to manipulate the composition of both the intra- and extravesicular solutions. The results presented here, based on this experimental approach, show that the transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine is mediated by specific carriers and that the driving force for this process is a proton electrochemical potential difference. Furthermore, an analysis of inter-amino acid competition experiments suggests at least four amino acid transport systems can be resolved. MATERIALS AND METHODS Plant Material Sugar beet (Beta vulgaris L. cv Great Western) seedlings in a 1:1 mixture of vermiculite and sand. When the seedlings were 1 inch tall, they were transferred to a hydroponic system, 2 plants per 13 L, containing one-half strength Hoagland solution (12) and 1 mM NaCl. Plants were maintained under a 10 h photoperiod (400 ,uE m-2 s-1) at 24°C, 70% RH, and 14 h night period at 18°C, 70% RH. Only fully expanded leaves were harvested for experimental analysis. were grown Plasma Membrane Vesicle Isolation Plasma membrane vesicles were prepared at 4°C as described previously (4). Briefly, 35 g of leaves were homogenized with a polytron homogenizer in 350 mL of 240 mM sorbitol, 50 mm Hepes, 10 mM KC1, 3 mm EGTA, 3 mM DTT, and 1% of BSA (final pH was adjusted to 8.0 with solid 1,3-bis[tris(hydroxymethyl)methylamino]propane). The homogenate was filtered through four layers of cheesecloth and the filtrate was centrifuged at 10,000g for 10 min. The supernatant was then centrifuged at 50,000g for 45 min. The resulting pellets, containing microsomal vesicles, were resuspended in 3 mL 340 mm sorbitol, 5 mM K-phosphate buffer (pH 7.8) and 0.1 mm DTT. Plasma membrane vesicles were purified from the resuspended microsomes with the aqueous phase partitioning method as described elsewhere (4). After the final phase separation, the plasma membrane vesicles were washed in 30 mL resuspension buffer containing 330 mM sorbitol, 2 mm Hepes (pH to 8.0 as above), 10 mm KCI, and 0.1 mM DTT and pelleted with a 45 min centrifugation at 50,000g. The final vesicle pellet was resuspended in 1 mL resuspension buffer (z5 mg protein/mL). Amino Acid Transport Assay All amino acids were L-form in this study. Amino acid transport was carried out in an acidic transport solution (200400 tL) at 10°C. The transport solution contained resuspension buffer whose pH was adjusted to 6.0 with solid Mes, 5 ,AM valinomycin, 0.1 to 0.5 ,Ci of labeled amino acid and unlabeled amino acid to the desired final concentration. Experiments were initiated by diluting 10 to 20 ,L of the plasma membrane vesicles into the acidic transport solution. At predetermined time points membrane vesicles were collected and 269 washed with the Millipore' filtration technique as previously described (4). Accumulated radioactivity was measured with scintillation spectrometry. ApH-Dependent transport was calculated from the difference between the transport activity in the absence and that in the presence of 5 ALM CCCP2. Each experiment was repeated at least three times with multiple time points sampled for each treatment. All rates were taken from the best fit lines generated with linear regression analysis. The results presented are from a single representative experiment. It has been suggested that, for the analysis of amino acid transport kinetics and inter-amino acid competition, it is preferable to avoid extremely high substrate concentrations (20, 25). This is especially true in competition experiments if the competing amino acid shifts the total amino acid concentration into a new concentration range (20). Therefore, in the experiments reported here, substrate and competitor amino acid concentrations have been maintained near apparent Km values for transport systems that are active in the 1 mM concentration range. We have not explored transport systems that may be active at concentrations exceeding 2 mm. Inhibition of Amino Acid Transport by Protein Modifiers To examine the effect of protein modifiers on amino acid transport activity, 10 to 25 ,L plasma membrane vesicles were mixed with an equal volume of uptake buffer containing twice the desired inhibitor concentration. Control samples were treated in parallel in the absence of inhibitors. After incubating at 10°C for 15 min, the samples were diluted into the standard transport solution and transport activity was measured as described above. Inter-Amino Acid Competition Experiments Transport solutions were prepared as above except that, in addition to the radiolabeled amino acid whose transport was monitored, a competing amino acid was included at 1 or 10 times the concentration of the transported substrate. Preliminary experiments showed that the relative activity of specific amino acids as transport antagonists was unaltered when the results were computed as either a function of total transport activity or as a function of ApH-dependent transport activity. Therefore, to conserve radiolabeled stocks, the results reported here are based on total transport activity. Protein Determinations Proteins were determined by the method of Markwell et al. (16) after solubilization in 0.02% Na-deoxycholate and precipitation in 6.2% TCA. The detergent and TCA step quan'Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Illinois and does not imply its approval to the exclusion of other products or vendors which may be suitable. 