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1.
Tonoplast and Soluble Vacuolar Proteins Are Targeted by Different Mechanisms 总被引:15,自引:6,他引:9
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The delivery of proteins to the vacuole and its limiting membrane (the tonoplast) by the secretory system is thought to be a dissociative process in which vesicles bud from one compartment and fuse with another. We studied the transport kinetics of phytohemagglutinin (PHA) and tonoplast intrinsic protein (TIP) in mesophyll protoplasts obtained from transgenic tobacco plants transformed with genes encoding these two proteins. In pulse-chase experiments, arrival of PHA in the vacuole was found to be slower (completed 24 hr after synthesis) than the arrival of TIP in the tonoplast (completed 6 hr after synthesis). Brefeldin A and monensin block protein transport by interfering in specific vesicle transport steps. Brefeldin A prevents anterograde vesicle transport between the endoplasmic reticulum and the Golgi, whereas monensin inhibits correct sorting in the trans-Golgi network by disrupting the proton gradient across the membrane. Both inhibitors blocked the transport of PHA to the vacuole and altered the rate at which its complex glycan is processed by Golgi enzymes. Neither drug stopped the arrival of TIP in the tonoplast, suggesting that the flow of vesicles continues in the presence of these inhibitors. We suggest that soluble proteins like PHA and membrane proteins like TIP reach their vacuolar destinations by different paths. 相似文献
2.
Functions of the Vacuole in Higher Plant Cells 总被引:2,自引:0,他引:2
Current views of the activities inherent in the vacuole as a multifunctional compartment in higher plant cells are outlined. The available data indicate that the vacuole is involved in ion homeostasis of the cytosol, storing products of the primary and secondary metabolism, osmoregulation, generation of defense responses of plant cells under biotic and abiotic stress, and programmed cell death. Transport of diverse molecules and ions across the vacuolar membrane, i.e., the tonoplast, plays the major role in all these functional activities of the vacuole. Much progress toward the identification of the transport systems located in this membrane and the elucidation of the mechanisms underlying their functioning has been achieved in the last 10–15 years and has given new insights into the role of the vacuole in the integration and regulation of plant cell metabolism. 相似文献
3.
Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms. 相似文献
4.
Voltage sensitivity of H+/Ca2+ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation 总被引:8,自引:0,他引:8
The electrogenicity of H+/Ca2+ exchange in vacuolar membrane (tonoplast) vesicles from Beta was studied to elucidate the role of this transport system in vacuolar Ca2+ accumulation. To overcome the inherently high proton permeability of tonoplast vesicles, the pH difference established by the primary H(+)-ATPase was titrated to a uniform value by variation of the concentration either of ATP or of a permanent anion (Cl-). This enabled manipulation of membrane potential independently of the transmembrane pH difference, with a higher inside-positive membrane potential produced at lower Cl- concentrations. The rate and the extent of uncoupler-sensitive Ca2+ uptake are both stimulated about 2-fold in conditions of more positive membrane potential, suggesting that the transport system translocates positive charge outward during Ca2+ uptake. A minimum integral H+:Ca2+ stoichiometry of 3 results in a driving force for Ca2+ accumulation in the vacuole amounting to -140 mV in typical physiological conditions. It is concluded that the antiporter is thermodynamically competent to account for Ca2+ accumulation in plant vacuoles and that its reversal in vivo is unlikely. 相似文献
5.
Because of its membrane-impermeant-properties Lucifer Yellow-CH (LY) is regarded by animal cell biologists as an ideal tracer for fluid-phase endocytosis. When presented to plant cells or protoplasts this fluoroprobe accumulates in the vacuole. On the other hand there are many cases where LY does not enter the vacuole when loaded into the plant cytosol. These, superficially divergent, results have previously been explained in terms of endocytosis whereby access to the vacuole is considered to occur through vesicle transport. This interpretation has now been challenged in three recent papers where the benzoic acid derivative, probenecid, has been shown to prevent vacuolar LY accumulation in plants. Since probenecid is a well-known inhibitor of organic anion transport in animal cells it has been argued that anion carriers capable of transporting LY might also exist at the plasma membrane and tonoplast of plant cells. Unfortunately probenecid has rarely, if ever, been used in plant transport studies. The fact that it is a weak acid, whose inhibitory effects are observed at concentrations of around 1 mM suggests that caution should prevail when interpreting results obtained with probenecid. The purpose of this article is therefore to highlight the current controversy surrounding LY uptake by plants and to critically evaluate the recent probenecid data. 相似文献
6.
Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [3H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane. 相似文献
7.
New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress 总被引:1,自引:0,他引:1
Background
The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp. 相似文献8.
Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica
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Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4(+) and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 microM for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine. 相似文献
9.
Loss of Tonoplast Integrity Programmed in Tracheary Element Differentiation 总被引:7,自引:2,他引:5
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Kuriyama H 《Plant physiology》1999,121(3):763-774
A tracheary element (TE) is a typical example of a cell type that undergoes programmed cell death in the developmental processes of vascular plants. The loss of the selective permeability of the tonoplast, which corresponds to tonoplast disintegration, occurred after the cells commenced secondary wall thickening and played a pivotal role in the programmed cell death of TEs in a zinnia (Zinnia elegans L.) cell culture. A search for events specifically associated with the TE vacuole provided an important clue to the understanding of the cell death mechanism. The transport of fluorescein, a fluorescent organic anion, across the tonoplast declined drastically in differentiating TEs. The capacity of the vacuole to accumulate the probe was also impaired. Treatment with probenecid, an inhibitor of organic anion transport, caused rapid cell death of TEs and led to the ultimate disruption of the vacuole even in other types of cultured cells. These changes in vacuolar properties during TE development were suppressed by cycloheximide. Specific mRNA accumulation in cells cultured in a TE differentiation-inductive condition was abolished by probenecid. These results suggest that a change in vacuolar membrane permeability promotes programmed cell death in TEs. 相似文献
10.
Ultrastructural transitions associated with the development of the bladder cells of the trichomes of Atriplex 总被引:1,自引:0,他引:1
Early in development, bladder cells are characterized by the absence of a vacuole or vacuoles, the presence of autophagic vesicles, and numerous, unaggregated ribosomes. With the formation and expansion of the central vacuole, the ribosomes become aggregated and elements of rough endoplasmic reticulum become apparent. This developmental transition is probably related to the production of proteins involved in ion accumulation in the vacuole. Throughout expansion, invaginations of the tonoplast and membraneous structures are associated with the vacuole. These may be indicative of a continued lytic function for this compartment. Also, dictyosomes are continuously present and dictyosome vesicles are associated with both the plasmalemma and tonoplast, which suggest that they contribute to both membrane systems during expansion of the cell and vacuole. 相似文献
11.
E. Martinoia 《Plant biology (Stuttgart, Germany)》1992,105(4):232-245
The vacuole is the largest compartment of a mature plant cell and serves as an internal reservoir of metabolites and nutrients. In the last years transport of solutes across the tonoplast has been intensively investigated. It was shown that two different proton pumps reside in the tonoplast. These pumps generate an electrochemical gradient which can be used as an energy-source to accumulate solutes. Cation uptake is driven by an H+ antiport mechanism. Anions are accumulated in response to the inside positive membrane potential. In addition, the existence of ion channels was shown using the patch clamp technique. The aim of this review is to compare and to discuss the present state of our knowledge of solute transport across the tonoplast. 相似文献
12.
