Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc⁺Nis⁻ strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc⁻ derivatives from Suc⁺Nis⁻ strains after conjugation and curing experiments.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport 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 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.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na⁺. Other monovalent cations such as K⁺, Li⁺, NH₄⁺ could not replace Na⁺. Experiments with energy-depleted cells have shown that succinate uptake against its concentration gradient can be energized by artificially imposed sodium gradients (ΔpNa).An artificial membrane potential (inside negative) inhibited ΔpNa-driven succinate uptake at pH 7.0 but stimulated it at pH 9.0.The results confirm the suggestion that succinate uptake in E. shaposhnikovii is carried out in symport with Na⁺.     

Sucrose Transport into Plasma Membrane Vesicles from Tobacco Leaves by H<Superscript>+</Superscript> Symport or Counter Exchange Does not Display a Linear Component

Uptake of [¹⁴C]sucrose by plasma membrane vesicles from leaves of tobacco (Nicotiana tabacum L.) was measured after the imposition of an inwardly directed proton gradient (ΔpH = 2) and an electrical gradient (Δψ = −68 mV, inside negative) across the vesicle membrane. The vesicles were isolated from a microsomal fraction by two-phase partitioning using media that contained 330 mM of either sorbitol or sucrose. Sucrose transport into vesicles isolated using the sorbitol-containing media showed the hallmarks of electrogenic H⁺ -symport, as it was highly dependent on ΔpH, could be increased three- to four-fold by Δψ, and was abolished by carbonylcyanide m-chlorophenylhydrazone (CCCP). Transport of [¹⁴C]sucrose into vesicles that were isolated using the sucrose-containing media apparently occurred by counter exchange. Its initial influx also depended on a low external pH, but it was insensitive to CCCP and hardly stimulated by Δψ. Both symport and counter exchange obeyed simple Michaelis-Menten kinetics. Transport that depends linearly on the external sucrose concentration could not be detected, indicating that the ‘linear’ component that has been observed in sucrose uptake by leaf tissues does not represent a transport route that is provided by the sucrose symporter. The potential role of H⁺/sucrose-symporters in phloem unloading is briefly discussed.This revised version was published online in August 2005 with a corrected cover date.     

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H⁺ symport system and via facilitated diffusion.To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H⁺ binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H⁺ binding first, yielding six sets of kinetic parameters.Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H⁺ symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H⁺.In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.     

Involvement of Protons as a Substrate for the Sucrose Carrier during Phloem Loading in Vicia faba Leaves

The effect of pH on uptake of exogenous sucrose by broadbean (Vicia faba L.) leaf discs without the lower epidermis has been investigated at various sucrose concentrations. The concentration dependence of sucrose uptake showed a biphasic saturation response. At high sucrose concentrations (>20 millimolar), sucrose uptake showed no pH dependence. At low sugar concentrations (<5 millimolar), plots of 1/V against 1/H⁺ give straight lines which all intercept at the same point at the left of the ordinal axis. Calculations show that these data agree well with two-substrate kinetics for the carrier, the substrates being the protons and the sucrose molecules. Our results provide further evidence that protonation/deprotonation processes of the carrier are involved in phloem loading, especially for low sucrose concentrations of the apoplast.     

Sucrose Loading in Isolated Veins of Pisum sativum: Regulation by Abscisic Acid, Gibberellic Acid, and Cell Turgor

Enzymatically isolated vein networks from mature pea (Pisum sativum L. cv Alaska) leaves were employed to investigate the properties of sucrose loading and the effect of phytohormones and cell turgor on this process. The sucrose uptake showed two components: a saturable and a first-order kinetics system. The high affinity system (K_m, 3.3 millimolar) was located at the plasmalemma (p-chloromercuriphenylsulfonic acid and orthovanadate sensitivity). Further characterization of this system, including pH dependence and effects of energy metabolism inhibitors, supported the H⁺-sugar symport concept for sucrose loading. Within a physiological range (0.1-100 micromolar) and after 90 min, abscisic acid (ABA) inhibited and gibberellic acid (GA₃) promoted 1 millimolar sucrose uptake. These responses were partially (ABA) or totally (GA₃) turgor-dependent. In experiments of combined hormonal treatments, ABA counteracted the GA₃ positive effects on sucrose uptake. The abolishment of these responses by p-chloromercuriphenylsulfonic acid and experiments on proton flux suggest that both factors (cell turgor and hormones) are modulating the H⁺ ATPase plasmalemma activity. The results are discussed in terms of their physiological relevance.     

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.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

Cytochrome changes in control and concanavalin A-stimulated lymphocytes

α-Aminoisobutyrate (AIB) serves as a transportable, nonmetabolizable alanine analog in the purple sulfur bacterium Chromatium vinosum. AIB transport in C. vinosum appears to be catalyzed by an electrogenic Na⁺-alanine (AIB) symport without any direct participation of ATP-driven or H⁺-symport systems. In addition to Na⁺ being cotransported with AIB via the symport, a transmembrane Na⁺ gradient appears to increase the affinity of the symport of AIB. It appears that these two effects of Na⁺ involve different Na⁺-binding sites.     

Coupling of Na<Superscript>+</Superscript>/α-ketoglutarate symport and PAH/α-ketoglutarate antiport in epithelial cells. Estimation of probability of the anion exchanger spontaneous re-orientation

In this work, dynamics was studied of uptake of p-aminohippurate by basolateral membrane vesicles isolated from rat kidney proximal tubules. The uphill PAH transport into the basolateral membrane vesicles was shown to occur in the presence of α-ketoglutarate and Na⁺-gradient. Based on mathematical model of symport and antiport cooperation, the mechanism of energy coupling of PAH transport via exchanger with Na⁺-dicarboxylate symport is discussed. Based on comparison of our own and literature data, the data analysis shows adequacy of the proposed mathematical model to describe the symport and antiport cooperation. This model has been shown to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.     

Proton-lactate co transport in basolateral membrane vesicles from rat jejunum

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H⁺ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN^–: this finding could explain results recently reported in the literature in which H⁺-lactate symport was not evidenced in basolateral membranes from rat jejunum.     

Phloem Loading of Sucrose: pH Dependence and Selectivity

Autoradiographic, plasmolysis, and ¹⁴C-metabolite distribution studies indicate that the majority of exogenously supplied ¹⁴C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent K_m of the loading process increases at the alkaline pH while the maximum velocity, V_max, is pH-independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose ¹⁴C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport.