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Vacuoles and prevacuolar compartments
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Vacuoles and prevacuolar compartments Paul C Bethke* and Russell L Jones Plant vacuoles are complex and dynamic organelles. Important advances have been made in our understanding of the transporters present in the tonoplast and of the molecular interactions that allow targeting to vacuoles. Despite these advances, markers that permit vacuoles to be defined unambiguously have not yet been identified.
compartments. Particular attention is focused on integral membrane transporters in the tonoplast and vesicle trafficking of proteins to the vacuole. This review is limited to literature published in 1999 to mid-2000 that has particular relevance to these topics.
Transport through the tonoplast Addresses Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720, USA *e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:469–475 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette CAX1 Ca2+/H+ antiporter from Arabidopsis thaliana det3 deetiolated3 ER endoplasmic reticulum TIP tonoplast-intrinsic protein V-ATPase vacuolar H+-ATPase V-PPase vacuolar H+-pyrophosphatase
Introduction The diversity of plant vacuoles
Plant vacuoles are morphologically and functionally diverse organelles, and recent reports have emphasized that there are different kinds of plant vacuoles. Some function primarily as storage organelles, others as lytic compartments. More than one kind of vacuole has been observed in cells undergoing differentiation [1], maturation [2], and autophagy [3], and in fully differentiated cells [4,5]. Vacuole function depends on a suite of soluble and membranebound proteins. These are specifically tailored for each cell type at every developmental stage, and the import and destruction of vacuolar proteins is carefully orchestrated. The paucity of endomembrane markers
A major impediment to our understanding of plant vacuoles is the lack of specific markers for these organelles and other endomembrane components. Different kinds of vacuoles may be morphologically similar, and prevacuolar compartments may be indistinguishable from other singlemembrane-bound organelles. Yet, an accurate interpretation of many kinds of data requires precise identification of endomembrane compartments. Our view of plant vacuoles and prevacuolar compartments is clouded because dependable markers are not available and agreed upon. Markers for these compartments have been proposed, but have not been shown to be unambiguous. Indeed, a unique marker may not exist for some compartments. In this review, we consider recent developments in our understanding of plant vacuoles and prevacuolar
Aquaporins are abundant constituents of all vacuoles
The tonoplast-intrinsic proteins (TIPs) are often the most abundant vacuolar transporters (for review see [6•]). Many of these proteins function as water channels (i.e. aquaporins) when expressed in Xenopus laevis oocytes. Individual plant species typically have several TIP genes, with representatives in evolutionarily conserved classes (e.g. see [7•]). TIP genes within species are differentially regulated, suggesting that different TIPs may be utilized under specific conditions. Two recent reports have shown that water stress affects the abundance of TIP mRNAs and proteins [8•,9•]. In cauliflower florets, the amount of BobTIP26-1 transcript increased rapidly in response to osmotic stress or desiccation [8•]. Transcripts were especially abundant in meristems and vascular tissues. The BobTIP26-1 protein also increased with desiccation. Sunflowers have at least five TIPs, and each has a different tissue-specific expression pattern [9•]. Each of the three sunflower δ-TIPs display a different response to water stress when sunflower roots are exposed to air. In this case, the differential regulation of TIP gene expression in response to changing environmental conditions was gene-specific rather than class-specific. The in vivo function of TIPs remains unclear. Many TIPs transport water (e.g. see [8•,9•]) and TIPs give the tonoplast a high permeability to water [10•]. A role for TIPs in water transport, therefore, seems likely. In a surprising development, Nt-TIPa from tobacco cells was shown to transport urea and glycerol as well as water [11••], leading to the proposal that some plant TIPs function in both water and solute transport. As such, TIPs, in conjunction with their plasmamembrane counterparts, might participate in the long-term regulation of relative cytosolic and vacuolar volumes. TIPs have been proposed to be markers of vacuole function. Jauh et al. [12••] probed pea and barley root tips, and pea cotyledons with antibodies against the carboxy-terminus of α-TIP, γ-TIP and δ-TIP. They determined that different kinds of vacuoles were labeled with different combinations of TIPs. Lytic vacuoles had γ-TIP alone, autophagic vacuoles only α-TIP, and storage vacuoles δ-TIP. Protein storage vacuoles with vegetative storage proteins had either δ-TIP alone or δ-TIP and γ-TIP. Protein storage vacuoles with seed storage proteins had either δ-TIP and α-TIP or δ-TIP, α-TIP and γ-TIP. Jauh et al. [12••] suggested that TIPs may also be markers for vacuole development and
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change. This is an intriguing proposal, but it provokes some concerns and further experimentation is required before it is generally accepted [7•]. These concerns can be outlined as follows: first, because the antibodies used by Jauh et al. to identify TIPs were generated against a part of the protein that is poorly conserved within classes of TIPs and between plant species, it remains to be confirmed that these antibodies identify TIP orthologs in other species; second, the proposal is made on the basis of a small dataset, with only a few cell types and plant species examined; and third, expression patterns for δ-TIPS from spinach, Arabidopsis, sunflower, and radish are different from each other at the cell and tissue levels, calling into question the idea that physiological roles for the different classes of TIPs can be extrapolated from one species to another. ATP- and pyrophosphate-dependent proton pumps
Plant vacuoles are unique among eukaryotic organelles in having two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase). Both H+-pumps are abundant in the tonoplast. The V-ATPase is found throughout the endomembrane system, including the endoplasmic reticulum (ER) and Golgi apparatus, and in the plasma membrane [13]. Recent data indicate that the V-PPase may also be present in the plasma membrane of some tissues [14], though it’s function as a plasma-membrane proton pump has been questioned on thermodynamic grounds [15]. V-PPase is a single polypeptide that acts as a dimer, but V-ATPase is a multi-subunit enzyme made up of integral membrane (V0) and peripheral (V1) domains that are assembled in the ER. The Vo domain attaches the V1 domain to the cytoplasmic face of the membrane, and functions in the translocation of protons into the lumen of the endomembrane compartment. The V1 domain is the catalytic domain and contains three pairs of A and B subunits, and at least five other subunits (i.e. subunits C, D, E, F, and G). The D subunit of V1 has been cloned from Arabidopsis [16], the C subunit from barley [17], and the A subunit from cotton [18•]. The function of the cotton A subunit was demonstrated in yeast, and domain-swapping experiments indicated that the carboxyterminal and amino-terminal domains contain structural information that effects V-ATPase function [18•]. It is not clear why plants have two tonoplast H+-pumps, but experiments with the deetiolated3 (det3) mutant of Arabidopsis have provided some clues. The DET3 gene encodes the C-subunit of the V-ATPase [19••]. When grown in the light, the det3 mutant has an organ-specific reduction in size that results in plants with short hypocotyls but normal leaf blades and roots. This phenotype was attributed to deficiencies in cell expansion in the hypocotyl. det3 mutant plants have reduced amounts of C-subunit mRNA and reduced V-ATPase activity compared to wild-type plants. Because the phenotype reflects a tissue-specific reduction in growth, the authors propose that maximal V-ATPase activity is required in the affected tissues. Additional data suggest that decreased