Schulze WX Schneider T Starck S Martinoia E Trentmann O 《The Plant journal : for cell and molecular biology》2012,69(3):529-541
Because they are immotile organisms, higher plants have developed efficient strategies for adaptation to temperature changes. During cold acclimation, plants accumulate specific types of solutes to enhance freezing tolerance. The vacuole is a major solute storage organelle, but until now the role of tonoplast proteins in cold acclimation has not been investigated. In a comparative tonoplast proteome analysis, we identified several membrane proteins with altered abundance upon cold acclimation. We found an increased protein abundance of the tonoplast pyrophosphatase and subunits of the vacuolar V-ATPase and a significantly increased V-ATPase activity. This was accompanied by increased vacuolar concentrations of dicarbonic acids and soluble sugars. Consistently, the abundance of the tonoplast dicarbonic acid transporter was also higher in cold-acclimatized plants. However, no change in the protein abundance of tonoplast monosaccharide transporters was detectable. However, a generally higher cold-induced phosphorylation of members of this sugar transporter sub-group was observed. Our results indicate that cold-induced solute accumulation in the vacuole is mediated by increased acidification of this organelle. Thus solute transport activity is either modulated by increased protein amounts or by modification of proteins via phosphorylation. 相似文献
13.
An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF). 总被引:17,自引:2,他引:17
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The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues. 相似文献
14.
Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass
fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully
turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate,
the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents
7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent
products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate
and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid
and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein
diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent
products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism
has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring
unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal
endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole
system inP. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems. 相似文献
15.
Summary A range of drugs was applied to developing pea seed cotyledons in an attempt to perturb the intracellular transport of newly synthesized vicilin through the endoplasmic reticulum and Golgi vesicles to its site of storage, the vacuole. The most pronounced effects, produced by the ionophores monensin and nigericin, were on Golgi-mediated transport. Unlike the situation in most other tissues that have been studied the number of Golgi vesicles did not increase, suggesting that their movement is not slowed or stopped. However, the Golgi-mediated transport of vicilin was redirected from the vacuole tonoplast to the plasmalemma and the newly synthesized vicilin was released from the cotyledon cells to accumulate between the plasmalemma and the cell wall. 相似文献
16.
Ion transport in Nitellopsis obtusa 总被引:22,自引:0,他引:22
The distribution and rates of exchange of the ions sodium, potassium, and chloride in single internodal cells of the ecorticate characean, Nitellopsis obtusa, have been studied. In tracer experiments three kinetic compartments were found, the outermost "free space" of the cell, a compartment we have called "protoplasmic non-free space", and the cell sap. The concentrations in the vacuole were 54 mM Na(+), 113 mM K(+), and 206 mM Cl(-). The steady state fluxes across the vacuolar membrane were 0.4 pmole Na(+)/cm.(2) sec., 0.25 pmole K(+)/cm.(2) sec., and 0.5 pmole Cl(-)/cm.(2) sec. The protoplasmic Na/K ratio is equal to that in the vacuole but protoplasmic chloride is relatively much lower. Osmotic considerations suggest a layer 4 to 6 micro thick with sodium and potassium concentrations close to those in the vacuole. The fluxes between protoplasm and external solution were of the order of 8 pmoles Na(+)/cm.(2) sec. and 4 pmoles K(+)/cm.(2) sec. We suggest that the protoplasm is separated from the cell wall by an outer protoplasmic membrane at which an outward sodium transport maintains the high K/Na ratio of the cell interior, and from the vacuole by the tonoplast at which an inward chloride transport maintains the high vacuolar chloride. The tonoplast appears to be the site of the principal diffusion resistance of the cell, but the outer protoplasmic membrane probably of the main part of the potential. 相似文献
17.
The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress. 相似文献
18.
Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-linked H+ efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO4, significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity. 相似文献
19.
Abstract. Ascorbate free radical (AFR) induced cell elongation in merislems of Allium cepa roots by promoting a high vacuolization as shown by the increased vacuole volume, vacuole volume density, tonoplast surface and tonoplast surface density. Accordingly, both plasma membrane- and tonoplast-associated ATPases and vacuole soluble acid phosphatase of meristematic cells were also increased. Neither the other subcellular organelles nor cell proliferation appeared to be significantly affected. It is suggested that AFR may be involved in some plasma membrane events related to the initiation of plant cell elongation. 相似文献
20.
Biagini GA Bray PG Spiller DG White MR Ward SA 《The Journal of biological chemistry》2003,278(30):27910-27915
The acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance. 相似文献