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Plant vacuoles are complex and dynamic organelles. Importantadvances have been made in our understanding of thetransporters present in the tonoplast and of the molecularinteractions that allow targeting to vacuoles. Despite theseadvances, markers that permit vacuoles to be definedunambiguously have not yet been identified.

AddressesDepartment of Plant and Microbial Biology, 111 Koshland Hall,University of California, Berkeley, California 94720, USA*e-mail: pcbethke@nature.berkeley.edu

Current Opinion in Plant Biology 2000, 3:469–475

1369-5266/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsABC ATP-binding cassetteCAX1 Ca2+/H+ antiporter from Arabidopsis thalianadet3 deetiolated3ER endoplasmic reticulumTIP tonoplast-intrinsic proteinV-ATPase vacuolar H+-ATPaseV-PPase vacuolar H+-pyrophosphatase

IntroductionThe diversity of plant vacuolesPlant vacuoles are morphologically and functionally diverseorganelles, and recent reports have emphasized that thereare different kinds of plant vacuoles. Some function pri-marily as storage organelles, others as lytic compartments.More than one kind of vacuole has been observed in cellsundergoing differentiation [1], maturation [2], andautophagy [3], and in fully differentiated cells [4,5]. Vacuolefunction depends on a suite of soluble and membrane-bound proteins. These are specifically tailored for each celltype at every developmental stage, and the import anddestruction of vacuolar proteins is carefully orchestrated.

The paucity of endomembrane markersA major impediment to our understanding of plant vacuolesis the lack of specific markers for these organelles and otherendomembrane components. Different kinds of vacuolesmay be morphologically similar, and prevacuolar compart-ments may be indistinguishable from other single-membrane-bound organelles. Yet, an accurate interpretationof many kinds of data requires precise identification ofendomembrane compartments. Our view of plant vacuolesand prevacuolar compartments is clouded because depend-able markers are not available and agreed upon. Markers forthese compartments have been proposed, but have not beenshown to be unambiguous. Indeed, a unique marker maynot exist for some compartments.

In this review, we consider recent developments in ourunderstanding of plant vacuoles and prevacuolar

compartments. Particular attention is focused on integralmembrane transporters in the tonoplast and vesicle traf-ficking of proteins to the vacuole. This review is limitedto literature published in 1999 to mid-2000 that has particular relevance to these topics.

Transport through the tonoplastAquaporins are abundant constituents of all vacuolesThe tonoplast-intrinsic proteins (TIPs) are often themost abundant vacuolar transporters (for review see [6•]).Many of these proteins function as water channels(i.e. aquaporins) when expressed in Xenopus laevisoocytes. Individual plant species typically have severalTIP genes, with representatives in evolutionarily con-served classes (e.g. see [7•]). TIP genes within species aredifferentially regulated, suggesting that different TIPsmay be utilized under specific conditions. Two recentreports have shown that water stress affects the abundanceof TIP mRNAs and proteins [8•,9•]. In cauliflower florets,the amount of BobTIP26-1 transcript increased rapidly inresponse to osmotic stress or desiccation [8•]. Transcriptswere especially abundant in meristems and vascular tis-sues. The BobTIP26-1 protein also increased withdesiccation. Sunflowers have at least five TIPs, and eachhas a different tissue-specific expression pattern [9•]. Eachof the three sunflower δ-TIPs display a different responseto water stress when sunflower roots are exposed to air. Inthis case, the differential regulation of TIP gene expres-sion in response to changing environmental conditions wasgene-specific rather than class-specific.

The in vivo function of TIPs remains unclear. Many TIPstransport water (e.g. see [8•,9•]) and TIPs give the tonoplasta high permeability to water [10•]. A role for TIPs in watertransport, therefore, seems likely. In a surprising develop-ment, Nt-TIPa from tobacco cells was shown to transporturea and glycerol as well as water [11••], leading to the pro-posal that some plant TIPs function in both water and solutetransport. As such, TIPs, in conjunction with their plasma-membrane counterparts, might participate in the long-termregulation 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 peacotyledons with antibodies against the carboxy-terminus ofα-TIP, γ-TIP and δ-TIP. They determined that differentkinds of vacuoles were labeled with different combinationsof TIPs. Lytic vacuoles had γ-TIP alone, autophagic vac-uoles only α-TIP, and storage vacuoles δ-TIP. Proteinstorage vacuoles with vegetative storage proteins had eitherδ-TIP alone or δ-TIP and γ-TIP. Protein storage vacuoleswith seed storage proteins had either δ-TIP and α-TIP orδ-TIP, α-TIP and γ-TIP. Jauh et al. [12••] suggested thatTIPs may also be markers for vacuole development and