V-ATPase activity limits the accumulation of vacuolar solutes and, hence, the osmotic driving force for growth. It is clear from the det3 phenotype that the V-ATPase and the V-PPase are not redundant in hypocotyl cells. How the activities of these two vacuolar proton pumps are coordinated remains unknown. Solute transporters in the tonoplast
The proton gradient established by the tonoplast H+-pumps is used to energize H+-coupled transport systems. One of these is the Ca2+/H+ antiporter. Ca2+/H+ antiporters from Arabidopsis (CAX1) [20] and mung bean (VCAX1) [21•] have been cloned, and their function has been demonstrated by complementation of yeast mutants [20,22•]. The subcellular location of VCAX1 was determined by immunoblotting mung-bean microsomal-membrane fractions with antiVCAX1 antibodies, and by visualizing VCAX1 linked to synthetic green fluorescent protein in transgenic tobacco cells [22•]. In both cases, the transporter was predominantly found in the tonoplast, although some appeared to be in the Golgi apparatus. The importance of the Ca2+/H+ antiporter for normal plant growth and development may be inferred from the phenotype of transgenic tobacco plants constitutively expressing the Arabidopsis antiporter CAX1 [23••]. Transformed plants were chlorotic and developed necrotic lesions, dead terminal buds, and stunted roots at much higher frequencies than control plants. Increasing the concentration of Ca2+ in the nutrient solution used to water these plants delayed the onset of visible symptoms. CAX1-expressing lines were hypersensitive to high concentrations of Mg2+ and K+, and had increased sensitivity to chilling temperatures. Plants overexpressing CAX1, therefore, appeared as if they were suffering from a calcium deficiency. The Ca2+-transport activity of tonoplast-enriched vesicles from CAX1-expressing plants was higher than that of control plants, as was the amount of calcium in roots or shoots. On the basis of these results Hirschi et al. suggested that CAX1 activity is regulated improperly in the transformed tobacco plants [23••]. Excess CAX1 activity leads to increased vacuolar Ca2+ accumulation and decreased availability of Ca2+ elsewhere in the plant. These data make it clear that the vacuolar Ca2+/H+ antiporter plays a key role in calcium homeostasis, calcium signaling or both. The importance of a second H+ antiporter, the Na+/H+ antiporter, AtNHX1, from Arabidopsis, was also demonstrated in transgenic plants [24••]. Antibodies against AtNHX1 recognized a protein in Arabidopsis that co-fractionated with markers for the tonoplast, Golgi and ER. The protein was more abundant in transformed Arabidopsis plants than in wild-type plants, and vacuoles isolated from transgenic plants had higher rates of Na+/H+ exchange than vacuoles from wild-type plants. Transgenic plants were virtually unaffected by watering with 200 mM NaCl, whereas control plants were stunted and chlorotic. In light of this dramatic difference, Apse et al. [24••] proposed that
Vacuoles and prevacuolar compartments Bethke and Jones
the salt tolerance of crop plants might be increased by enhancing their ability to sequester Na+ in the vacuole. ATP-binding cassette (ABC)-transporters are ATP-dependent, proton-gradient-independent transporters that are found in the tonoplast of plant cells, where they facilitate the vacuolar accumulation of secondary metabolites and xenobiotics [25,26•]. Like other tonoplast transport proteins, ABC transporters are found throughout the endomembrane system as well as in the plasma membrane [27]. How many kinds of ABC-transporters are present on the tonoplast, what substrates they transport, and how their activity is regulated are important, unanswered questions. Our understanding of how these pumps function has been broadened, however, by a report by Klein et al. [28••] that showed that a multi-drug-resistance-associated protein (MRP)-class ABC transporter from rye has unusual properties. Vacuolar uptake of 7-O-diglucuronyl-4′O-glucuronide was found to be dependent on tonoplast membrane potential but did not require conjugation to glutathione. These findings hint at the diversity of substrates carried by this important class of vacuolar transporters and suggest a potential regulatory mechanism.
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vesicles traffic storage proteins and α-TIP to protein storage vacuoles, whereas clathrin-coated vesicles traffic other proteins to acidic vacuoles [33••]. The interpretation of these data has been extended to suggest that a pathway equivalent to the dense-vesicle-pathway exists even in cells in which dense vesicles are not observed by electron microscopy [34], but this interpretation has been questioned [35]. Precursor-accumulating vesicles in developing pumpkin cotyledons and castor bean endosperm are 200–400 nm in diameter with an electron-dense core and electron-translucent outer layer [36]. Some have additional vesicle-like structures within. In developing pumpkin cotyledons, the core of the precursor-accumulating vesicle contains aggregates of water-insoluble seed-storage proteins, including proforms of 11S globulin and 2S albumin. Aggregates of storage protein precursors are also seen in the ER. A model for precursor-accumulating vesicle formation is that the ER around these aggregates buds off to form vesicles that then bypass the Golgi. The observation that proteins in the periphery of castor bean precursor-accumulating vesicles are glycosylated indicates that Golgi-derived vesicles may fuse with precursor-accumulating vesicles prior to delivery of their contents to protein-storage vacuoles.
Vesicular transport to the vacuole Routes for protein transport to the vacuole
Proteins destined for the vacuole are synthesized on the ER and delivered by vesicles to the vacuole (for reviews see [29–32]). Most soluble proteins transported to the vacuole pass through the Golgi apparatus, where they are sorted into vesicles. These vesicles may then fuse with a prevacuolar compartment or directly with the tonoplast. Alternative routes that bypass the Golgi or begin as endocytotic vesicles are likely to exist. Morphologically distinct transport vesicles carry proteins to the vacuole
Three classes of morphologically distinct vesicles that transport proteins from the Golgi or ER to the vacuole have been identified: dense vesicles, clathrin-coated vesicles and precursor-accumulating vesicles. Dense vesicles in developing pea cotyledons are 130 nm in diameter, uncoated when released from the Golgi, and contain an electron-dense core. Clathrin-coated vesicles have a clathrin coat when they leave the Golgi. In an elegant series of experiments, Hinz et al. [33••] presented compelling evidence that suggests that dense vesicles and clathrin-coated vesicles participate in two different vesicular sorting pathways. They used cell fractionation to produce samples that were enriched in either dense vesicles or clathrin-coated vesicles. The densevesicle-enriched fraction contained prolegumin but little BP-80 (the putative vacuolar-sorting signal receptor of 80 kiloDaltons [kDa]), whereas the clathrin-coated-vesicleenriched fraction contained BP-80 but no prolegumin. Immuno-electron microscopy provided complimentary data: antibodies to BP-80 labeled Golgi stacks but not dense vesicles, whereas antibodies against α-TIP labeled Golgi stacks and dense vesicles. Hinz et al. concluded that dense
Precursor-accumulating vesicles have not been observed in vegetative tissues of Arabidopsis. In transgenic Arabidopsis expressing a truncated form of pumpkin 2S albumin linked to phosphinothricin acetyltransferase, however, vesicles similar to precursor-accumulating vesicles accumulated in the cotyledons and leaves [37•]. These vesicles contained 2S albumin and phosphinothricin acetyltransferase. Because only a proform of the protein was detected, Hayashi et al. [37•] suggested that these vesicles were not delivered to vacuoles in these vegetative cells. These experiments raise interesting questions about the signals required for the formation of precursor-accumulating vesicles and their delivery. They also point out that care must be exercised when interpreting data from experiments that use transgenic plants to study protein trafficking. Overexpressed proteins, or proteins from heterologous systems, may be transported via alternative or novel pathways if the normal transport mechanisms are overwhelmed or absent.
