Sec-independent protein translocation by the maize Hcf106 protein.

The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (DeltapH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.

Sugar transporters in plant biology.

Sugar transporters are key players in many fundamental processes in plant growth and development. Recent results have identified several new transporters that contribute to a wide array of physiological activities, and detailed molecular analysis has provided exciting insights into the structure and regulation of these essential membrane proteins.

Amino acid transporters in plants.

Amino acid transporters are essential participants in the resource allocation processes that support plant growth and development. Recent results have identified several new transporters that contribute to a wide array of physiological activities, and detailed molecular analysis has provided fundamental insights into the structure, function and regulation of these integral membrane proteins.

Surfactant-Increased Glyphosate Uptake into Plasma Membrane Vesicles Isolated from Common Lambsquarters Leaves.

Plasma membrane vesicles were isolated from mature leaves of lambsquarters (Chenopodium album L.) to investigate whether this membrane is a barrier to glyphosate uptake and whether surfactants possess differential abilities to enhance glyphosate permeability. Amino acids representing several structural classes showed [delta]pH-dependent transport, indicating that the proteins necessary for active, proton-coupled amino acid transport were present and functional. Glyphosate uptake was very low compared to the acidic amino acid glutamate, indicating that glyphosate is not utilizing an endogenous amino acid carrier to enter the leaf cells and that the plasma membrane appears to be a significant barrier to cellular uptake. In addition, glyphosate flux was much lower than that measured for either bentazon or atrazine, both lipid-permeable herbicides that diffuse through the bilayer. Glyphosate uptake was stimulated by 0.01% (v:v) MON 0818, the cationic surfactant used in the commercial formulation of this herbicide for foliar application. This concentration of surfactant did not disrupt the integrity of the plasma membrane vesicles, as evidenced by the stability of imposed pH gradients and active amino acid transport. Nonionic surfactants that disrupt the cuticle but that do not promote glyphosate toxicity in the field also increased glyphosate transport into the membrane vesicles. Thus, no correlation was observed between whole plant toxicity and surfactant-aided uptake. Current data suggest that surfactant efficacy may be the result of charged surfactants' ability to diffuse away from the cuticle into the subtending apoplastic space, where they act directly on the plasma membrane to increase glyphosate uptake.

Expression and transcriptional regulation of amino acid transporters in plants.

Recent studies have shown that there are more than 50 amino acid transporter genes in the Arabidopsis genome. This abundance of amino acid transporters implies that they play a multitude of fundamental roles in plant growth and development. Current research on the expression and regulation (i.e., tissue-specific expression and regulation of expression in response to nutrient and environmental changes) of these genes has provided useful information about the functional significance of plant amino acid transport systems.

Inhibitors of the proton-sucrose symport.

Sucrose transporters are important components of the assimilate partitioning pathway in many plants. In the results reported here, we examined the effect of several inhibitors on proton-coupled sucrose transport into plasma membrane vesicles isolated from sugar beet leaf tissue. Three compounds that are reversible inhibitors of glucose transporters, phlorizin, cytochalasin B, and forskolin, also inhibited the proton-sucrose symport. Additionally, several reagents that covalently modify specific amino acid residues, including p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide (NEM), diethyl pyrocarbonate (DEPC), and Hg2+, were also examined. NEM was not an effective inhibitor of the symport under both energized (pH 6.0) and unenergized (pH 7.7) conditions. In contrast, PCMBS, DEPC, and Hg2+ blocked sucrose transport activity. However, in control experiments it was discovered that Hg2+, but not PCMBS or DEPC, dissipated the proton electrochemical potential difference (delta mu H) that drives sucrose accumulation. It was further demonstrated that Hg2+ dissipated an imposed delta mu+H in protein-free liposomes, thus obscuring its effect on the sucrose symport. In time- and concentration-dependent inactivation experiments, it was shown that DEPC binding was substrate protectable, thereby implicating binding at or near the active site of the carrier. In contrast, PCMBS activity was not linked to substrate binding. DEPC activity was partially reversed with hydroxylamine. This is consistent with specific modification of a histidine residue. Preloading purified vesicles with free histidine did not slow the DEPC-dependent inactivation kinetics. Since these membrane vesicles are predominantly right-side out, the last observation is consistent with a DEPC-sensitive site which is accessible from the outside face of the vesicle. The results with DEPC suggest that a histidine residue is at or near the active site of the sucrose symport and that this amino acid plays a critical role in the reaction mechanism.

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport.

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on DeltapH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V(max) only with no apparent change in K(m). Sucrose transport rates driven by membrane potential only, i.e. in the absence of DeltapH, were comparable to DeltapH-driven flux. Both membrane potential and DeltapH-driven sucrose transport were used to examine proton binding to the symport and the apparent K(m) for H(+) was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.

The proton-sucrose symport.

The heterotrophic tissues of the plant are dependent upon carbon and nitrogen import for normal growth and development. In general, oxidized forms of these essential elements are reductively assimilated in the leaf and, subsequently, sucrose and amino acids are transported to the heterotrophic cells in a process known as assimilate partitioning. In many plant species, a critical component of the assimilate partitioning pathway is the proton-sucrose symport. This active transport system couples sucrose translocation across the plasma membrane to the proton motive force generated by the H(+)-pumping ATPase. To date, the proton-sucrose symport is the only known system that can account for sucrose accumulation in the vascular tissue of the plant. This review focuses on recent advances describing the transport properties and bioenergetics of the proton-sucrose symport.

