Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport

The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT-green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter-beta-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type-specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH(4)(+), fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

Transport processes of solutes across the vacuolar membrane of higher plants

The central vacuole is the largest compartment of a mature plant cell and may occupy more than 80% of the total cell volume. However, recent results indicate that beside the large central vacuole, several small vacuoles may exist in a plant cell. These vacuoles often belong to different classes and can be distinguished either by their contents in soluble proteins or by different types of a major vacuolar membrane protein, the aquaporins. Two vacuolar proton pumps, an ATPase and a PPase energize vacuolar uptake of most solutes. The electrochemical gradient generated by these pumps can be utilized to accumulate cations by a proton antiport mechanism or anions due to the membrane potential difference. Uptake can be catalyzed by channels or by transporters. Growing evidence shows that for most ions more than one transporter/channel exist at the vacuolar membrane. Furthermore, plant secondary products may be accumulated by proton antiport mechanisms. The transport of some solutes such as sucrose is energized in some plants but occurs by facilitated diffusion in others. A new class of transporters has been discovered recently: the ABC type transporters are directly energized by MgATP and do not depend on the electrochemical force. Their substrates are organic anions formed by conjugation, e.g. to glutathione. In this review we discuss the different transport processes occurring at the vacuolar membrane and focus on some new results obtained in this field.     

Flavone glucoside uptake into barley mesophyll and Arabidopsis cell culture vacuoles. Energization occurs by H(+)-antiport and ATP-binding cassette-type mechanisms

In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a K(m) of 50 to 100 microM. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H(+) antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta.     

Transport of primary metabolites across the plant vacuolar membrane

Mesophyll cells and most types of storage cells harbor large central vacuoles representing the main cellular store for sugars and other primary metabolites like carboxylic- or and amino acids. The general biochemical characteristics of sugar transport across the vacuolar membrane are already known since a couple of years but only recently the first tonoplast sugar carriers have been identified on the molecular level. A candidate sucrose carrier has been identified in a proteomic approach. In Arabidopsis, the tonoplast monosaccharide transporters (TMT) represent a small protein family comprising only three members, which reside in the vacuolar membrane. Two of three tmt genes are induced upon cold, drought or salt stress and tmt knock out mutants exhibit altered monosaccharide levels upon cold induction. These observations indicate that TMT proteins represent the first examples of tonoplast sugar carriers involved in the cellular response upon osmotic stress stimuli.     

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.     

Vacuoles release sucrose via tonoplast-localised SUC4-type transporters

Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.     

Substrate specificity of the Arabidopsis thaliana sucrose transporter AtSUC2

The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant. A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates. By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides. Sucrose induced inward currents with a K0.5 of 1.44 mM at pH 5 and a membrane potential of -137 mV. Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis. These glucosides were maltose, arbutin (hydroquinone-beta-D-glucoside), salicin (2-(hydroxymethyl)phenyl-beta-D-glucoside), alpha-phenylglucoside, beta-phenylglucoside, alpha-paranitrophenylglucoside, beta-paranitrophenylglucoside, and paranitrophenyl-beta-thioglucoside. In addition, turanose and alpha-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2. The results indicate that AtSUC2 is not highly selective for alpha-over beta-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.     

Vacuoles from Sugarcane Suspension Cultures : II. CHARACTERIZATION OF SUGAR UPTAKE

Vacuoles, isolated from sugarcane (Saccharum sp.) cells, took up 3-O methylglucose and sucrose and the evidence suggests specific transport systems for these sugars. There was no evidence of sugar efflux from preloaded vacuoles. Vacuoles in situ accumulated 3-O methylglucose, sucrose, glucose, and fructose, as shown by incubation of protoplasts with labeled sugar and subsequent analysis of vacuolar and cytoplasmic radio-activity. During the initial minutes of incubation, the amount and concentration of labeled sugar was higher in the cytoplasm than in the vacuole, but subsequently there was active uptake and accumulation into the vacuole. The rate of hexose transfer into the vacuole in situ approached that of hexose uptake by isolated vacuoles; however, the rate of sucrose uptake by isolated vacuoles was below the in situ rate. The site of sucrose synthesis was in the cytoplasm.     

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning,Sugar Signaling,and Seed Yield in Arabidopsis

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyll_ab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO₂ release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development.Sugars fulfill an extraordinarily wide range of functions in plants as well as in other organisms. They serve as valuable energy resources that are easy to store and remobilize. Sugars are required for the synthesis of cell walls and carbohydrate polymers. They are also necessary for starch accumulation and serve as precursors for a range of primary and secondary plant intermediates. From a chemical point of view, sugars represent a large class of metabolites. Among the prominent members in higher plants are the monosaccharides Glc and Fru and the disaccharide Suc (ap Rees, 1994).In contrast to heterotrophic organisms, plants are able to synthesize sugars de novo and to degrade them via oxidative or fermentative metabolism (Heldt, 2005). Net sugar accumulation in plants takes place during the day, whereas net degradation of stored carbohydrate reserves takes place the following night. In higher plants, autotrophic and heterotrophic organs appear to be interconnected by phloem for long-distance transport of sugars (Ruiz-Medrano et al., 2001). Accordingly, sugars must be transported within cells, between cells, and between plant organs. Given these factors, along with the outstanding importance of sugars, it is not surprising that plants sense intracellular sugar availability and use this information to coordinate the expression of many genes (Koch, 1996; Moore et al., 2003).In Arabidopsis (Arabidopsis thaliana), about 60 genes code for putative monosaccharide transport proteins and about 10 genes encode predicted disaccharide carriers (Lalonde et al., 2004). Transport of neutral sugars has been monitored across the plasma membrane, the chloroplast envelope, and the vacuolar membrane (Weber et al., 2000; Niittylä et al., 2004; Martinoia et al., 2007). So far, all sugar carriers residing in the plant plasma membrane have been characterized to catalyze proton-coupled sugar movement (Sauer, 1992; Büttner and Sauer, 2000; Carpaneto et al., 2005). In contrast, both facilitated diffusion and proton-driven antiport mechanisms have been described for monosaccharide and Suc transport across the vacuolar membrane (Thom and Komor, 1984; Daie and Wilusz, 1987; Martinoia et al., 1987; Shiratake et al., 1997; Neuhaus, 2007).In plants, vacuoles fulfill critical functions in the long-term and temporary storage of sugars, sugar alcohols, and other primary metabolites such as carboxylates and amino acids (Dietz et al., 1990; Rentsch and Martinoia, 1991; Martinoia and Rentsch, 1992; Emmerlich et al., 2003). Recently, the first solute carriers responsible for vacuolar Suc and inositol transport have been identified (Endler et al., 2006; Schneider et al., 2008). In addition, TMT (for tonoplast monosaccharide transporter) and VGT (for vacuolar Glc transporter) were the first vacuolar carrier proteins proven to have transport capacity for both Glc and Fru (Wormit et al., 2006; Aluri and Büttner, 2007).TMT exists in three isoforms in Arabidopsis (TMT1–TMT3), and orthologs have been found in other plant species like grapevine (Vitis vinifera), barley (Hordeum vulgare), and rice (Oryza sativa; Wormit et al., 2006). In Arabidopsis, the genes TMT1 and TMT2 are expressed in various tissues, whereas TMT3 is hardly expressed throughout the entire plant life cycle (Wormit et al., 2006). Interestingly, TMT1 and TMT2 are induced by Glc, salt, drought, and cold stress (Wormit et al., 2006), and vacuoles isolated from a TMT1 loss-of-function (T-DNA) Arabidopsis mutant showed reduced Glc import capacity in comparison with corresponding wild-type organelles (Wormit et al., 2006). Moreover, after transfer into the cold, these mutant leaves showed impaired ability to accumulate Glc and Fru, underscoring the in vivo function of TMT under selected conditions (Wormit et al., 2006).However, it is unknown to what extent overexpression of a vacuolar sugar carrier affects subcellular sugar allocation in Arabidopsis. In addition, whether increased vacuolar sugar transport influences sugar signaling, plant development, or organ properties has not been determined. Thus, it is unknown how important controlled activity of vacuolar monosaccharide transport is to plant development or physiological properties. To reveal whether TMT activity affects these processes, we created TMT1-overexpressing Arabidopsis lines and analyzed their physiological and molecular feedbacks.     

Arabidopsis sucrose transporter AtSUC1 is important for pollen germination and sucrose-induced anthocyanin accumulation

The Arabidopsis (Arabidopsis thaliana) sucrose transporter AtSUC1 (At1g71880) is highly expressed in pollen; however, its function has remained unknown. Here, we show that suc1 mutant pollen is defective in vivo, as evidenced by segregation distortion, and also has low rates of germination in vitro. AtSUC1-green fluorescent protein was localized to the plasma membrane in pollen tubes. AtSUC1 is also expressed in roots and external application of sucrose increased AtSUC1 expression in roots. AtSUC1 is important for sucrose-dependent signaling leading to anthocyanin accumulation in seedlings. suc1 mutants accumulated less anthocyanins in response to exogenous sucrose or maltose and microarray analysis revealed reduced expression of many genes important for anthocyanin biosynthesis. The results indicate that AtSUC1 is important for sugar signaling in vegetative tissue and for normal male gametophyte function.     

Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells.     

Analysis of the transport activity of barley sucrose transporter HvSUT1

Localization studies indicate that barley (Hordeum vulgare) sucrose transporter HvSUT1 functions in sucrose uptake into seeds during grain filling. To further understand the physiological function of HvSUT1, we have expressed the HvSUT1 cDNA in Xenopus laevis oocytes and analyzed the transport activity by two-electrode voltage clamping. Consistent with a H(+)-coupled transport mechanism, sucrose induced large inward currents in HvSUT1-expressing oocytes with a K (0.5) of 3.8 mM at pH 5.0 and a membrane potential of -157 mV. Of 21 other sugars tested, four glucosides were also transported by HvSUT1. These glucosides were maltose, salicin (2-(hydroxymethyl) phenyl beta-D-glucoside), alpha-phenylglucoside and alpha-paranitrophenylglucoside. Kinetic analysis of transport of these substrates by HvSUT1 was performed and K (0.5) values were measured. The apparent affinity for all substrates was dependent on membrane potential and pH with lower K (0.5) values at lower external pH and more negative membrane potentials. HvSUT1 was more selective for alpha-glucosides over beta-glucosides than the Arabidopsis sucrose transporter AtSUC2. Several substrates transported by AtSUC2 (beta-phenylglucoside, beta-paranitrophenylglucoside, alpha-methylglucoside, turanose, and arbutin (hydroquinone beta-D-glucoside)) showed low or undetectable transport by HvSUT1. Of these, beta-paranitrophenylglucoside inhibited sucrose transport by HvSUT1 indicating that it interacts with the transporter while arbutin and alpha-methyl glucoside did not inhibit. The results demonstrate significant differences in substrate specificity between HvSUT1 and AtSUC2.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

Novel tonoplast transporters identified using a proteomic approach with vacuoles isolated from cauliflower buds

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane. These results demonstrate that proteomic analysis of highly purified vacuoles from specific tissues allows the identification of new vacuolar proteins and provides an additional view of tonoplastic proteins.     

The role of the vacuole in the accumulation and mobilization of sucrose

The contribution that isolated vacuoles have made to understanding sucrose storage and mobilization is reviewed briefly, with particular reference to the storage root of red beet (Beta vulgaris L.). Work with isolated vacuoles has shown that in this tissue sucrose is confined to the vacuole and some progress has been made in elucidating the possible mechanism of sucrose transport into the vacuole. The evidence that this is a H⁺: sucrose antiport, dependent on the activity of a proton-translocating ATPase is examined. It is concluded that while there is some evidence for the presence of a proton pump, a link between this and sucrose uptake has still to be established. Using isolated vacuoles it has been demonstrated that during mobilization of sucrose, hydrolysis occurs within the vacuole but the mechanism of unloading of hexoses from the vacuole remains to be elucidated.     

Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.     

Na+/H+逆向转运蛋白和植物耐盐性

Na^ /H^ 逆向转运蛋白对植物耐盐起着重要作用,它利用质膜H^ -ATPase或液泡膜H^ -ATPase及PPiase泵H^ 产生的驱动力把Na^ 排出细胞或在液泡中区隔化以消除Na^ 的毒害。主要讨论植物中Na^ /H^ 逆向转运蛋白研究在分子水平的最新进展。     

The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis

The Arabidopsis AtSUC1 protein has previously been characterized as a plasma membrane H+-sucrose symporter. This paper describes the sites of AtSUC1 gene expression and AtSUC1 protein localization and assigns specific functions to this sucrose transporter in anther development and pollen tube growth. RNase protection assays revealed AtSUC1 expression exclusively in floral tissue, which was confirmed by analyses of AtSUC1 promoter-beta-glucuronidase (GUS) plants. In situ hybridizations identified AtSUC1 expression in anther connective tissue, in funiculi and in fully developed pollen grains. Indirect immuno-fluorescence analyses with anti-AtSUC1 antiserum confirmed AtSUC1 protein localization in the connective tissue and funiculi. In mature pollen grains, however, despite high AtSUC1 mRNA levels no AtSUC1 protein was found. Only after pollination of stylar papillae was AtSUC1 protein detected inside the pollen and later inside the growing pollen tubes, suggesting a translation of pre-existing AtSUC1 mRNA after pollination. Pollen germination analyses underlined the important role of sucrose for pollen tube growth. The data presented suggest a role of AtSUC1 in the controlled dehiscence of Arabidopsis anthers. It is postulated that an important function of AtSUC1 is the cell-specific modulation of water potentials.