2 Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DEPC, diethyl pyrocarbonate; AAH', proton electrochemical potential difference; A'I, membrane potential Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 94, 1990 Li AND BUSH 270 titatively recover low concentrations of membrane protein (1). RESULTS c tE 0- Carrier Mediated and ApH-Dependent Amino Acid Transport It has been shown previously that plasma membrane vehicles isolated from mature leaves of sugar beet by the aqueous two phase partitioning method are highly purified (4). When these plasma membrane vehicles (equilibrated at pH 8.0) were diluted into an acidic transport solution at pH 6.0, a stable trans-membrane proton gradient was established and this proton electrochemical potential was successfully used to drive a proton-sucrose symport (4). In this report, the same experimental approach was used to examine proton-amino acid symport activity. Amino acid transport was linear for at least 4 min and exhibited the same ApH-dependence as previously demonstrated for sucrose flux and for alanine transport in zucchini plasma membrane vehicles (4, 6). Although transport was linear as a function of time over the course of each experiment, occasionally a positive, nonzero intercept of the y axis was observed. We attribute this to a rapid, initial influx through porters that are poised for substrate binding and translocation. ApH-dependent alanine, leucine, glutamine, and glutamate transport exhibited saturable, concentration dependent influx (Figs. 1, 2, 3, and 4) that is consistent with carrier-mediated transport. The Lineweaver-Burk plots of these data were linear, yielding apparent Km values of 272, 346, 258, and 1981 AM, respectively (Figs. 1, 2, 3, and 4, insets). In contrast, ApH-dependent isoleucine and arginine transport was bi- _ tCLE o c Co Y-' Q a C- 4 E S 0 200 400 600 800 1000 Alanine (IM) Figure 1. Concentration dependence of ApH-dependent alanine transport into plasma membrane vesicles isolated from sugar beet leaves. Concentrated plasma membrane vesicles were diluted into an acidic transport solution and subsequently collected and washed with the Millipore filtration technique. ApH-Dependent transport was defined as the difference between transport activity in the absence and in the presence of of 5 AM CCCP. The inset shows a LineweaverBurk plot of the data with an apparent Km = 276 + 46 AM. Coe 0. .- 0 C E 00 Cj 0 200 400 600 Leucine (pM) 800 1000 Figure 2. Concentration dependence of ApH-dependent leucine transport into plasma membrane vesicles. Apparent Km = 346 + 25 JIM. phasic (Figs. 5A and 6A) and the Lineweaver-Burk plots of these data yielded evidence of high affinity (apparent Km values 140 Mm) and low affinity (apparent Km values, 1.61.9 mM) transport systems, suggesting more than one carrier is capable of transporting these two amino acids (Figs. 5B and 6B). ~ Electrogenicity of Alanine Transport Since ApH-dependent amino acid transport is coupled to the cotransport of protons into the vesicle, this transport process should be electrogenic. To explore this possibility, the affect of membrane potential on ApH-dependent amino acid transport was examined. The membrane potential of the isolated plasma membrane vesicles was altered with potassium gradients and valinomycin. In the presence of 10 mm KCI on both sides of the vesicle membrane a low rate of alanine transport was recorded (Fig. 7A). If, however, valinomycin was included in the transport solution, the resulting increase in potassium conductance clamped the membrane potential at zero and a fourfold stimulation in transport activity was observed (Fig. 7A). When KCl-loaded vesicles were diluted into a potassium free transport solution in the presence of valinomycin, a negative membrane potential developed, as previously demonstrated (5), due to the steep potassium diffusion potential. Under these conditions, alanine transport was stimulated sixfold (Fig. 7A). We conclude from these results that alanine transport was electrogenic. Similar changes in transport activity as a function of membrane potential were also noted for leucine. A Lineweaver-Burk plot of these data showed that the apparent Km for alanine was unaffected by changes in the membrane potential (Fig. 7B). Inhibition of Amino Acid Transport by Protein Modifiers Several reagents that modify protein amino acid residues were tested for their ability