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves.

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. DeltapH-dependent sucrose transport exhibited saturation kinetics with an apparent K(m) of 1.20 +/- 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. DeltapH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because DeltapH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K(+), Mg(2+)-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.

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 beta-carbon position that places a dense electron cloud close to the alpha-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.

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. DeltapH-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. DeltapH-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 (DeltaPsi) on DeltapH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When DeltaPsi was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative DeltaPsi, 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 DeltaPsi 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.

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells.

Two active calcium (Ca(2+)) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca(2+) transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl(-)-stimulated, NO(3) (-)-inhibited ATPase activity on sucrose density gradients. Ca(2+) transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The K(m) for MgATP and Ca(2+) were 0.1 mm and 21 micromolar, respectively. The predominant Ca(2+) transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca(2+) transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I(50) = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca(2+) dependent kinetics were complex with an apparent K(m) ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca(2+)/H(+) antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca(2+) transport systems are proposed to restore and maintain cytoplasmic Ca(2+) homeostasis under changing cellular and environmental conditions.

Potassium transport in suspension culture cells and protoplasts of carrot.

The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.

Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves.

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). D-Isomers of alanine and isoleucine were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the alpha-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that beta-alanine and analogues with methylation at the alpha-carbon, at the carboxyl group, or at the alpha-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the alpha-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the beta-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.

Sucrose is a signal molecule in assimilate partitioning.

The proton-sucrose symporter mediates the key transport step in the resource distribution system that allows many plants to function as multicellular organisms. In the results reported here, we identify sucrose as a signaling molecule in a previously undescribed signal-transduction pathway that regulates the symporter. Sucrose symporter activity declined in plasma membrane vesicles isolated from leaves fed exogenous sucrose via the xylem transpiration stream. Symporter activity dropped to 35-50% of water controls when the leaves were fed 100 mM sucrose and to 20-25% of controls with 250 mM sucrose. In contrast, alanine symporter and glucose transporter activities did not change in response to sucrose treatments. Decreased sucrose symporter activity was detectable after 8 h and reached a maximum by 24 h. Kinetic analysis of transport activity showed a decrease in Vmax. RNA gel blot analysis revealed a decrease in symporter message levels, suggesting a drop in transcriptional activity or a decrease in mRNA stability. Control experiments showed that these responses were not the result of changing osmotic conditions. Equal molar concentrations of hexoses did not elicit the response, and mannoheptulose, a hexokinase inhibitor, did not block the sucrose effect. These data are consistent with a sucrose-specific response pathway that is not mediated by hexokinase as the sugar sensor. Sucrose-dependent changes in the sucrose symporter were reversible, suggesting this sucrose-sensing pathway can modulate transport activity as a function of changing sucrose concentrations in the leaf. These results demonstrate the existence of a signaling pathway that can control assimilate partitioning at the level of phloem translocation.

Topology of NAT2, a prototypical example of a new family of amino acid transporters.

Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N or C terminus of the protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group of oriented peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to estimate the location of protease accessible cleavage sites. We independently identified the location of the N and C termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. We also investigated the glycosylation status of several sites of potential N-linked glycosylation. Based on the combined data, we propose a novel 11 transmembrane domain model with the N terminus in the cytoplasm and C terminus facing outside the cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis.

LHT1, a lysine- and histidine-specific amino acid transporter in arabidopsis.

We have identified a new amino acid transporter from the Arabidopsis thaliana expressed sequence tag cDNA collection by functional complementation of a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 microM, respectively. This transporter (LHT1, lysine histidine transporter) has little affinity for arginine when measured directly in uptake experiments or indirectly with substrate competition. The cDNA is 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHT1 is an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT1 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter is present in all tissues, with the strongest expression in young leaves, flowers, and siliques. Wholemount, in situ hybridization revealed that expression is further localized on the surface of roots in young seedlings and in pollen. Overall, LHT1 belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given its substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains.

Molecular cloning, immunochemical localization to the vacuole, and expression in transgenic yeast and tobacco of a putative sugar transporter from sugar beet.

Several plant genes have been cloned that encode members of the sugar transporter subgroup of the major facilitator superfamily of transporters. Here we report the cloning, expression, and membrane localization of one of these porters found in sugar beet (Beta vulgaris L.). This clone, cDNA-1, codes for a protein with 490 amino acids and an estimated molecular mass of 54 kD. The predicted membrane topology and sequence homology suggest that cDNA-1 is a member of the sugar transporter family. RNA gel blot analysis revealed that this putative sugar transporter is expressed in all vegetative tissues and expression increases with development in leaves. DNA gel blot analysis indicated that multiple gene copies exist for this putative sugar transporter in the sugar beet genome. Antibodies directed against small peptides representing the N- and C-terminal domains of the cDNA1 protein identified a 40-kD polypeptide in microsomes isolated from cDNA-1-transformed yeast (Saccharomyces cerevisiae). Moreover, the same protein was identified in sugar beet and transgenic tobacco (Nicotaina tobacum L.) membrane fractions. Detailed analysis of the transporter's distribution across linear sucrose gradients and flotation centrifugations showed that it co-migrates with tonoplast membrane markers. We conclude that this carrier is located on the tonoplast membrane and that it may mediate sugar partitioning between the vacuole and cytoplasmic compartments.

His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity.

The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.

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-beta-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. DeltapH-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(+)-Mg(2+)-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.