Targeting and delivery of proteins to vacuoles requires specific molecular interactions Vacuolar sorting signals
The orderly sorting of proteins into transport vesicles requires recognition of vacuolar sorting signals by vesicleassociated receptors. Three classes of vacuolar signals have been characterized in plants: first, short sequences within an amino-terminal propeptide containing a consensus sequence of NPIR or NPIXL (using the single-letter code for amino acids); second, short sequences with no identified consensus sequence at the carboxyl terminus of a carboxy-terminal propeptide; and third, structural domains within the mature protein. Evidence suggests that the amino-terminal vacuolar sorting signal targets proteins to
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lytic vacuoles, and that proteins with amino- and carboxyterminal vacuolar sorting signals are sorted by different mechanisms [38•]. Candidate receptors for amino-terminal vacuolar sorting signals have been identified, and binding to synthetic peptides containing amino-terminal vacuolar sorting signals has been demonstrated for BP-80 [39], PV72 (for putative vacuolar-sorting receptor of 72 kDa) and PV82 [40]. Domains that are important for the interaction of BP-80 with the amino-terminal vacuolar sorting signal of proaleurain have also been identified [41•]. The Nicotiana alata proteinase inhibitor precursor protein (Na-PI) undergoes proteolytic processing to produce protease inhibitors that are located in the vacuole. Sorting determinants that direct Na-PI to the vacuole have been characterized [42•]. The carboxy-terminus of Na-PI is required for targeting to the vacuole and does not contain an NPIR or NPIXL consensus sequence. Pre-NaPI co-localizes with proteins having antigenic properties similar to BP-80 and the t-SNARE AtPEP12p. In transgenic plants overexpressing Na-PI, an association of pre-Na-PI with BP-80 was demonstrated in immunoprecipitation experiments. These data led the authors to conclude that BP-80 binds to a carboxy-terminal vacuolar sorting signal that is substantially different from NPIR/NPIXL. This is an important finding that needs confirmation. In particular, binding of BP-80 to the carboxy-terminus needs to be demonstrated. As some Na-PI may have been incorrectly targeted to the BP-80-containing pathway in these transgenic plants, traffic through alternative pathways needs to be quantified [34]. Some vacuolar proteins are present in more than one kind of vacuole, suggesting that these proteins have multiple vacuolar sorting signals. This was demonstrated for the 20 kDa potato-tuber protein, PT20 [43•]. The amino-terminal prepropeptide of PT20 contains a vacuolar sorting signal (NPINL) that targeted sweet potato sporamin to vacuoles. This transport was relatively insensitive to wortmanin, a compound that preferentially blocks the transport of dense vesicles relative to that of clathrin-coated vesicles. The carboxy-terminal 13 amino acids of PT20 also targeted sporamin to the vacuole. This transport was sensitive to wortmanin and greatly reduced by the addition of one or more glycine residues at the carboxyl terminus. Further experiments showed that the carboxy-terminal sequence SFKQVQ functions as a vacuolar sorting signal. Whether the amino- or carboxy-terminal vacuolar sorting signal is used preferentially in planta remains unknown. SNARES are involved in transport to vacuoles
Vesicle transport of proteins requires the docking and fusion of transport vesicles with target organelles. In the SNARE hypothesis, docking is accomplished through a specific interaction between an integral membrane protein on the vesicle (v-SNARE) and an integral membrane protein on the target organelle (t-SNARE). Each kind of vacuole or intermediate compartment must have one or
more t-SNAREs, and each kind of vacuole is likely to have a unique v-SNARE (for review see [44]). A few v- and t-SNAREs have been identified in plants, but many more remain undiscovered. Two v-SNAREs, AtVTI1a and AtVTI1b, were recently cloned from Arabidopsis because of their homology to the yeast v-SNARE Vti1p [45•]. Vti1p interacts with the yeast prevacuolar t-SNARE PEP12p, and AtVTI1a was immunoprecipitated with the putative Arabidopsis t-SNARE AtPEP12p. AtVTI1a could also substitute for Vti1p in Golgi-to-prevacuole transport in yeast. When visualized by immuno-electron microscopy, an AtVTI1a-containing construct that was overexpressed in transgenic Arabidopsis was found in the Golgi and electron-dense vesicles near the Golgi with equal frequency. AtVTI1b substituted for Vti1p in yeast vacuolar import pathways other than the Golgi-toprevacuole pathway. In yeast, therefore, AtVTI1a and AtVTI1b act as v-SNAREs with different functions. Whether this is true in plants is unknown. Several t-SNAREs have been identified in plants. AtPEP12p, a homolog of the yeast t-SNARE PEP12p, was one of the first t-SNAREs cloned from plants, and recent attempts have been made to demonstrate its function [46]. α-SNAP (i.e. α-soluble N-ethylmaleimide-sensitive factor attachment protein) binds to the SNARE complex and facilitates the formation of a 20S complex that dissociates in the presence of ATP. AtPEP12p was shown to bind recombinant mammalian α-SNAP. When detergent-solubilized Arabidopsis root proteins were separated on a glycerol density gradient, AtPEP12p was present in complexes of less than 4S to more than 20S. In the presence of ATP, the size distribution of these complexes shifted to less than 11S. These data suggest functional parallels between AtPEP12p and t-SNARES such as PEP12p. A homolog of AtPEP12p, AtVAM3p, has also been characterized [47•]. AtVAM3p interacts with AtVTI1a in immunoprecipitation experiments, suggesting that it has a role in post-Golgi trafficking. On sucrose gradients, AtVAM3p was not separated from AtPEP12p, leaving open the question of whether these two proteins play redundant or separate roles in vesicle trafficking.
The uncertain nature of prevacuolar compartments Although there is agreement that plant cells might contain a prevacuolar compartment, the nature of this organelle remains unclear. Prevacuoles are defined as organelles that receive cargo from transport vesicles and subsequently deliver that cargo to the vacuole by fusion with the tonoplast. Alternatively, a prevacuole can be defined as an organelle that contains t-SNAREs and v-SNAREs, which bind to v-SNAREs on transport vesicles and t-SNAREs on vacuoles, respectively. To date, a prevacuolar compartment that fits either of these definitions has not been unequivocally identified [48•].
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The multi-vesicular body in developing pea cotyledons has been proposed to be a prevacuolar compartment, largely on the basis of results from immuno-electron microscopy. In this tissue, multi-vesicular bodies are strongly labelled with antibodies to legumin and α-TIP, both vacuolar proteins. Multi-vesicular bodies also accumulate tracers for endocytosis. Interestingly, monensin prevents dense-vesicle traffic in pea cotyledons and results in swelling of multi-vesicular bodies and Golgi. These are tantalizing observations, but further support is needed to define the function of the multi-vesicular body and to assign to it the role of prevacuolar compartment. An AtPEP12p-containing compartment in Arabidopsis has also been called a prevacuolar compartment [45•,46]. Electron micrographs show this putative prevacuolar compartment to be a vesicular/tubular compartment of less than 100 nm in diameter. Cell fractionation and biochemical data showed that AtPEP12p was located in a post-Golgi compartment [49], and this compartment has subsequently been called a prevacuolar compartment [45•,46,50]. Unambiguous interpretation of these biochemical data is difficult, however, because the AtPEP12p-containing compartment was not cleanly resolved on density gradients. Likewise, the electron micrographs are difficult to interpret because the subcellular structures were poorly preserved. Additional data are needed to confirm that the AtPEP12p-containing compartment is a prevacuolar compartment.
Conclusions Plant vacuoles and prevacuolar compartments are part of a continuum of endomembrane compartments. All of these compartments are specialized for individual functions. Most of them are dynamic and can change morphologically and functionally to suit the needs of the cell. Although our understanding of plant vacuoles remains rudimentary, we are beginning to appreciate the plasticity of this organelle. Rapid progress is being made in the areas of tonoplast transport and the regulation and import of vacuolar proteins. The same cannot be said for other, equally fundamental aspects of vacuole biology. The mechanisms that control vacuole identity are unknown, so too are the means by which one vacuole fuses with another or fragments into many. The challenge for plant biologists is to merge views of vacuole function, morphology and biochemistry into a clear, unified picture that encompasses the dynamic nature of the vacuole.