to inhibit ApH-dependent amino Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. ' PROTON-AMINO ACID TRANSPORT oE * 0. Co E- 0 0_ = E 200 400 600 800 Glutamine (pM) Figure 3. Concentration dependence of ApH-dependent glutamine transport into plasma membrane vesicles. Apparent Km = 258 + 59 JIM. acid transport. DEPC was the only compound that inhibited the transport of Ala, Leu, Gln, Glu, and Ile. In contrast, the transport of these amino acids was insensitive to 15 min pretreatments at 10°C with N-ethylmalemide (2.0 mM), 4,4'diisothiocyano-2,2'-stilbene disulfonic acid (2.0 mM), p-chloromercuribenzenesulfonic acid (1.0 mM), N,N'-dicyclohexylcarbodiimide (0.1 mM), or phenylglyoxal (1.0 mM). The inactivation of alanine transport by DEPC was concentration dependent (Fig. 8) and the I1o ofinactivation was 1 mm DEPC. These results suggest that the imidazole ring of a histidine residue is important in proton-coupled amino acid transport. Amino Acid Transport Competition Alanine, leucine, glutamate, arginine, isoleucine, and glutamine were chosen as the focus of a broad study of interamino acid transport competition. These amino acids were selected as representatives of the neutral, acidic, and basic amino acids. The inhibition of alanine and leucine transport by the other 20 common amino acids is shown in Table I. From these results, the 20 amino acids can be divided into two groups. The first group includes the acidic amino acids (Glu, Asp), basic amino acids (Arg, Lys, and His), and five neutral amino acids (Thr, Ile, Val, Pro, and OH-Pro). In this group, little or no inhibition was observed at equimolar concentrations, although some inhibition (from 10 to 20%) was observed when the concentration of the competing amino acid was an order of magnitude higher than the transported substrate. The second group identified in this experiment (Table I) included the remaining neutral amino acids (Ala, Leu, Ser, Gly, Met, Cys, Gln, Asn, Tyr, Phe, and Trp) that exhibited intermediate to strong inhibition of alanine and leucine transport. When glutamate and arginine transport were measured in the presence of other amino acids, the acidic and basic amino acids can be separated into two distinct groups (Table II). The basic amino acids had no effect on glutamate transport, 271 whereas aspartate showed strong inhibition. Generally, those amino acids that significantly decreased alanine and leucine transport showed little inhibition of glutamate transport, although methionine, alanine, leucine, glutamine, and asparagine exhibited some activity. In contrast, the acidic and aromatic amino acids showed no inhibition of arginine transport and a moderate inhibition of arginine transport was observed for the remaining neutral amino acids, with the exception of asparagine. These results (Table II) suggest that the acidic and basic amino acids are transported through independent transport systems and that each system exhibits some sensitivity to different groups of neutral amino acids. To examine whether the neutral amino acids are transported by more than one transport system, the effect of the other common amino acids on isoleucine and glutamine transport was also examined (Table III). All neutral amino acids were effective inhibitors of isoleucine transport while the basic and acidic amino acids had little impact. Although the pattern of sensitivity was similar for glutamine transport, it is noteworthy that several neutral amino acids (Thr, Val, Ile, and Pro) exhibited little transport inhibition. These neutral amino acids also displayed little activity against alanine and leucine transport (Table I). Based on these observations, we conclude two neutral amino acid transport systems are active in the isolated plasma membrane vesicles. DISCUSSION Isolated plasma membrane vesicles and imposed proton electrochemical potential differences (AAH+) were used in the present investigation to study the transport of alanine, leucine, glutamate, arginine, glutamine, and isoleucine. This group of amino acids includes representatives of the various structural and charge configurations displayed by this biologically important class of compounds. The transport of each of these amino acids was driven by an imposed proton concentration difference and each exhibited concentration dependent influx that was consistent with one or two saturable transport sys800 c 600 0E c 0 - C. 400 E E a C. 200 = 0 0 400 800 Glutamate (pM) 1200 1600 Figure 4. Concentration dependence of ApH-dependent glutamate transport into plasma membrane vesicles. Apparent Km = 1981 + 272 AM. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. 272 Plant Physiol. Vol. 94, 1990 Li AND BUSH 800 , s E 600 a. c (go "L 400 ._ m 0) <,o E0. 