Vacuoles and prevacuolar compartmentsPaul C Bethke* and Russell L Jones

change. This is an intriguing proposal, but it provokes someconcerns and further experimentation is required before it isgenerally accepted [7•]. These concerns can be outlined asfollows: first, because the antibodies used by Jauh et al. toidentify TIPs were generated against a part of the proteinthat is poorly conserved within classes of TIPs and betweenplant species, it remains to be confirmed that these antibod-ies identify TIP orthologs in other species; second, theproposal is made on the basis of a small dataset, with only afew 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 thecell and tissue levels, calling into question the idea thatphysiological roles for the different classes of TIPs can beextrapolated from one species to another.

ATP- and pyrophosphate-dependent proton pumpsPlant vacuoles are unique among eukaryotic organelles inhaving 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 endomembranesystem, including the endoplasmic reticulum (ER) andGolgi apparatus, and in the plasma membrane [13]. Recentdata indicate that the V-PPase may also be present in theplasma membrane of some tissues [14], though it’s func-tion as a plasma-membrane proton pump has beenquestioned on thermodynamic grounds [15]. V-PPase is asingle polypeptide that acts as a dimer, but V-ATPase is amulti-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 cytoplasmicface of the membrane, and functions in the translocation ofprotons into the lumen of the endomembrane compart-ment. The V1 domain is the catalytic domain and containsthree pairs of A and B subunits, and at least five other sub-units (i.e. subunits C, D, E, F, and G). The D subunit of V1has been cloned from Arabidopsis [16], the C subunit frombarley [17], and the A subunit from cotton [18•]. The func-tion of the cotton A subunit was demonstrated in yeast, anddomain-swapping experiments indicated that the carboxy-terminal and amino-terminal domains contain structuralinformation that effects V-ATPase function [18•].

It is not clear why plants have two tonoplast H+-pumps,but experiments with the deetiolated3 (det3) mutant ofArabidopsis have provided some clues. The DET3 geneencodes the C-subunit of the V-ATPase [19••]. Whengrown in the light, the det3 mutant has an organ-specificreduction in size that results in plants with shorthypocotyls but normal leaf blades and roots. This pheno-type was attributed to deficiencies in cell expansion inthe hypocotyl. det3 mutant plants have reduced amountsof C-subunit mRNA and reduced V-ATPase activity com-pared to wild-type plants. Because the phenotype reflectsa tissue-specific reduction in growth, the authors proposethat maximal V-ATPase activity is required in theaffected tissues. Additional data suggest that decreased

V-ATPase activity limits the accumulation of vacuolarsolutes and, hence, the osmotic driving force for growth.It is clear from the det3 phenotype that the V-ATPase andthe V-PPase are not redundant in hypocotyl cells. Howthe activities of these two vacuolar proton pumps arecoordinated remains unknown.

Solute transporters in the tonoplastThe proton gradient established by the tonoplast H+-pumpsis used to energize H+-coupled transport systems. One ofthese is the Ca2+/H+ antiporter. Ca2+/H+ antiporters fromArabidopsis (CAX1) [20] and mung bean (VCAX1) [21•]have been cloned, and their function has been demonstratedby complementation of yeast mutants [20,22•]. The subcel-lular location of VCAX1 was determined by immunoblottingmung-bean microsomal-membrane fractions with anti-VCAX1 antibodies, and by visualizing VCAX1 linked tosynthetic green fluorescent protein in transgenic tobaccocells [22•]. In both cases, the transporter was predominantlyfound in the tonoplast, although some appeared to be in theGolgi apparatus.