Acknowledgements The authors thank Masayoshi Maeshima and Christophe Maurel for critical review of this manuscript. Eleanor Crump assisted in editing the manuscript and her help is gratefully acknowledged.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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6. Kaldenhoff R, Eckert M: Features and function of plant aquaporins. • J Photochem Photobiol B Biol 1999, 52:1-6. This brief review of plant aquaporins is wonderfully inclusive and up to date. It is an excellent introduction to the field. 7. •
Karlsson M, Johansson I, Bush M, McCann MC, Maurel C, Larsson C, Kjellbom P: An abundant TIP expressed in mature highly vacuolated cells. Plant J 2000, 21:83-90. A TIP from spinach leaf, So-δTIP, was cloned and characterized. The expression pattern of So-δTIP was unlike those of the δ-TIPs in Arabidopsis, sunflower and radish. This raises questions about the hypothesis proposed in [12••] that the TIPs present on the tonoplast are markers for vacuoles with different functions. 8. •
Barrieu F, Marty-Mazars D, Thomas D, Chaumont F, Charbonnier M, Marty F: Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells. Planta 1999, 209:77-86. In response to severe water-stress treatments, mRNA abundance for the aquaporin BobTIP26 increased within 30 minutes of the onset of osmotic stress and 2 hours of the onset of desiccation. These data show that TIP genes can be rapidly upregulated by water stress. 9. •
Sarda X, Tousch D, Ferrare K, Cellier F, Alcon C, Dupuis JM, Casse F, Lamaze T: Characterization of closely related delta-TIP genes encoding aquaporins which are differentially expressed in sunflower roots upon water deprivation through exposure to air. Plant Mol Biol 1999, 40:179-191. Sunflower roots were exposed to air and the transcript abundance for each gene determined as the roots dried. SunTIP7, SunTIP18 and SunTIP20 are homologs of Arabidopsis δ-TIP. There was an increase in SunTIP7 transcript, a decrease in SunTIP18 transcript and a transient increase in SunTIP20 transcript. These data clearly show that TIP genes are responsive to environmental conditions and that closely related TIP-genes can be differentially regulated. 10. Morillon R, Lassalles J-P: Osmotic water permeability of isolated • vacuoles. Planta 1999, 210:80-84. Osmotic permeability values were calculated for isolated vacuoles from various species and tissues. The data suggest that all vacuoles contain aquaporins and that water transport through the lipid part of the tonoplast is relatively low. 11. Gerbeau P, Guclu J, Ripoche P, Maurel C: Aquaporin Nt-TIPa can •• account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes. Plant J 1999, 18:577-587. A cDNA for the novel TIP Nt-TIPa was cloned and the protein localized to the tonoplast. Nt-TIPa expressed in Xenopus oocytes is permeable to water as well as to urea and glycerol. It was suggested that the Nt-TIPa protein could account for the permeability of the tonoplast to neutral solutes. A dual role for aquaporins in water and solute transport is proposed. 12. Jauh G-Y, Phillips TE, Rogers JC: Tonoplast intrinsic protein •• isoforms as markers for vacuolar functions. Plant Cell 1999, 11:1867-1882. Antipeptide antibodies to the tonoplast-intrinsic proteins α-TIP, γ-TIP and δ-TIP were used to label vacuoles in pea and barley roots and in pea cotyledons. Combinations of TIPs labeled vacuoles in a tissue- and developmental-stage-specific manner. These data gave rise to the intriguing hypothesis that the TIPs present on the tonoplast are markers for vacuoles with different functions. The applicability of this hypothesis to other species and tissues should be determined quickly, as there is a pressing need to identify markers for endomembrane compartments. 13. Sze H, Li X, Palmgren MG: Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. Plant Cell 1999, 11:677-689. 14. Ratajczak R, Hinz G, Robinson DG: Localization of pyrophosphatase in membranes of cauliflower inflorescence cells. Planta 1999, 208:205-211.
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Tavakoli N, Eckerskorn C, Golldack D, Dietz K-J: Subunit C of the vacuolar H+-ATPase of Hordeum vulgare. FEBS Lett 1999, 465:68-72.
18. Kim W, Wan C-Y, Wilkins TA: Functional complementation of yeast • vma1DELTA cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+-ATPase activity. Plant J 1999, 17:501-510. The A subunit from the cotton H+-ATPase was expressed in yeast vma1∆ cells, and shown to rescue the mutant phenotype even though V-ATPase activity was only partially restored. These data show that A subunits of plants and yeast have conserved functional domains. 19. Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J: •• The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes Devel 1999, 13:3259-3270. The DET3 gene from Arabidopsis was isolated by positional cloning and found to encode the C subunit of the V-ATPase. Det3 mutant plants have only half the amount of wild-type C-subunit protein. In the Det3 mutants, tissuespecific defects in growth, particularly reduced elongation of hypocotyls, petioles and inflorescence stems, were attributed to reduced cell expansion. A link between the V-ATPase and cell expansion was envisioned in which the proton-coupled accumulation of vacuolar solutes maintains the osmotic potential of the enlarging vacuole and permits growth. 20. Hirschi KD, Zhen R-G, Cunningham KW, Rea PA, Fink GR: CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proc Natl Acad Sci USA 1996, 93:8782-8786. 21. Ueoka-Nakanishi H, Nakanishi Y, Tanaka Y, Maeshima M: Properties • and molecular cloning of Ca2+/H+ antiporter in the vacuolar membrane of mung bean. Eur J Biochem 1999, 262:417-425. Ca2+-ATPase and Ca2+/H+antiport activities were characterized in tonoplast vesicles. The authors propose an attractive model in which the Ca2+/H+ antiporter and the Ca2+-ATPase work together to lower cytosolic Ca2+ concentrations following a stimulus-induced rise in cytosolic free calcium. 22. Ueoka-Nakanishi H, Tsuchiya T, Sasaki M, Nakanishi Y, • Cunningham KW, Maeshima M: Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells. Eur J Biochem 2000, 267:3090-3098. VCAX1p is shown to be a functional Ca2+/H+ antiporter that is predominantly located in the tonoplast. The possibility that VCAX1p also functions in the Golgi is discussed. 23. Hirschi KD: Expression of Arabidopsis CAX1 in tobacco: altered •• calcium homeostasis and increased stress sensitivity. Plant Cell 1999, 11:2113-2122. Transgenic tobacco plants expressing the Ca2+/H+ antiporter CAX1 appear to suffer from calcium deficiency. The data reported here indicate that the Ca2+/H+ antiporter is important for calcium homeostasis or signaling in wild-type plants. 24. Apse MP, Aharon GS, Snedden WA, Blumwald E: Salt tolerance •• conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 1999, 285:1256-1258. Overexpression of the vacuolar Na+/H+ antiporter is shown to be a potential mechanism for engineering salt tolerance into plants. 25. Rea PA, Li Z-S, Lu Y-P, Drozdowicz YM: From vacuolar GS-X pumps to multispecific ABC transporters. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:727-760. 26. Rea PA: MRP subfamily ABC transporters from plants and yeast. • J Exp Botany 1999, 50:895-913. The MRP subfamily of ABC transporters may be the most important subfamily of these transporters in plants. The structural and functional characteristics of these transporters in yeast and plants are discussed in this highly detailed review. 27.
Sidler M, Hassa P, Hasan S, Ringli C, Dudler R: Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light. Plant Cell 1998, 10:1623-1636.
28. Klein M, Martinoia E, Hoffmann-Thoma G, Weissenboeck G: A •• membrane-potential dependent ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides: regulation of glucuronide uptake by glutathione and its conjugates. Plant J 2000, 21:289-304. The energy-dependent uptake of two flavones by an MRP-like transporter with broad specificity was characterized. In experiments with isolated tonoplast vesicles, application of valinomycin doubled the uptake rate of one flavone in the presence of a 10-fold K +-gradient, but had little effect in the
absence of a K+-gradient. These data suggest that flavone transport is dependent on membrane potential. This finding raises the possibility that the activity of some MRP-like transporters may be coordinated with the activity of other tonoplast transporters, especially the proton pumps. 29. Jiang L, Rogers JC: Sorting of membrane proteins to vacuoles in plant cells. Plant Sci 1999, 146:55-67. 30. Hawes CR, Brandizzi F, Andreeva AV: Endomembranes and vesicle trafficking. Curr Opin Plant Biol 1999, 2:454-461. 31. Marty F: Plant vacuoles. Plant Cell 1999, 11:587-599. 32. Miller EA, Anderson MA: Uncoating the mechanisms of vacuolar protein transport. Trends Plant Sci 1999, 4:46-48. 33. Hinz G, Hillmer S, Baumer M, Hohl I: Vacuolar storage proteins and •• the putative vacuolar sorting receptor BP-80 exit the Golgi apparatus of developing pea cotyledons in different transport vesicles. Plant Cell 1999, 11:1509-1524. Localization studies using cell fractionation and immuno-electron microscopy show that Golgi-derived dense vesicles contain storage proteins and that clatherin-coated vesicles contain BP-80. These data lend support to a model for protein import in which proteins destined for lytic vacuoles are sorted into separate transport vesicles from proteins destined for storage vacuoles. 34. Jiang L, Rogers JC: The role of BP-80 and homologs in sorting proteins to vacuoles. Plant Cell 1999, 11:2069-2071. 35. Miller E, Anderson M: The role of BP-80 and homologs in sorting proteins to vacuoles. Plant Cell 1999, 11:2071-2073. 36. Hara-Nishimura I, Shimada T, Hatano K, Takeuchi Y, Nishimura M: Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell 1998, 10:825-836. 37. •
Hayashi M, Toriyama K, Kondo M, Hara-Nishimura I, Nishimura M: Accumulation of a fusion protein containing 2S albumin induces novel vesicles in vegetative cells of Arabidopsis. Plant Cell Physiol 1999, 40:263-272. A chimeric gene was constructed that contained a truncated pumpkin 2S albumin linked to a selectable marker. When expressed in vegetative cells of Arabidopsis, the protein product accumulated in novel vesicles that had characteristics of precursor-accumulating vesicles. These data suggest that the formation of precursor-accumulating vesicles is driven by the synthesis of specific proteins. They also highlight the risk of using transgenic plants to study protein targeting.