2000 _ _ 0 0 400 800 1200 1600 Isoleucine (gM) ,- 0.01 -0.00 0.02 0.01 0.03 0.04 11S Figure 5. Concentration dependence of ApH-dependent isoleucine transport into plasma membrane vesicles. (A) Concentration-dependent uptake. (B) Lineweaver-Burk plot. Transport was biphasic with a high affinity component (K, = 156 ± 27 gM) and a low affinity component (Km = 1925 ± 579 ltM). potential would contribute to the total proton electrochemical potential difference. These results are consistent with electrogenic transport and support previous electrophysiological studies using intact cells (10, 13, 15). Interestingly, the impact of membrane potential on alanine transport altered Vmax with no apparent effect on Km (Fig. 7B). This suggests that the binding affinity of the porter was not sensitive to AI. We conclude from these results that AI exerts its influence over a critical charge translocation step, which may be rate limiting, in the translocation process. Similar results were also observed for the proton-sucrose transport system in plasma membrane vesicles isolated from sugar beet leaves (5). The inhibition of amino acid transport by various compounds that chemically modify specific amino acid residues within proteins was investigated. Only DEPC, which forms covalent bonds with the imidazole ring of histidine, inhibited the transport of each amino acid examined. Inhibition of amino acid transport by DEPC suggests that a histidine residue may play an important role in the reaction sequence which couples proton and amino acid transport. Similar sensitivity to DEPC has been observed for the leaf protonsucrose symport and for the proton-lactose symport of Escherichia coli (4, 19). Although N,N'-dicyclohexylcarbodiimide was an effective inhibitor of alanine transport in zucchini hypocotyl plasma membrane vesicles (6) and p-chloromer0.40 0. tems (Figs. 1-6). Since we limited our study to substrate concentrations less than 2 mm, linear rates of flux in a higher concentration range (e.g. 5-50 mM) cannot be excluded. The data presented here are similar to those previously reported for alanine transport into membrane vesicles isolated from zucchini hypocotyls (6) and, taken together, we conclude that amino acid transport across the plant plasma membrane is mediated by proton-coupled cotransport systems. ApH-dependent alanine transport was electrogenic. This conclusion was based on experiments wherein the membrane potential of the isolated vesicles was altered with potassium concentration differences and valinomycin. Under conditions of minimum charge compensation, low rates of amino acid transport were recorded (Fig. 7A). When the membrane potential was clamped at zero or set at a negative potential, alanine transport was stimulated four- and sixfold, respectively. These data are consistent with an electrogenic transport process which results in the net flux of positive charge into the vesicle. In the absence of significant charge compensation, lower relative rates of flux would be expected due to the build up of positive charge as protons enter the vesicle with alanine. Charge accumulation results in the generation of a positive membrane potential that thermodynamically brakes the symport. In vesicles clamped at zero potential, where potassium ions exit the vesicle as protons enter, this would not occur. Similarly, in vesicles clamped with a negative potential, increased rates of flux would occur since a negative membrane co .=E ZsE 4 308 1 1 400 1200 1600 0.200.10 0.000 800 Arginine (gM) B 12.0- 8.0 4.0 0.0- . -0.00 l/S . 0.01 0.02 Figure 6. Concentration dependence of ApH-dependent arginine transport into plasma membrane vesicles. (A) Concentration-dependent uptake. (B) Lineweaver-Burk plot. Transport was biphasic with a high affinity component (Km = 136 ± 26 AM) and a low affinity component (Km = 1657 ± 1458 jAM). Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. PROTON-AMINO ACID TRANSPORT U A c E -Q a -C 6.0 00 -0. s0 'c0 cm / 4.0 'E E 2.0 / c w/ 0.0 0 200 400 600 8t 10 200 400 600 800 Alanine (pM) 2.0 response to changes in At, was not examined in these intact cell systems. In contrast, such indirect interactions were easily controlled for in the experiments reported here and, based on our observations with isolated plasma membrane vesicles, we conclude that four independent amino acid transport systems can be resolved. Two neutral amino acid symport systems were identified in this study. One system appears to transport all neutral amino acids while the second system, which also exhibits broad specificity, does not transport Thr, Val, Ile, and Pro. These four neutral amino acids displayed a unique pattern of transport inhibition. Although they were effective transport inhibitors within their own group (i.e. Val, Thr, and Pro inhibition of Ile: Table III), they were not competitive against alanine, leucine, and glutamine transport (Tables I and III). In contrast, isoleucine transport was sensitive to all neutral amino acids. Therefore, we conclude two neutral amino acid transport systems are active in isolated plasma membrane vesicles. It is noteworthy that threonine, isoleucine, and valine also exhibited a unique pattern of transport characteristics in B Chlorella vulgaris (21). 