Proton-coupled Symport Research Articles

Biochemical Characterization of the C 4 -Dicarboxylate Transporter DctA from Bacillus subtilis

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C(4)-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C(4)-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt(Ph) from Pyrococcus horikoshii.

Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus

Sucrose transporters in the SUT family are important for phloem loading and sucrose uptake into sink tissues. The recent localization of type III SUTs AtSUT4 and HvSUT2 to the vacuole membrane suggests that SUTs also function in vacuolar sucrose transport. The transport mechanism of type III SUTs has not been analyzed in detail. LjSUT4, a type III sucrose transporter homolog from Lotus japonicus, is expressed in nodules and its transport activity has not been previously investigated. In this report, LjSUT4 was expressed in Xenopus oocytes and its transport activity assayed by two-electrode voltage clamping. LjSUT4 transported a range of glucosides including sucrose, salicin, helicin, maltose, sucralose and both alpha- and beta-linked synthetic phenyl glucosides. In contrast to other sucrose transporters, LjSUT4 did not transport the plant glucosides arbutin, fraxin and esculin. LjSUT4 showed a low affinity for sucrose (K(0.5)=16 mM at pH 5.3). In addition to inward currents induced by sucrose, other evidence also indicated that LjSUT4 is a proton-coupled symporter: (14)C-sucrose uptake into LjSUT4-expressing oocytes was inhibited by CCCP and sucrose induced membrane depolarization in LjSUT4-expressing oocytes. A GFP-fusion of LjSUT4 localized to the vacuole membrane in Arabidopsis thaliana and in the roots and nodules of Medicago truncatula. Based on these results we propose that LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole.

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

The Cl − ATPase pump

Seven widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium symport, sodium potassium chloride symport, potassium chloride symport, proton-coupled symport, an electrochemical coupling process, and chloride channels. No direct genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl −-stimulated ATPases coexisting in the same tissue with uphill chloride transport that could not be accounted for by the four common active chloride transport processes. Cl −-stimulated ATPases are a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl −-stimulated ATPase activity. Recent studies of Cl −-stimulated ATPase activity and chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl −-stimulated ATPases.

ZmYS1 Functions as a Proton-coupled Symporter for Phytosiderophore- and Nicotianamine-chelated Metals

Among higher plants graminaceous species have the unique ability to efficiently acquire iron from alkaline soils with low iron solubility by secreting phytosiderophores, which are hexadentate metal chelators with high affinity for Fe(III). Iron(III)-phytosiderophores are subsequently taken up by roots via YS1 transporters, that belong to the OPT oligopeptide transporter family. Despite its physiological importance at alkaline pH, uptake of Fe-phytosiderophores into roots of wild-type maize plants was greater at acidic pH and sensitive to the proton uncoupler CCCP. To access the mechanism of Fe-phytosiderophore acquisition, ZmYS1 was expressed in an iron uptake-defective yeast mutant and in Xenopus oocytes, where ZmYS1-dependent Fe-phytosiderophore transport was stimulated at acidic pH and sensitive to CCCP. Electrophysiological analysis in oocytes demonstrated that Fephytosiderophore transport depends on proton cotransport and on the membrane potential, which allows ZmYS1-mediated transport even at alkaline pH. We further investigated substrate specificity and observed that ZmYS1 complemented the growth defect of the zinc uptake-defective yeast mutant zap1 and transported various phytosiderophore-bound metals into oocytes, including zinc, copper, nickel, and, at a lower rate, also manganese and cadmium. Unexpectedly, ZmYS1 also transported Ni(II), Fe(II), and Fe(III) complexes with nicotianamine, a structural analog of phytosiderophores, which has been shown to act as an intracellular metal chelator in all higher plants. Our results show that ZmYS1 encodes a proton-coupled broad-range metal-phytosiderophore transporter that additionally transports Fe- and Ni-nicotianamine. These biochemical properties indicate a novel role of YS1 transporters for heavy metal homeostasis in plants.

Bidirectional electrogenic transport of peptides by the proton-coupled carrier PEPT1 in Xenopus laevis oocytes: its asymmetry and symmetry.

1. The giant patch clamp technique in the inside-out configuration and the two-electrode voltage clamp technique were used to characterize the bidirectional transport properties of the proton-coupled peptide carrier PEPT1 expressed in Xenopus laevis oocytes. 2. The addition of the neutral dipeptide Gly-L-Gln to the cytoplasmic solution induced a net outward transport current in a membrane potential range between -80 and +60 mV, even in the absence of a pH gradient. 3. The concentration dependency of the outwardly directed transport currents followed Michaelis-Menten-type kinetics, with an apparent K0.5 of 3.28 mM (at pH 7.5 and +60 mV membrane potential). This apparent affinity is around fivefold lower than the apparent affinity measured for the inward transport mode (K0.5 of 0.70 mM (at pH 7.5 and -60 mV) under identical experimental conditions). 4. Apparent K0.5 values were strongly pH and potential dependent only on the external face for inward transport. The transport currents were potential dependent, but essentially pH independent for inward transport and only modestly altered by pH in the reverse direction. In addition to the membrane potential, the transmembrane substrate gradient acts as a driving force and contributes significantly to total transport currents. 5. The differences in apparent substrate affinity under identical experimental conditions suggest major differences in the conformation of the substrate binding pocket of PEPT1 when exposed to the external versus the internal face of the membrane. The lower affinity on the internal face allows the substrate to be released into the cytosolic compartment even in the absence of a proton-motive force. 6. Our study demonstrates for the first time that PEPT1 can transport dipeptides bidirectionally in an electrogenic and proton-coupled symport mode. When substrates are present on both sides of the membrane in sufficiently high concentrations, the direction and rate of transport are solely dependent on the membrane potential, and transport occurs symmetrically.

Characterization of Glucosinolate Uptake by Leaf Protoplasts of Brassica napus

The uptake of radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) by protoplasts isolated from leaves of Brassica napus was detected using silicone oil filtration technique. The uptake was pH-dependent with higher uptake rates at acidic pH. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of p-OHBG, which was inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone, indicating that the uptake is dependent on a proton motive force. Dissipation of the internal positive membrane potential generated a small influx as compared with that seen for pH gradient (DeltapH). Kinetic studies demonstrated the presence of two uptake systems, a saturable and a linear component. The saturable kinetics indicated carrier-mediated translocation with a K(m) of 1.0 mm and a V(max) of 28.7 nmol/microl/h. The linear component had very low substrate affinity. The carrier-mediated transport had a temperature coefficient (Q(10)) of 1.8 +/- 0.2 in the temperature range from 4-30 degrees C. The uptake was against a concentration gradient and was sensitive to protonophores, uncouplers, H(+)-ATPase inhibitors, and the sulfhydryl group modifier p-chloromercuriphenylsulfonic acid. The carrier-mediated uptake system had high specificity for glucosinolates because glucosinolate degradation products, amino acids, sugars, or glutathione conjugates did not compete for p-OHBG uptake. Glucosinolates with different side chains were equally good competitors of p-OHBG uptake, which indicates that the uptake system has low specificity for the glucosinolate side chains. Our data provide the first evidence of an active transport of glucosinolates by a proton-coupled symporter in the plasma membrane of rape leaves.

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.