38. Matsuoka K, Neuhaus J-M: Cis-elements of protein transport to the • plant vacuoles. J Exp Botany 1999, 50:165-174. This is an excellent review containing detailed sequence comparisons of vacuolar sorting signals. These are discussed in the context of protein-sorting receptors and putative transport pathways. 39. Kirsch T, Paris N, Butler JM, Beevers L, Rogers JC: Purification and initial characterization of a potential plant vacuolar targeting receptor. Proc Natl Acad Sci USA 1994, 91:3403-3407. 40. Shimada T, Kuroyanagi M, Nishimura M, Hara-Nishimura I: A pumpkin 72-kDa membrane protein of precursor-accumulating vesicles has characteristics of a vacuolar sorting receptor. Plant Cell Physiol 1997, 38:1414-1420. 41. Cao XF, Rogers SW, Butler J, Beevers L, Rogers JC: Structural • requirements for ligand binding by a probable plant vacuolar sorting receptor. Plant Cell 2000, 12:493-506. A soluble, truncated form of BP-80 was produced, and the structural domains required for binding to a synthetic proaleurain peptide were identified. A model for the interaction of BP-80 with proaleurain is presented that may indicate how BP-80 binds amino-terminal vacuolar sorting signals in general. 42. Miller EA, Lee MCS, Anderson MA: Identification and • characterization of a prevacuolar compartment in stigmas of Nicotiana alata. Plant Cell 1999, 11:1499-1508. Data are presented that show an association between the vacuolar sorting signal receptor BP-80 and the precursor form of Na-PI. The suggestion is made that BP-80 may bind to a carboxy-terminal vacuolar sorting signal. This is a novel finding that challenges prevailing views of how vacuolar proteins are sorted into transport vesicles. 43. Koide Y, Matsuoka K, Ohto M-A, Nakamura K: The N-terminal • propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals. Plant Cell Physiol 1999, 40:1152-1159. Putative targeting signals from the amino and carboxyl terminus of the 20-kDa potato tuber protein were linked to sweet potato sporamin in order to assess their effectiveness in targeting sporamin to the vacuole. Both a canonical aminoterminal vacuolar sorting signal and a carboxy-terminal sorting signal functioned
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in vacuolar targeting. The conditions under which each is used, and whether targeting is developmentally regulated remain important, unanswered questions. 44. Gerst JE: SNAREs and SNARE regulators in membrane fusion and exocytosis. Cell Mol Life Sci 1999, 55:707-734. 45. Zheng H, Fischer von Mollard G, Kovaleva V, Stevens TH, Raikhel NV: • The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment. Mol Biol Cell 1999, 10:2251-2264. Two Arabidopsis homologs of the yeast v-SNARE vti1p were identified. Complementation analysis in yeast vti1 mutants indicated that AtVTI1a functioned in yeast Golgi-to-prevacuole transport and AtVTI1b in two alternative pathways. AtVTI1a co-localized with the putative vacuolar sorting signal receptor AtELP and the t-SNARE AtPEP12p. A model is presented in which proteins recognized by AtELP are sorted into vesicles containing AtVTI1a, which are subsequently targeted to a post-Golgi compartment through an interaction with AtPEP12p. 46. Bassham DC, Raikhel NV: The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP. Plant J 1999, 19:599-603. 47. •
Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV: The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells. Plant Physiol 1999, 121:929-938. AtVAM3p, like its homolog AtPEP12p, was shown to interact with the v-SNARE AtVTI1a. AtVAM3p was not separated from AtPEP12p on
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sucrose density gradients, and antibodies to both t-SNAREs labeled the same compartments as seen by immuno-electron microscopy. The authors propose that plant endomembrane compartments may have more than one t-SNARE. Whether AtVAM3p and AtPEP12p are redundant, or act in cell-specific or pathway-specific transport requires further experimentation. 48. Robinson DG, Hinz G: Golgi-mediated transport of seed storage • proteins. Seed Sci Res 1999, 9:267-283. This is a comprehensive review of Golgi morphology and function, and of Golgi-mediated transport of seed storage proteins, that is illustrated with examples from the authors’ research. Particular emphasis is given to vesicular transport and the sorting of storage proteins from proteins destined for lytic vacuoles. The evidence for prevacuolar compartments is briefly reviewed. The authors suggest that multivesicular bodies may be prevcuolar compartments. 49. Conceicao ADS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, Raikhel NV: The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants. Plant Cell 1997, 9:571-582. 50. Sanderfoot AA, Ahmed SU, Marty-Mazars D, Rapoport I, Kirchhausen T, Marty F, Raikhel NV: A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots. Proc Natl Acad Sci USA 1998, 95:9920-9925.
Vacuoles and prevacuolar compartments Paul C Bethke* and Russell L Jones Plant vacuoles are complex and dynamic organelles. Important advances have been made in our understanding of the transporters present in the tonoplast and of the molecular interactions that allow targeting to vacuoles. Despite these advances, markers that permit vacuoles to be defined unambiguously have not yet been identified.
compartments. Particular attention is focused on integral membrane transporters in the tonoplast and vesicle trafficking of proteins to the vacuole. This review is limited to literature published in 1999 to mid-2000 that has particular relevance to these topics.
Transport through the tonoplast Addresses Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720, USA *e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:469–475 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette CAX1 Ca2+/H+ antiporter from Arabidopsis thaliana det3 deetiolated3 ER endoplasmic reticulum TIP tonoplast-intrinsic protein V-ATPase vacuolar H+-ATPase V-PPase vacuolar H+-pyrophosphatase
Introduction The diversity of plant vacuoles
Plant vacuoles are morphologically and functionally diverse organelles, and recent reports have emphasized that there are different kinds of plant vacuoles. Some function primarily as storage organelles, others as lytic compartments. More than one kind of vacuole has been observed in cells undergoing differentiation [1], maturation [2], and autophagy [3], and in fully differentiated cells [4,5]. Vacuole function depends on a suite of soluble and membranebound proteins. These are specifically tailored for each cell type at every developmental stage, and the import and destruction of vacuolar proteins is carefully orchestrated. The paucity of endomembrane markers
A major impediment to our understanding of plant vacuoles is the lack of specific markers for these organelles and other endomembrane components. Different kinds of vacuoles may be morphologically similar, and prevacuolar compartments may be indistinguishable from other singlemembrane-bound organelles. Yet, an accurate interpretation of many kinds of data requires precise identification of endomembrane compartments. Our view of plant vacuoles and prevacuolar compartments is clouded because dependable markers are not available and agreed upon. Markers for these compartments have been proposed, but have not been shown to be unambiguous. Indeed, a unique marker may not exist for some compartments. In this review, we consider recent developments in our understanding of plant vacuoles and prevacuolar
Aquaporins are abundant constituents of all vacuoles