1.0 00o 273 ~ - , -0.000 0.004 0.008 Vs Figure 7 Concentration dependence of ApH-dependent alanine transport as a function of membrane potential. Plasma membrane vesicles Mvere diluted into an acidic transport solution in the presence (0, U) or absence (0) of 10 mm KCI and in the presence (, 0) or absence (0) of 5 mM valinomycin. These treatments resulted in rh_r rmraneainrI_i 1 Arejlm"rwi meMDrane poILeniLai m1nimum cnarge ciampea compensaxlontm), ae r%mnmkr,%no (U), and a negative membrane potential (0). (A) Concentration-dependence of Aph-dependent alanine transport. (B) Lineweaver-Burk plots of the data from A. at 0 mV curibenzenesulfonic acid was a potent inhibitor of the protonsucrose symport of sugar beet (4), these compounds exhibited little activity against the amino acid transport systems found in plasma membrane vesicles isolated from sugar beet leaves. The number of carriers which mediate amino acid transport into the higher plant cell has yet to be determined and, in fact, this number is rather controversial. Some workers suggest a single carrier is responsible for the influx of all amino acids while others have proposed two or three separate systems (2, 8, 11, 14, 18, 20, 21, 25). This situation is not surprising because in many of the experiments these conclusions are based on utilized intact tissues or cultured cell systems. Intact tissues and cells can contribute to experimental ambiguity because it is difficult sometimes to differentiate between specific mechanisms of interaction (7, 20). For example, the observation that the transport of any single amino acid is inhibited in the presence of any one of the other 20 amino acids has been interpreted as evidence for a single carrier with broad specificity ( 1 8). However, the possibility that decreased transport in the presence of another amino acid may be the result of indirect effects, such as competition for ApH or in The specific activity of the neutral amino acid transport systems varies with tissue and species (Z-C Li, DR Bush, unpublished data). In sugar beet leaf plasma membrane vesicles, d , .alanine transport is >10 times higher than isoleucine transport. In cucumber leaf plasma membrane vesicles, alanine transport is only twice as high. This variation in the relative abundance of alanine and isoleucine transport provides further evidence in support of the notion that multiple neutral amino acid transport systems are active in the plant plasma membrane. Whether these systems are differentially expressed within single cells or between specific tissue types cannot be deduced from the heterogeneous plasma membrane vesicles used in these experiments. Acidic amino acids were transported by a third system. This is supported by the fact that glutamate transport was not inhibited by the basic amino acids (Table II) and little or no inhibition was exerted by most of the neutral amino acids. In contrast, aspartate was an effective inhibitor of glutamate 100 - 0- a.. (U 0 C - -o o- 0.0 1.0 3.0 DEPC (mM) 2.0 4.0 5.0 Figure 8. Concentration dependence of DEPC inactivation of ApHdependent alanine transport. Plasma membrane vesicles were pretreated with DEPC for 15 min and then diluted into the acidic transport solution containing 100 yM alanine, 0.2 ACi [14C]L-alanine, 5 4M valinomycin, and plus or minus 5 Mm CCCP. Transport activity was expressed as a percentage of the control. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. 274 LI AND BUSH Plant Physiol. Vol. 94, 1990 Table I. Inhibition of Alanine and Leucine Transport by Different Amino Acids Plasma membrane vesicles were diluted into the transport solution containing 100 AM of [14C]alanine or [14C]leucine and the antagonist amino acid at 1 or 10 times the concentration of the radiolabeled substrate. Transport activity was determined after 1 min as described in the "Materials and Methods." Antagonist Amino Acid Controlb Glutamate Aspartate Lysine Histidine Arginine Threonine Proline OH-Proline Valine Isoleucine Asparagine Glutamine Serine Cysteine Methionine Glycine Alanine Antagonist Concentration 1x lOx % alanine transport' 100 99 ± 4 65 ± 10 94 ± 3 71 ± 3 100 ± 6 87 ± 9 94 ± 6 85 ± 12 93 ±13 65 ± 12 96 ± 1 88 ± 8 98±2 91±2 102 ± 5 95 ± 4 101±3 89±4 107 ± 2 91 ± 4 85 ± 1 48 ± 7 75 ± 16 33 ± 5 84± 11 41±7 80 ± 6 36 ± 7 55 ± 5 20 ± 5 97 ± 5 56 ± 5 1x lOx % leucine transport' 100 93 ± 3 83 ± 11 106 ± 4 95 ± 16 97 ± 2 105 ± 7 106 ± 2 92 ± 5 99 ± 2 81 ± 6 96 ± 4 83 ± 16 104±6 90±8 108 ± 8 89 ± 4 99±4 94± 14 103 ± 3 87 ± 16 90 ± 5 47 ± 8 77 ± 5 38 ± 6 85+7 53±9 80 ± 6 40 ± 7 52 ± 4 23 ± 9 92 ± 2 54 ± 5 74 ± 5 36 ± 4 Leucine 83 ± 10 36 ± 6 Tyrosine 91 ± 2 67 ± 2 86 ± 4 53 ± 2 Tryptophan 89 ± 7 52 ± 9 90 ± 2 47 ± 7 Phenylalanine 