Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H(+) efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H(+) antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.

Isolated sugarcane (Saccharum spp. hybrid H50-7209) vacuoles incorporate radioactivity during incubation with labeled UDP-glucose by a mechanism which was postulated to be responsible for sucrose storage in the vacuoles (UDP-glucose group translocator). Analysis of the reaction products in the medium revealed that several enzymic processes are going on during incubation with UDP-glucose such as production of hexose phosphates, UMP, and sugars, all of which seem unrelated to the incorporation of radioactivity into vacuoles. The incorporated radioactiv- (3) found a sucroseproton antiport system in sugar beet vacuoles and Thom and Maretzki (25) detected sucrose formation in sugarcane vacuoles by incubation of isolated vacuoles with UDP-glucose. They postulated a "UDP-glucose group translocator," a tonoplastbound enzyme complex that could achieve formation offructose-6-phosphate from UDP-glucose and subsequently a vectorial synthesis of sucrose-phosphate from the generated fructose-6-phosphate and UDP-glucose. This postulated mechanism of sucrose storage in sugarcane vacuoles attracted much attention because it constituted an entirely new type of transport through membranes. Furthermore, the operation of this mechanism in sugar beet vacuoles was also shown (8,23).According to the postulated scheme of Thom and Maretzki (25) no reaction that required transmembrane energization of the tonoplast was involved, neither a pH-gradient nor a membrane potential. On one side, addition of sucrose in presence of MgATP to vacuoles from sugarcane stalk gave barely perceptible uptake of sucrose (14), while on the other hand MgATP even inhibited the UDP-glucose group translocator (26). The delicate coexistence of tonoplast energization by the ATPase (22) and sucrose storage by the UDP-glucose group translocator was puzzling, especially in the case of sugar beet vacuoles, where the coexistence of the proton-driven sucrose-proton antiport system and the UDP-glucose group translocator was visualized (23). Therefore, as a first step to obtain ideas about the mutual interaction of energization and sucrose storage, a careful analysis of the reaction products, which are produced by incubation of sugarcane vacuoles with UDP-glucose, was performed. MATERIALS AND METHODSIsolation of Protoplasts and Vacuoles. Protoplasts were isolated from 8-to 1 -d-old sugarcane suspension cells (a subclone of Saccharum spp. hybrid H50-7209 grown in supplemented White's basal salt medium) as described by Thom et al. (24) and suspended in White's basal salt medium containing 0.5 M mannitol at pH 5.6. For isolation of vacuoles, the protoplast suspension was layered over a cushion of 12% Ficoll made up in protoplast suspension medium and centrifuged for 1 h in a Kontron TST 54 Rotor at 45,000 rpm. Vacuoles were recovered at the 0/12% Ficoll interface and washed three times in White's basal salt medium containing 0.5 M mannitol at pH 6.5.Uptake Measurements. For uptake measurements, vacuoles were suspended in White's basal salt medium plus 0.5 M manni...

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl2 to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by (31)P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na(+) and Mg(2+) are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K(+) is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg(2+) and Na(+), show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K(+), the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.

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The tonoplast-bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100-fold with two ammonium sulfate precipitation steps and a (3-200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 1 I0 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1 O h sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/ polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54-kDa and the 66-kDa polypeptides exist in multiple copies. N o close resemblance to the membrane-bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody crossreaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase.Many properties of the tonoplast-bound ATPase such as pH-dependence, substrate specificity, ion-dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP-hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, which are strong inhibitors of membrane-bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results arc interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anionThe latex of Hevea Drasiliensis (rubber tree) contains a membrane fraction called lutoids, which according to enzymatic and cytological criteria can be regarded as equivalent to the tonoplast of higher plant cells [l]. An ATPhydrolyzing enzyme, which is bound to the membrane, translocated protons into the intravesicular space [2]. The function of the ATPase is presumably the energization of transport of citrate, lysine and other solutes (31. The ATPase of lutoids is in many respects, such as inhibitor sensitivity and stimulation by chloride ions and ionophores, similar to the ATPase of vacuoles from sugar beet, tulip petals, maize roots and sugarcane [4-71. The lutoid ATPase can therefore be used as a model system for tonoplast ATPase of higher plants with the merits of a vacuolar material, which can be obtained in high yield by simple differential centrifugation of caoutchouc sap.Despite lack of chemical critera it has been suggested that the tonoplast-bound ATPase is more closely related to mitochondrial and chloroplast ATPase than to plasmalemma ATPase [8]...