The tonoplast-intrinsic proteins (TIPs) are often the most abundant vacuolar transporters (for review see [6•]). Many of these proteins function as water channels (i.e. aquaporins) when expressed in Xenopus laevis oocytes. Individual plant species typically have several TIP genes, with representatives in evolutionarily conserved classes (e.g. see [7•]). TIP genes within species are differentially regulated, suggesting that different TIPs may be utilized under specific conditions. Two recent reports have shown that water stress affects the abundance of TIP mRNAs and proteins [8•,9•]. In cauliflower florets, the amount of BobTIP26-1 transcript increased rapidly in response to osmotic stress or desiccation [8•]. Transcripts were especially abundant in meristems and vascular tissues. The BobTIP26-1 protein also increased with desiccation. Sunflowers have at least five TIPs, and each has a different tissue-specific expression pattern [9•]. Each of the three sunflower δ-TIPs display a different response to water stress when sunflower roots are exposed to air. In this case, the differential regulation of TIP gene expression in response to changing environmental conditions was gene-specific rather than class-specific. The in vivo function of TIPs remains unclear. Many TIPs transport water (e.g. see [8•,9•]) and TIPs give the tonoplast a high permeability to water [10•]. A role for TIPs in water transport, therefore, seems likely. In a surprising development, Nt-TIPa from tobacco cells was shown to transport urea and glycerol as well as water [11••], leading to the proposal that some plant TIPs function in both water and solute transport. As such, TIPs, in conjunction with their plasmamembrane counterparts, might participate in the long-term regulation of relative cytosolic and vacuolar volumes. TIPs have been proposed to be markers of vacuole function. Jauh et al. [12••] probed pea and barley root tips, and pea cotyledons with antibodies against the carboxy-terminus of α-TIP, γ-TIP and δ-TIP. They determined that different kinds of vacuoles were labeled with different combinations of TIPs. Lytic vacuoles had γ-TIP alone, autophagic vacuoles only α-TIP, and storage vacuoles δ-TIP. Protein storage vacuoles with vegetative storage proteins had either δ-TIP alone or δ-TIP and γ-TIP. Protein storage vacuoles with seed storage proteins had either δ-TIP and α-TIP or δ-TIP, α-TIP and γ-TIP. Jauh et al. [12••] suggested that TIPs may also be markers for vacuole development and
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change. This is an intriguing proposal, but it provokes some concerns and further experimentation is required before it is generally accepted [7•]. These concerns can be outlined as follows: first, because the antibodies used by Jauh et al. to identify TIPs were generated against a part of the protein that is poorly conserved within classes of TIPs and between plant species, it remains to be confirmed that these antibodies identify TIP orthologs in other species; second, the proposal is made on the basis of a small dataset, with only a few cell types and plant species examined; and third, expression patterns for δ-TIPS from spinach, Arabidopsis, sunflower, and radish are different from each other at the cell and tissue levels, calling into question the idea that physiological roles for the different classes of TIPs can be extrapolated from one species to another. ATP- and pyrophosphate-dependent proton pumps
Plant vacuoles are unique among eukaryotic organelles in having two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase). Both H+-pumps are abundant in the tonoplast. The V-ATPase is found throughout the endomembrane system, including the endoplasmic reticulum (ER) and Golgi apparatus, and in the plasma membrane [13]. Recent data indicate that the V-PPase may also be present in the plasma membrane of some tissues [14], though it’s function as a plasma-membrane proton pump has been questioned on thermodynamic grounds [15]. V-PPase is a single polypeptide that acts as a dimer, but V-ATPase is a multi-subunit enzyme made up of integral membrane (V0) and peripheral (V1) domains that are assembled in the ER. The Vo domain attaches the V1 domain to the cytoplasmic face of the membrane, and functions in the translocation of protons into the lumen of the endomembrane compartment. The V1 domain is the catalytic domain and contains three pairs of A and B subunits, and at least five other subunits (i.e. subunits C, D, E, F, and G). The D subunit of V1 has been cloned from Arabidopsis [16], the C subunit from barley [17], and the A subunit from cotton [18•]. The function of the cotton A subunit was demonstrated in yeast, and domain-swapping experiments indicated that the carboxyterminal and amino-terminal domains contain structural information that effects V-ATPase function [18•]. It is not clear why plants have two tonoplast H+-pumps, but experiments with the deetiolated3 (det3) mutant of Arabidopsis have provided some clues. The DET3 gene encodes the C-subunit of the V-ATPase [19••]. When grown in the light, the det3 mutant has an organ-specific reduction in size that results in plants with short hypocotyls but normal leaf blades and roots. This phenotype was attributed to deficiencies in cell expansion in the hypocotyl. det3 mutant plants have reduced amounts of C-subunit mRNA and reduced V-ATPase activity compared to wild-type plants. Because the phenotype reflects a tissue-specific reduction in growth, the authors propose that maximal V-ATPase activity is required in the affected tissues. Additional data suggest that decreased
V-ATPase activity limits the accumulation of vacuolar solutes and, hence, the osmotic driving force for growth. It is clear from the det3 phenotype that the V-ATPase and the V-PPase are not redundant in hypocotyl cells. How the activities of these two vacuolar proton pumps are coordinated remains unknown. Solute transporters in the tonoplast
The proton gradient established by the tonoplast H+-pumps is used to energize H+-coupled transport systems. One of these is the Ca2+/H+ antiporter. Ca2+/H+ antiporters from Arabidopsis (CAX1) [20] and mung bean (VCAX1) [21•] have been cloned, and their function has been demonstrated by complementation of yeast mutants [20,22•]. The subcellular location of VCAX1 was determined by immunoblotting mung-bean microsomal-membrane fractions with antiVCAX1 antibodies, and by visualizing VCAX1 linked to synthetic green fluorescent protein in transgenic tobacco cells [22•]. In both cases, the transporter was predominantly found in the tonoplast, although some appeared to be in the Golgi apparatus. The importance of the Ca2+/H+ antiporter for normal plant growth and development may be inferred from the phenotype of transgenic tobacco plants constitutively expressing the Arabidopsis antiporter CAX1 [23••]. Transformed plants were chlorotic and developed necrotic lesions, dead terminal buds, and stunted roots at much higher frequencies than control plants. Increasing the concentration of Ca2+ in the nutrient solution used to water these plants delayed the onset of visible symptoms. CAX1-expressing lines were hypersensitive to high concentrations of Mg2+ and K+, and had increased sensitivity to chilling temperatures. Plants overexpressing CAX1, therefore, appeared as if they were suffering from a calcium deficiency. The Ca2+-transport activity of tonoplast-enriched vesicles from CAX1-expressing plants was higher than that of control plants, as was the amount of calcium in roots or shoots. On the basis of these results Hirschi et al. suggested that CAX1 activity is regulated improperly in the transformed tobacco plants [23••]. Excess CAX1 activity leads to increased vacuolar Ca2+ accumulation and decreased availability of Ca2+ elsewhere in the plant. These data make it clear that the vacuolar Ca2+/H+ antiporter plays a key role in calcium homeostasis, calcium signaling or both. The importance of a second H+ antiporter, the Na+/H+ antiporter, AtNHX1, from Arabidopsis, was also demonstrated in transgenic plants [24••]. Antibodies against AtNHX1 recognized a protein in Arabidopsis that co-fractionated with markers for the tonoplast, Golgi and ER. The protein was more abundant in transformed Arabidopsis plants than in wild-type plants, and vacuoles isolated from transgenic plants had higher rates of Na+/H+ exchange than vacuoles from wild-type plants. Transgenic plants were virtually unaffected by watering with 200 mM NaCl, whereas control plants were stunted and chlorotic. In light of this dramatic difference, Apse et al. [24••] proposed that
Vacuoles and prevacuolar compartments Bethke and Jones
the salt tolerance of crop plants might be increased by enhancing their ability to sequester Na+ in the vacuole. ATP-binding cassette (ABC)-transporters are ATP-dependent, proton-gradient-independent transporters that are found in the tonoplast of plant cells, where they facilitate the vacuolar accumulation of secondary metabolites and xenobiotics [25,26•]. Like other tonoplast transport proteins, ABC transporters are found throughout the endomembrane system as well as in the plasma membrane [27]. How many kinds of ABC-transporters are present on the tonoplast, what substrates they transport, and how their activity is regulated are important, unanswered questions. Our understanding of how these pumps function has been broadened, however, by a report by Klein et al. [28••] that showed that a multi-drug-resistance-associated protein (MRP)-class ABC transporter from rye has unusual properties. Vacuolar uptake of 7-O-diglucuronyl-4′O-glucuronide was found to be dependent on tonoplast membrane potential but did not require conjugation to glutathione. These findings hint at the diversity of substrates carried by this important class of vacuolar transporters and suggest a potential regulatory mechanism.