93 ± 7 56 ± 7 88 ± 2 52 ± 6 Sucrose (1 mM) 91 + 3 96 + 4 a b The results are presented as mean % ± SD (n = 3) of alanine or leucine transport. Control rates: Ala = 2.2 nmol/mg protein/min; Leu = 1.1 nmol/mg protein min. transport, suggesting this pathway is specific for acidic amino acids. When the competing amino acid concentration was increased 10-fold higher than that of glutamate, however, a few neutral amino acids partially inhibited glutamate uptake, suggesting that these amino acids have a measurable affinity for the acidic transport system. Asparagine and glutamine, at 10 times the concentration of glutamate, were especially competitive against glutamate transport (Table II). These two neutral amino acids exhibit some structural similarity to glutamate and, thus, they may share the glutamate transport system. The fourth transport system appears to be mainly responsible for the basic amino acids. Since glutamate and aspartate do not inhibit arginine transport (Table II), this porter is not shared by the acidic amino acids. However, this system is shared differentially by the neutral amino acids. Most neutral amino acids exhibited little evidence of transport competition, yet alanine and methionine were very effective. The two groups of neutral amino acids that were evident in Table I can not be distinguished based on the inhibition of arginine transport (Table II). Such differential sensitivity is consistent with multiple transporters. The inhibition of arginine transport by lysine and histidine was low at the concentrations tested. One explanation for this is that there may be more than one system for the basic amino acids. Further study is needed to resolve this observation. Sucrose was used as an internal control in each competition experiment and it showed little or no inhibition of amino acid transport (Tables I-III). Testing the effect of sucrose on amino acid transport was important because it demonstrates that competition for the proton motive force is not a reasonable explanation for the amino acid-dependent transport inhibition observed in the competition studies reported here. Since sucrose is actively transported by a proton-symport in the plasma membrane vesicles used in these experiments, any significant sucrose-dependent decrease in AILH+ would have resulted in a change in amino acid flux. The absence of such a decrease suggests symport activity is too slow to significantly alter AAH' over the time course of the competition experiments. Since these experiments were also performed with the membrane potential clamped at zero, we conclude that amino acid-dependent decreases in transport activity is due to direct interactions at the transport proteins and cannot be assigned to indirect effects on ApH or membrane potential. The resolution of four transport systems was based on the results of inter-amino acid competition (Tables I-III). It should be noted, however, that considerable variation exists within these broad classifications. For example, some amino Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. PROTON-AMINO ACID TRANSPORT 275 Table II. Inhibition of Arginine and Glutamate Transport by Different Amino Acids Plasma membrane vesicles were diluted into the transport solution containing 50 lM [14C]arginine or 25 /M [14C]glutamate and the antagonist amino acid at 1 or 10 times the concentration of the radiolabeled substrate. Transport activity was determined after 2 min of uptake. Antagonist Concentration Antagonist lx lOx % arginine transporta 1x lox % glutamate transporta 100 Controlb 100 Glutamate 96 ± 2 90 ± 3 Aspartate 95 ± 1 104 ± 11 76 ± 2 63 ± 3 77±4 104±16 Lysine 93±1 95±2 87 ± 2 Histidine 88 ± 3 83 ± 1 96 ± 3 94 ± 1 111 ± 12 Arginine 84 ± 1 Threonine 70 ± 8 75 ± 14 83 ± 2 97 ± 4 88 ± 7 93 ± 4 105 ± 11 Proline 97 ± 3 105 ± 8 104 ± 7 OH-proline 111 ± 10 91±2 84±3 100±16 Valine 98±2 94 ± 8 85 ± 4 96 ± 2 Isoleucine 111 + 11 91 ± 4 101 ± 10 83 ± 7 63 ± 12 Asparagine 62 ± 9 77 ± 10 66 ± 2 Glutamine 90 ± 1 91 ± 1 58 ± 7 87 ± 7 Serine 106 ± 15 94 ± 2 56 ± 8 92 ± 9 82 ± 12 Cysteine 79 ± 7 48 ± 7 79 ± 5 72 ± 15 Methionine 89±3 61±7 89±7 77±14 Glycine 82 ± 1 77 ± 4 77 ± 8 Alanine 46 ± 2 84± 16 Leucine 56±6 91±3 91±3 Tyrosine 95±2 70±3 103±1 88±5 87 ± 7 93 ± 3 92 ± 6 124 ± 17 Tryptophan 95 ± 1 89 ± 8 98 ± 2 122 ± 9 Phenylalanine 91 2 Sucrose ( 1 mM) 100 4 b ± Control Results are presented as mean % SD (n = 3) of glutamine or isoleucine transport. rates: Arg = 0.06 nmol/mg protein min; Glu = 0.02 nmol/mg protein/min acids inhibited the transport of a given amino acid to a lesser extent than other amino acids within the same group. Methionine, alanine, and cysteine were strong inhibitors of leucine and glutamine transport (Tables I and III) while glycine and serine were not as effective. Similarly, glutamine and asparagine were stronger inhibitors ofglutamate transport than were the other neutral amino