The importance of the Ca2+/H+ antiporter for normal plantgrowth and development may be inferred from the pheno-type of transgenic tobacco plants constitutively expressingthe Arabidopsis antiporter CAX1 [23••]. Transformed plantswere chlorotic and developed necrotic lesions, dead termi-nal buds, and stunted roots at much higher frequenciesthan control plants. Increasing the concentration of Ca2+ inthe nutrient solution used to water these plants delayedthe onset of visible symptoms. CAX1-expressing lines werehypersensitive to high concentrations of Mg2+ and K+, andhad increased sensitivity to chilling temperatures. Plantsoverexpressing CAX1, therefore, appeared as if they weresuffering from a calcium deficiency. The Ca2+-transportactivity of tonoplast-enriched vesicles from CAX1-express-ing plants was higher than that of control plants, as was theamount of calcium in roots or shoots. On the basis of theseresults Hirschi et al. suggested that CAX1 activity is regu-lated improperly in the transformed tobacco plants [23••].Excess CAX1 activity leads to increased vacuolar Ca2+

accumulation and decreased availability of Ca2+ elsewherein the plant. These data make it clear that the vacuolarCa2+/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 demon-strated in transgenic plants [24••]. Antibodies againstAtNHX1 recognized a protein in Arabidopsis that co-frac-tionated with markers for the tonoplast, Golgi and ER.The protein was more abundant in transformed Arabidopsisplants than in wild-type plants, and vacuoles isolated fromtransgenic plants had higher rates of Na+/H+ exchangethan vacuoles from wild-type plants. Transgenic plantswere virtually unaffected by watering with 200 mM NaCl,whereas control plants were stunted and chlorotic. In lightof this dramatic difference, Apse et al. [24••] proposed that

470 Cell biology

the salt tolerance of crop plants might be increased byenhancing their ability to sequester Na+ in the vacuole.

ATP-binding cassette (ABC)-transporters are ATP-depen-dent, proton-gradient-independent transporters that arefound in the tonoplast of plant cells, where they facilitatethe vacuolar accumulation of secondary metabolites andxenobiotics [25,26•]. Like other tonoplast transport proteins, ABC transporters are found throughout theendomembrane system as well as in the plasmamembrane [27]. How many kinds of ABC-transporters arepresent on the tonoplast, what substrates they transport,and how their activity is regulated are important, unan-swered questions. Our understanding of how these pumpsfunction has been broadened, however, by a report by Kleinet al. [28••] that showed that a multi-drug-resistance-associ-ated protein (MRP)-class ABC transporter from rye hasunusual properties. Vacuolar uptake of 7-O-diglucuronyl-4′-O-glucuronide was found to be dependent on tonoplastmembrane potential but did not require conjugation to glu-tathione. These findings hint at the diversity of substratescarried by this important class of vacuolar transporters andsuggest a potential regulatory mechanism.

Vesicular transport to the vacuoleRoutes for protein transport to the vacuoleProteins destined for the vacuole are synthesized on theER and delivered by vesicles to the vacuole (for reviewssee [29–32]). Most soluble proteins transported to thevacuole pass through the Golgi apparatus, where they aresorted into vesicles. These vesicles may then fuse with aprevacuolar compartment or directly with the tonoplast.Alternative routes that bypass the Golgi or begin asendocytotic vesicles are likely to exist.

Morphologically distinct transport vesicles carry proteinsto the vacuoleThree classes of morphologically distinct vesicles that trans-port proteins from the Golgi or ER to the vacuole have beenidentified: dense vesicles, clathrin-coated vesicles and pre-cursor-accumulating vesicles. Dense vesicles in developingpea cotyledons are 130 nm in diameter, uncoated whenreleased from the Golgi, and contain an electron-dense core.Clathrin-coated vesicles have a clathrin coat when theyleave the Golgi. In an elegant series of experiments, Hinzet al. [33••] presented compelling evidence that suggeststhat dense vesicles and clathrin-coated vesicles participatein two different vesicular sorting pathways. They used cellfractionation to produce samples that were enriched ineither dense vesicles or clathrin-coated vesicles. The dense-vesicle-enriched fraction contained prolegumin but littleBP-80 (the putative vacuolar-sorting signal receptor of80 kiloDaltons [kDa]), whereas the clathrin-coated-vesicle-enriched fraction contained BP-80 but no prolegumin.Immuno-electron microscopy provided complimentarydata: antibodies to BP-80 labeled Golgi stacks but not densevesicles, whereas antibodies against α-TIP labeled Golgistacks and dense vesicles. Hinz et al. concluded that dense

vesicles traffic storage proteins and α-TIP to protein storagevacuoles, whereas clathrin-coated vesicles traffic other pro-teins to acidic vacuoles [33••]. The interpretation of thesedata has been extended to suggest that a pathway equivalentto the dense-vesicle-pathway exists even in cells in whichdense vesicles are not observed by electron microscopy [34],but this interpretation has been questioned [35].