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vesicles traffic storage proteins and α-TIP to protein storage vacuoles, whereas clathrin-coated vesicles traffic other proteins to acidic vacuoles [33••]. The interpretation of these data has been extended to suggest that a pathway equivalent to the dense-vesicle-pathway exists even in cells in which dense vesicles are not observed by electron microscopy [34], but this interpretation has been questioned [35]. Precursor-accumulating vesicles in developing pumpkin cotyledons and castor bean endosperm are 200–400 nm in diameter with an electron-dense core and electron-translucent outer layer [36]. Some have additional vesicle-like structures within. In developing pumpkin cotyledons, the core of the precursor-accumulating vesicle contains aggregates of water-insoluble seed-storage proteins, including proforms of 11S globulin and 2S albumin. Aggregates of storage protein precursors are also seen in the ER. A model for precursor-accumulating vesicle formation is that the ER around these aggregates buds off to form vesicles that then bypass the Golgi. The observation that proteins in the periphery of castor bean precursor-accumulating vesicles are glycosylated indicates that Golgi-derived vesicles may fuse with precursor-accumulating vesicles prior to delivery of their contents to protein-storage vacuoles.
Vesicular transport to the vacuole Routes for protein transport to the vacuole
Proteins destined for the vacuole are synthesized on the ER and delivered by vesicles to the vacuole (for reviews see [29–32]). Most soluble proteins transported to the vacuole pass through the Golgi apparatus, where they are sorted into vesicles. These vesicles may then fuse with a prevacuolar compartment or directly with the tonoplast. Alternative routes that bypass the Golgi or begin as endocytotic vesicles are likely to exist. Morphologically distinct transport vesicles carry proteins to the vacuole
Three classes of morphologically distinct vesicles that transport proteins from the Golgi or ER to the vacuole have been identified: dense vesicles, clathrin-coated vesicles and precursor-accumulating vesicles. Dense vesicles in developing pea cotyledons are 130 nm in diameter, uncoated when released from the Golgi, and contain an electron-dense core. Clathrin-coated vesicles have a clathrin coat when they leave the Golgi. In an elegant series of experiments, Hinz et al. [33••] presented compelling evidence that suggests that dense vesicles and clathrin-coated vesicles participate in two different vesicular sorting pathways. They used cell fractionation to produce samples that were enriched in either dense vesicles or clathrin-coated vesicles. The densevesicle-enriched fraction contained prolegumin but little BP-80 (the putative vacuolar-sorting signal receptor of 80 kiloDaltons [kDa]), whereas the clathrin-coated-vesicleenriched fraction contained BP-80 but no prolegumin. Immuno-electron microscopy provided complimentary data: antibodies to BP-80 labeled Golgi stacks but not dense vesicles, whereas antibodies against α-TIP labeled Golgi stacks and dense vesicles. Hinz et al. concluded that dense
Precursor-accumulating vesicles have not been observed in vegetative tissues of Arabidopsis. In transgenic Arabidopsis expressing a truncated form of pumpkin 2S albumin linked to phosphinothricin acetyltransferase, however, vesicles similar to precursor-accumulating vesicles accumulated in the cotyledons and leaves [37•]. These vesicles contained 2S albumin and phosphinothricin acetyltransferase. Because only a proform of the protein was detected, Hayashi et al. [37•] suggested that these vesicles were not delivered to vacuoles in these vegetative cells. These experiments raise interesting questions about the signals required for the formation of precursor-accumulating vesicles and their delivery. They also point out that care must be exercised when interpreting data from experiments that use transgenic plants to study protein trafficking. Overexpressed proteins, or proteins from heterologous systems, may be transported via alternative or novel pathways if the normal transport mechanisms are overwhelmed or absent.
Targeting and delivery of proteins to vacuoles requires specific molecular interactions Vacuolar sorting signals
The orderly sorting of proteins into transport vesicles requires recognition of vacuolar sorting signals by vesicleassociated receptors. Three classes of vacuolar signals have been characterized in plants: first, short sequences within an amino-terminal propeptide containing a consensus sequence of NPIR or NPIXL (using the single-letter code for amino acids); second, short sequences with no identified consensus sequence at the carboxyl terminus of a carboxy-terminal propeptide; and third, structural domains within the mature protein. Evidence suggests that the amino-terminal vacuolar sorting signal targets proteins to
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lytic vacuoles, and that proteins with amino- and carboxyterminal vacuolar sorting signals are sorted by different mechanisms [38•]. Candidate receptors for amino-terminal vacuolar sorting signals have been identified, and binding to synthetic peptides containing amino-terminal vacuolar sorting signals has been demonstrated for BP-80 [39], PV72 (for putative vacuolar-sorting receptor of 72 kDa) and PV82 [40]. Domains that are important for the interaction of BP-80 with the amino-terminal vacuolar sorting signal of proaleurain have also been identified [41•]. The Nicotiana alata proteinase inhibitor precursor protein (Na-PI) undergoes proteolytic processing to produce protease inhibitors that are located in the vacuole. Sorting determinants that direct Na-PI to the vacuole have been characterized [42•]. The carboxy-terminus of Na-PI is required for targeting to the vacuole and does not contain an NPIR or NPIXL consensus sequence. Pre-NaPI co-localizes with proteins having antigenic properties similar to BP-80 and the t-SNARE AtPEP12p. In transgenic plants overexpressing Na-PI, an association of pre-Na-PI with BP-80 was demonstrated in immunoprecipitation experiments. These data led the authors to conclude that BP-80 binds to a carboxy-terminal vacuolar sorting signal that is substantially different from NPIR/NPIXL. This is an important finding that needs confirmation. In particular, binding of BP-80 to the carboxy-terminus needs to be demonstrated. As some Na-PI may have been incorrectly targeted to the BP-80-containing pathway in these transgenic plants, traffic through alternative pathways needs to be quantified [34]. Some vacuolar proteins are present in more than one kind of vacuole, suggesting that these proteins have multiple vacuolar sorting signals. This was demonstrated for the 20 kDa potato-tuber protein, PT20 [43•]. The amino-terminal prepropeptide of PT20 contains a vacuolar sorting signal (NPINL) that targeted sweet potato sporamin to vacuoles. This transport was relatively insensitive to wortmanin, a compound that preferentially blocks the transport of dense vesicles relative to that of clathrin-coated vesicles. The carboxy-terminal 13 amino acids of PT20 also targeted sporamin to the vacuole. This transport was sensitive to wortmanin and greatly reduced by the addition of one or more glycine residues at the carboxyl terminus. Further experiments showed that the carboxy-terminal sequence SFKQVQ functions as a vacuolar sorting signal. Whether the amino- or carboxy-terminal vacuolar sorting signal is used preferentially in planta remains unknown. SNARES are involved in transport to vacuoles
Vesicle transport of proteins requires the docking and fusion of transport vesicles with target organelles. In the SNARE hypothesis, docking is accomplished through a specific interaction between an integral membrane protein on the vesicle (v-SNARE) and an integral membrane protein on the target organelle (t-SNARE). Each kind of vacuole or intermediate compartment must have one or
more t-SNAREs, and each kind of vacuole is likely to have a unique v-SNARE (for review see [44]). A few v- and t-SNAREs have been identified in plants, but many more remain undiscovered. Two v-SNAREs, AtVTI1a and AtVTI1b, were recently cloned from Arabidopsis because of their homology to the yeast v-SNARE Vti1p [45•]. Vti1p interacts with the yeast prevacuolar t-SNARE PEP12p, and AtVTI1a was immunoprecipitated with the putative Arabidopsis t-SNARE AtPEP12p. AtVTI1a could also substitute for Vti1p in Golgi-to-prevacuole transport in yeast. When visualized by immuno-electron microscopy, an AtVTI1a-containing construct that was overexpressed in transgenic Arabidopsis was found in the Golgi and electron-dense vesicles near the Golgi with equal frequency. AtVTI1b substituted for Vti1p in yeast vacuolar import pathways other than the Golgi-toprevacuole pathway. In yeast, therefore, AtVTI1a and AtVTI1b act as v-SNAREs with different functions. Whether this is true in plants is unknown. Several t-SNAREs have been identified in plants. AtPEP12p, a homolog of the yeast t-SNARE PEP12p, was one of the first t-SNAREs cloned from plants, and recent attempts have been made to demonstrate its function [46]. α-SNAP (i.e. α-soluble N-ethylmaleimide-sensitive factor attachment protein) binds to the SNARE complex and facilitates the formation of a 20S complex that dissociates in the presence of ATP. AtPEP12p was shown to bind recombinant mammalian α-SNAP. When detergent-solubilized Arabidopsis root proteins were separated on a glycerol density gradient, AtPEP12p was present in complexes of less than 4S to more than 20S. In the presence of ATP, the size distribution of these complexes shifted to less than 11S. These data suggest functional parallels between AtPEP12p and t-SNARES such as PEP12p. A homolog of AtPEP12p, AtVAM3p, has also been characterized [47•]. AtVAM3p interacts with AtVTI1a in immunoprecipitation experiments, suggesting that it has a role in post-Golgi trafficking. On sucrose gradients, AtVAM3p was not separated from AtPEP12p, leaving open the question of whether these two proteins play redundant or separate roles in vesicle trafficking.