acids. The broad specificity of the four transport systems identified here has also been observed in the major amino acid transport systems described in animal cells (9). Ultimately, differential inhibition of specific symports or purification and characterization of individual systems will be necessary to unequivocally determine the number of amino acid transport systems in the plasma membrane of sugar beet leaves. Although the inter-amino acid transport competition studies reported here suggest four amino acid transport systems are present in isolated plasma membrane vesicles, this conclusion is not always consistent with the concentration-dependent transport kinetics reported in Figures 1 to 6. For example, concentration-dependent alanine transport exhibited simple saturation kinetics and yielded linear Lineweaver-Burk plots of the data that are consistent with a single transport system within the concentration range tested (Fig. 1). Yet, alanine was a potent transport inhibitor of amino acids representing three of the four symports reported here. If alanine is transported through all three systems, why did not this show up as complex, multiphasic transport kinetics in Figure 1 (note that the amino acid concentrations used in all inter-amino acid competition experiments were kept within the same range as those used in the kinetic experiments to avoid complications from low affinity porters). One possible explanation for these data is that the apparent Km for alanine for all three symports is approximately the same and/or that a high Vma for the dominant alanine symport overshadows the other transport systems. An alternative explanation is essentially a special case of the above wherein alanine binds to the active site of each symport but this binding is not translated into efficient translocation into the vesicle. As a small neutral amino acid it is possible that alanine can enter the active site of a symport system without binding in the necessary configuration that results in translocation across the membrane. Yet, the presence of alanine in the active site would competitively limit access to the natural substrate and result in a significant decrease in transport. This kind of binding would not show up in alanine transport kinetics and this explanation is consistent with reports that alanine is a potent, across the board inhibitor of amino acid transport in a variety of experimental system (14, 25). In contrast to the alanine data, the biphasic Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. Li AND BUSH 276 Plant Physiol. Vol. 94, 1990 Table ll. Inhibition of Glutamine and Isoleucine Transport by Different Amino Acids Plasma membrane vesicles were diluted into the transport solution containing 100 AM [14C]glutamine or 25 AM [14C]isoleucine and the antagonist amino acid at 1 or 10 times the concentration of the radiolabeled substrate. Transport activity was determined after 2 min of uptake. Antagonist Concentration Antagonist Controlb Glutamate Aspartate Lysine Histidine Arginine Threonine Proline OH-proline Valine Isoleucine Asparagine Glutamine Serine Cysteine Methionine Glycine Alanine Leucine Tyrosine Tryptophan lox 1x % glutamine transport' 100 100 ± 1 73±2 87±12 83±7 101 ±3 104±4 99 ±1 93±5 100±3 109±6 110±8 109 ± 1 98±3 101±8 92±5 77±1 81±1 86±2 97±3 89±4 89 ± 2 51±1 92±3 90 ±5 60±4 99 ± 6 81±3 90±1 93±1 57±4 57±4 24±2 61±1 97±4 35±0 47±2 58±0 57±3 61 ± 5 lox 1x % isoleucine transporta 100 91±2 98±2 100±5 103±13 102±7 88± 15 74±7 85±4 104±4 78±5 103±12 101±2 87±5 81±9 43±3 54±4 104±2 45±1 89±10 80±13 72±3 62±15 43±2 84±6 55±4 63±10 95±2 97±10 64 ± 4 70±1 46±2 47±7 34±6 22±2 44±4 27±2 31±3 60±2 80±3 40 ± 1 97 ± 4 Phenylalanine 104 1 Sucrose (1 mM) 98±6 ± = a Results are presented as mean % SD (n 3) of glutamine or isoleucine transport. rates: Gln = 1.1 nmol/mg protein/min; lie = 0.05 nmol /mg protein/min. transport kinetics of isoleucine and arginine are consistent with the inter-amino acid competition experiments that suggest more than one transport system is present in the isolated membrane vesicles. CONCLUSION Isolated plasma membrane vesicles and imposed proton electrochemical potential differences were used to examine proton-coupled amino acid transport. ApH-dependent alanine, leucine, glutamine, glutamate, isoleucine, and arginine transport exhibited saturable transport kinetics and sensitivity to protein modification. Alanine transport was electrogenic and both components of AAH', ApH and At, contributed to b Control transport activity. From these results, we conclude that amino acid transport is mediated by specific proton-amino acid symports. The results of inter-amino acid competition experiments suggest at least four transport systems can be resolved in plasma membrane vesicles isolated from sugar beet leaves (Table IV). Although a given transport system may be the primary porter for a specific