Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

Sucrose transport and hexose release in the maize scutellum

Since hexoses readily diffuse from maize scutellum cells, it should be possible to detect them if they are produced during sucrose transport at the tonoplast or the plasmalemma. To test this idea, scutellum slices were placed in dinitrophenol (DNP) (which inhibits hexose utilization while greatly increasing utilization of vacuolar sucrose), and the utilization, uptake and leakage of sugars were measured. Only negligible amounts of hexose appeared in the DNP solution during a 5-hr incubation during which the slices metabolized 72μmol of sucrose. Glucose and fructose, added at a concentration of 2 mM, were taken up by the slices at rates 33% and 14% (respectively) of the rate of vacuolar sucrose utilization. It is suggested, therefore, that sucrose transport at the tonoplast does not release free hexose into the cytoplasm. Sucrose transport at the plasmalemma was studied using DNP- and mannose-treated slices. During incubation of these slices in sucrose, the disappearance of sucrose resulted in the appearance of significant quantities of glucose and fructose in the bathing solution. Evidence is presented that sucrose is split into glucose and fructose during transport across the plasmalemma. It is concluded that free hexose is not normally a product of this splitting but is a result of an uncoupling in the transport system caused by the DNP or mannose treatments.     

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. ΔpH-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. ΔpH-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 ΔpH-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⁺, Mg²⁺-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.     

Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

Sucrose transport at the tonoplast

Sucrose that leaked from maize scutellum slices upon transfer of slices from a hexose or hexitol solution to water or upon placing the slices in a buffered EDTA solution was considered to be cytoplasmic in origin; residual (after leakage) tissue sucrose was considered to be stored in the vacuoles. This paper presents a study of the movement of sucrose across the tonoplast between the vacuoles and the cytoplasmic compartment. It is concluded that; (a) sucrose transport into the vacuoles is directly linked to sucrose synthesis in such a way that free sucrose is not an intermediate in the coupled process, (b) cytoplasmic sucrose is not (cannot be?) stored, (c) sucrose transport out of the vacuoles is linked to the metabolic demand for sugar, and (d) the transport process removing sucrose from the vacuoles does not release free sucrose into the cytoplasm. The sucrose fluxes at the plasmalemma and at the tonoplast are calculated, and the transport processes at the two membranes are compared.     

Electrogenic sucrose transport in developing soybean cotyledons

Addition of sucrose to a solution bathing an excised developing soybean cotyledon causes a transient depolarization of the membrane potential, as measured using standard electrophysiological techniques. The magnitude of the depolarization is dependent on the concentration of both sucrose and protons in a manner which suggests carrier mediation; this process has an apparent K_m for sucrose of about 10 millimolar. Agents interfering with the generation or maintenance of a proton electrochemical gradient eliminate these depolarizations. Electrogenic sugar transport is sensitive to sulfhydryl-modifying reagents; their effect appears to be through a direct interaction with the carrier protein and/or with the process establishing the proton electrochemical gradient across the plasma membrane. p-Chloromercuribenzene sulfonate appears to be a selective inhibitor of the carrier-mediated process itself.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.     

Maltose uptake in the Zea mays scutellum

Maltose transport in slices of the maize scutellum was demonstrated despite the presence of an active maltase situated at the cell surface. The maltase could be inhibited or destroyed by treatments (neutral pH during uptake, pretreatment in Tris buffer at pH 7·5, or in 0·01 N HCl) that allowed appreciable rates of maltose uptake to occur. Using Tris- and HCl-treated slices, it was found that at disaccharide concentrations of 50 and 100 mM, maltose and sucrose were taken up at very nearly the same rates. At sugar concentrations below 50 mM, sucrose was taken up at greater rates than maltose. The maltose content of the slices was directly proportional to the maltose concentration of the bathing solution, and about 4 hr were required for equilibration. From this, it is concluded that one way maltose enters the slices is by free or facilitated diffusion. However, endogenous maltose is utilized by the slices at rates that are much too low to account for the net rates of maltose uptake. Although the slices contain a high level of surface maltase activity, only a low level of endogenous maltase activity was found. This probably accounts for the slow utilization of endogenous maltose. Therefore, the existence of a specific maltose transport system is proposed; a system that contains a carrier saturable with maltose, but one that does not release free maltose into the cytoplasm.     

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

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

Phloem loading of sucrose: Update and opportunities in molecular biology

Sucrose accumulates in the phloem against a concentration gradient via a presumed sucrose-specific carrier protein located at the plasmalemma of the sieve elements/companion cells. Recent evidence suggests that sucrose carrier in soybean is a 62-kDa protein. Immunocytochemical localization has shown the protein to be exclusively at the plasmalemma, which is also the site of sucrose transport. To enhance our understanding of the phenomenon, the structural gene of the sucrose carrier must be cloned and sequenced. Furthermore, development of appropriate probes should help answer long-standing questions relative to the molecular nature of sugar transport and phloem loading, the mechanism of induction/activation of sugar carriers, and developmental regulation of expression of genes encoding such carriers.     

Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator $\text{m-}\text{chlorocarbonylcyanide}$ phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.     

Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli.

Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used. The orientation of the proton gradient was acid inside and alkaline outside. Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692). Phosphate accumulation was found to occur in conjunction with calcium accumulation. Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3). Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport. Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state. These results suggest the existence of a calcium/proton antiport.     

A model proteoliposomal system for proline transport using a purified proline carrier protein from Mycobacterium phlei

The proteoliposomes prepared from purified proline carrier protein isolated from membrane vesicles of Mycobacterium phlei exhibited an uptake of proline, which was dependent upon a proton gradient generated across the lipid bilayer. Although a proton gradient was generated by the reduction of the entrapped ferricyanide by ascorbate oxidation with benzoquinone serving as a lipid soluble hydrogen carrier, transport of proline was dependent on the addition of sodium ion. The movement of sodium and proline across the artificial membrane resulted in a simultaneous collapse of the proton gradient.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.     

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 ΔpH-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 ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-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.     

Mechanism of sugar retention by roots of intact maize and field bean plants

The re-uptake of sugars driven by the proton gradient was studied in sugar net-release and net-uptake experiments using roots of intact maize (Zea mays cv. Blizzard) and field bean (Vicia faba L. cv. Alfred) plants. The net release of sugars into the root medium (0.1 mM CaSO₄) was stimulated by: the protonophore CCCP (10 M); the sulfhydryl reagent NEM (300 M); the specific inhibitor of plasmalemma ATPase vanadate (0.5 mM); and the inhibitor of the glucose carrier phlorizin (2 mM). Net uptake of glucose, fructose and arabinose from 10 M external concentrations was also inhibited by these substances. Surprisingly fusicoccin, a stimulator of net proton release did not effect net sugar uptake. Medium pH values only influenced sugar net uptake if the pH was above 7. It is concluded that a degradation of the proton gradient across the plasmalemma stimulates net sugar release because of disturbed re-uptake of sugars (in particular glucose) via a proton/sugar cotransport system. Thus, the retention of sugars by root cells not only depends on the plasmalemma permeability but also on the electro-chemical proton gradient. If an electro-chemical proton gradient is established by plasmalemma ATPase activity the re-uptake of sugars by proton/sugar cotransport minimizes the release of sugars into the rhizosphere.     

Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force

The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.