The uncertain nature of prevacuolar compartments Although there is agreement that plant cells might contain a prevacuolar compartment, the nature of this organelle remains unclear. Prevacuoles are defined as organelles that receive cargo from transport vesicles and subsequently deliver that cargo to the vacuole by fusion with the tonoplast. Alternatively, a prevacuole can be defined as an organelle that contains t-SNAREs and v-SNAREs, which bind to v-SNAREs on transport vesicles and t-SNAREs on vacuoles, respectively. To date, a prevacuolar compartment that fits either of these definitions has not been unequivocally identified [48•].
Vacuoles and prevacuolar compartments Bethke and Jones
The multi-vesicular body in developing pea cotyledons has been proposed to be a prevacuolar compartment, largely on the basis of results from immuno-electron microscopy. In this tissue, multi-vesicular bodies are strongly labelled with antibodies to legumin and α-TIP, both vacuolar proteins. Multi-vesicular bodies also accumulate tracers for endocytosis. Interestingly, monensin prevents dense-vesicle traffic in pea cotyledons and results in swelling of multi-vesicular bodies and Golgi. These are tantalizing observations, but further support is needed to define the function of the multi-vesicular body and to assign to it the role of prevacuolar compartment. An AtPEP12p-containing compartment in Arabidopsis has also been called a prevacuolar compartment [45•,46]. Electron micrographs show this putative prevacuolar compartment to be a vesicular/tubular compartment of less than 100 nm in diameter. Cell fractionation and biochemical data showed that AtPEP12p was located in a post-Golgi compartment [49], and this compartment has subsequently been called a prevacuolar compartment [45•,46,50]. Unambiguous interpretation of these biochemical data is difficult, however, because the AtPEP12p-containing compartment was not cleanly resolved on density gradients. Likewise, the electron micrographs are difficult to interpret because the subcellular structures were poorly preserved. Additional data are needed to confirm that the AtPEP12p-containing compartment is a prevacuolar compartment.
Conclusions Plant vacuoles and prevacuolar compartments are part of a continuum of endomembrane compartments. All of these compartments are specialized for individual functions. Most of them are dynamic and can change morphologically and functionally to suit the needs of the cell. Although our understanding of plant vacuoles remains rudimentary, we are beginning to appreciate the plasticity of this organelle. Rapid progress is being made in the areas of tonoplast transport and the regulation and import of vacuolar proteins. The same cannot be said for other, equally fundamental aspects of vacuole biology. The mechanisms that control vacuole identity are unknown, so too are the means by which one vacuole fuses with another or fragments into many. The challenge for plant biologists is to merge views of vacuole function, morphology and biochemistry into a clear, unified picture that encompasses the dynamic nature of the vacuole.
Acknowledgements The authors thank Masayoshi Maeshima and Christophe Maurel for critical review of this manuscript. Eleanor Crump assisted in editing the manuscript and her help is gratefully acknowledged.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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38. Matsuoka K, Neuhaus J-M: Cis-elements of protein transport to the • plant vacuoles. J Exp Botany 1999, 50:165-174. This is an excellent review containing detailed sequence comparisons of vacuolar sorting signals. These are discussed in the context of protein-sorting receptors and putative transport pathways. 39. Kirsch T, Paris N, Butler JM, Beevers L, Rogers JC: Purification and initial characterization of a potential plant vacuolar targeting receptor. Proc Natl Acad Sci USA 1994, 91:3403-3407. 40. Shimada T, Kuroyanagi M, Nishimura M, Hara-Nishimura I: A pumpkin 72-kDa membrane protein of precursor-accumulating vesicles has characteristics of a vacuolar sorting receptor. Plant Cell Physiol 1997, 38:1414-1420. 41. Cao XF, Rogers SW, Butler J, Beevers L, Rogers JC: Structural • requirements for ligand binding by a probable plant vacuolar sorting receptor. Plant Cell 2000, 12:493-506. A soluble, truncated form of BP-80 was produced, and the structural domains required for binding to a synthetic proaleurain peptide were identified. A model for the interaction of BP-80 with proaleurain is presented that may indicate how BP-80 binds amino-terminal vacuolar sorting signals in general. 42. Miller EA, Lee MCS, Anderson MA: Identification and • characterization of a prevacuolar compartment in stigmas of Nicotiana alata. Plant Cell 1999, 11:1499-1508. Data are presented that show an association between the vacuolar sorting signal receptor BP-80 and the precursor form of Na-PI. The suggestion is made that BP-80 may bind to a carboxy-terminal vacuolar sorting signal. This is a novel finding that challenges prevailing views of how vacuolar proteins are sorted into transport vesicles. 43. Koide Y, Matsuoka K, Ohto M-A, Nakamura K: The N-terminal • propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals. Plant Cell Physiol 1999, 40:1152-1159. Putative targeting signals from the amino and carboxyl terminus of the 20-kDa potato tuber protein were linked to sweet potato sporamin in order to assess their effectiveness in targeting sporamin to the vacuole. Both a canonical aminoterminal vacuolar sorting signal and a carboxy-terminal sorting signal functioned
Vacuoles and prevacuolar compartments Bethke and Jones
in vacuolar targeting. The conditions under which each is used, and whether targeting is developmentally regulated remain important, unanswered questions. 44. Gerst JE: SNAREs and SNARE regulators in membrane fusion and exocytosis. Cell Mol Life Sci 1999, 55:707-734. 45. Zheng H, Fischer von Mollard G, Kovaleva V, Stevens TH, Raikhel NV: • The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment. Mol Biol Cell 1999, 10:2251-2264. Two Arabidopsis homologs of the yeast v-SNARE vti1p were identified. Complementation analysis in yeast vti1 mutants indicated that AtVTI1a functioned in yeast Golgi-to-prevacuole transport and AtVTI1b in two alternative pathways. AtVTI1a co-localized with the putative vacuolar sorting signal receptor AtELP and the t-SNARE AtPEP12p. A model is presented in which proteins recognized by AtELP are sorted into vesicles containing AtVTI1a, which are subsequently targeted to a post-Golgi compartment through an interaction with AtPEP12p. 46. Bassham DC, Raikhel NV: The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP. Plant J 1999, 19:599-603. 47. •
Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV: The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells. Plant Physiol 1999, 121:929-938. AtVAM3p, like its homolog AtPEP12p, was shown to interact with the v-SNARE AtVTI1a. AtVAM3p was not separated from AtPEP12p on
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sucrose density gradients, and antibodies to both t-SNAREs labeled the same compartments as seen by immuno-electron microscopy. The authors propose that plant endomembrane compartments may have more than one t-SNARE. Whether AtVAM3p and AtPEP12p are redundant, or act in cell-specific or pathway-specific transport requires further experimentation. 48. Robinson DG, Hinz G: Golgi-mediated transport of seed storage • proteins. Seed Sci Res 1999, 9:267-283. This is a comprehensive review of Golgi morphology and function, and of Golgi-mediated transport of seed storage proteins, that is illustrated with examples from the authors’ research. Particular emphasis is given to vesicular transport and the sorting of storage proteins from proteins destined for lytic vacuoles. The evidence for prevacuolar compartments is briefly reviewed. The authors suggest that multivesicular bodies may be prevcuolar compartments. 49. Conceicao ADS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, Raikhel NV: The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants. Plant Cell 1997, 9:571-582. 50. Sanderfoot AA, Ahmed SU, Marty-Mazars D, Rapoport I, Kirchhausen T, Marty F, Raikhel NV: A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots. Proc Natl Acad Sci USA 1998, 95:9920-9925.