group of amino acids, the data reported here also suggest that structural similarity between the various amino acids results in an unusual lack of specificity. Since many soluble enzymes that interact with amino acids do not exhibit such broad specificity, structural similarity is not an impenetrable biophysical barrier. We conclude, therefore, that evolutionary pressures have selected for gen- Table IV. Summary of Amino Acid Transport Systems in the plasma membrane of sugar beet leaves. Four amino acid transport systems were resolved here based on transport kinetics and inter-amino acid competition. Transport System Amino Acid Transported Neutral system (low abundance) All neutral amino acids Neutral system 11 (high abundance) All neutral amino acids, but low affinity for lie, Thr, Val, and Pro Acidic system Glu, Asp Basic system Arg, Lys, His Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved. PROTON-AMINO ACID TRANSPORT eralized amino acid transport systems rather than highly specific, single substrate porters. 12. ACKNOWLEDGMENT 13. We thank Cara Ripperda for her expert technical assistance. 14. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. LITERATURE CITED Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70: 241-250 Borstlap AC, Meenks JLD, van Eck WF, Briker JTE (1986) Kinetics and specificity of amino acid uptake by the duckweed Spirodela polyrhiza (L.) Schleiden. J Exp Bot 37: 1020-1035 Bown AW, Chung I, Snedden W, Shelp B (1989) Specific glutamate cotransport into mesophyll cells and efflux of the major metabolite 4-aminobutyric acid. In J Dainty, MI DeMichelis, E Marre, F. Rasi-Caldogno, eds, Plant Membrane Transport: The Current Position. Elsevier, Amsterdam, pp 329-334 Bush DR (1989) Proton-coupled sucrose transport in plasmalemma vesicles isolated from sugar beet (Beta vulgaris L.) leaves. Plant Physiol 89: 1318-1323 Bush DR (1990) Electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport. Plant Physiol 93: 15901596 Bush DR, Langston-Unkefer PJ (1988) Amino acid transport into membrane vesicles isolated from zucchini: evidence of a proton-amino acid symport in the plasmalemma. Plant Physiol 88: 487-490 Bush DR, Langston-Unkefer PJ (1987) Tabtoxinine-j3-lactam transport into plant cells: Uptake via an amino acid transport system. Plant Physiol 85: 845-849 Cheruel J, Jullien M (1979) Amino acid uptake into cultivated mesophyll cells from Asparagus officinalis L. Plant Physiol 63: 621-626 Christensen HN (1990) Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev 70: 4377 Etherton B, Rubinstein B (1 978) Evidence for amino acid-H' co- transport in oat coleoptiles. Plant Physiol 61: 933-937 11. Harrington HM, Henke RR (1981) Amino acid transport into 15. 16. 17. 18. 19. 20. 21. 277 cultured tobacco cells. I. Lysine transport. Plant Physiol 67: 373-378 Hoagland DR, Arnon DI (1938) The water culture method for growing plants. Agric Expt Sta Calif Circ 347 Jung K-D, Luttge U (1980) Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L- and D-amino acids. Planta 150: 230-235 Kinraide TB (1981) Interammino acid inhibition of transport in higher plants: evidence for two transport channels with ascertainable affinities for amino acids. Plant Physiol 68: 13271333 Kinraide TB, Etherton B (1980) Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles. Plant Physiol 65: 1085-1089 Markwell MK, Haas SM, Tolbert NE, Bieber LL (1981) Protein determinations in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol 72: 296-303 McCutcheon SL, Ciccarelli BW, Chung I, Shelp B, Bown AW (1988) L-Glutamate-dependent medium alkalization by Asparagus mesophyll cells. Plant Physiol 88: 1042-1047 McDaniel CN, Holterman RK, Bone RF, Wozniak PM (1982) Amino acid transport in suspension-cultured plant cells. III. Common carrier system for the uptake of L-arginine, L-aspartic acid, L-histidine, L-leucine, and L-phenylalanine. Plant Physiol 69: 246-249 Padan E, Patel L, Kaback HR (1979) Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles. Proc Natl Acad Sci USA 76: 6221-6225 Reinhold L, Kaplan A (1984) Membrane transport of sugars and amino acids. Annu Rev Plant Physiol 35: 45-83 Sauer N, Tanner W (1985) Selection and characterization of Chlorella mutants deficient in amino acid transport: further evidence for three independent systems. Plant Physiol 79: 760- 764 22. Schobert C, Komor E (1989) The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap. Planta 177: 342-349 23. Sze H (1985) H+-Translocating ATPase: advances using membrane vesicles. Annu Rev Plant Physiol 36: 175-208 24. Ver Nooy CD, Lin W (1986) Amino acid transport in protoplasts isolated from soybean leaves. Plant Physiol 81: 8-11 25. Wyse RE, Komor E (1984) Mechanism of amino acid uptake by sugarcane suspension cells. Plant Physiol 76: 865-870 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1990 American Society of Plant Biologists. All rights reserved.