Precursor-accumulating vesicles in developing pumpkincotyledons and castor bean endosperm are 200–400 nm indiameter with an electron-dense core and electron-translu-cent outer layer [36]. Some have additional vesicle-likestructures within. In developing pumpkin cotyledons, thecore of the precursor-accumulating vesicle contains aggre-gates of water-insoluble seed-storage proteins, includingproforms of 11S globulin and 2S albumin. Aggregates ofstorage protein precursors are also seen in the ER. A modelfor precursor-accumulating vesicle formation is that theER around these aggregates buds off to form vesicles thatthen bypass the Golgi. The observation that proteins inthe periphery of castor bean precursor-accumulating vesi-cles are glycosylated indicates that Golgi-derived vesiclesmay fuse with precursor-accumulating vesicles prior todelivery of their contents to protein-storage vacuoles.

Precursor-accumulating vesicles have not been observed invegetative tissues of Arabidopsis. In transgenic Arabidopsisexpressing a truncated form of pumpkin 2S albumin linkedto phosphinothricin acetyltransferase, however, vesiclessimilar to precursor-accumulating vesicles accumulated inthe cotyledons and leaves [37•]. These vesicles contained2S albumin and phosphinothricin acetyltransferase. Becauseonly a proform of the protein was detected, Hayashi et al.[37•] suggested that these vesicles were not delivered tovacuoles in these vegetative cells. These experiments raiseinteresting questions about the signals required for the for-mation of precursor-accumulating vesicles and theirdelivery. They also point out that care must be exercisedwhen interpreting data from experiments that use trans-genic plants to study protein trafficking. Overexpressedproteins, or proteins from heterologous systems, may betransported via alternative or novel pathways if the normaltransport mechanisms are overwhelmed or absent.

Targeting and delivery of proteins to vacuolesrequires specific molecular interactionsVacuolar sorting signalsThe orderly sorting of proteins into transport vesiclesrequires recognition of vacuolar sorting signals by vesicle-associated receptors. Three classes of vacuolar signals havebeen characterized in plants: first, short sequences withinan amino-terminal propeptide containing a consensussequence of NPIR or NPIXL (using the single-letter codefor amino acids); second, short sequences with no identi-fied consensus sequence at the carboxyl terminus of acarboxy-terminal propeptide; and third, structural domainswithin the mature protein. Evidence suggests that theamino-terminal vacuolar sorting signal targets proteins to

Vacuoles and prevacuolar compartments Bethke and Jones 471

lytic vacuoles, and that proteins with amino- and carboxy-terminal vacuolar sorting signals are sorted by differentmechanisms [38•]. Candidate receptors for amino-terminalvacuolar sorting signals have been identified, and bindingto synthetic peptides containing amino-terminal vacuolarsorting signals has been demonstrated for BP-80 [39],PV72 (for putative vacuolar-sorting receptor of 72 kDa) andPV82 [40]. Domains that are important for the interactionof BP-80 with the amino-terminal vacuolar sorting signal ofproaleurain have also been identified [41•].

The Nicotiana alata proteinase inhibitor precursor protein(Na-PI) undergoes proteolytic processing to produce pro-tease inhibitors that are located in the vacuole. Sortingdeterminants that direct Na-PI to the vacuole have beencharacterized [42•]. The carboxy-terminus of Na-PI isrequired for targeting to the vacuole and does not containan NPIR or NPIXL consensus sequence. Pre-NaPIco-localizes with proteins having antigenic propertiessimilar to BP-80 and the t-SNARE AtPEP12p. In trans-genic plants overexpressing Na-PI, an association ofpre-Na-PI with BP-80 was demonstrated in immunopre-cipitation experiments. These data led the authors toconclude that BP-80 binds to a carboxy-terminal vacuolarsorting signal that is substantially different fromNPIR/NPIXL. This is an important finding that needsconfirmation. In particular, binding of BP-80 to the car-boxy-terminus needs to be demonstrated. As some Na-PImay have been incorrectly targeted to the BP-80-contain-ing pathway in these transgenic plants, traffic throughalternative pathways needs to be quantified [34].

Some vacuolar proteins are present in more than one kindof vacuole, suggesting that these proteins have multiplevacuolar sorting signals. This was demonstrated for the20 kDa potato-tuber protein, PT20 [43•]. The amino-ter-minal prepropeptide of PT20 contains a vacuolar sortingsignal (NPINL) that targeted sweet potato sporamin tovacuoles. This transport was relatively insensitive to wort-manin, a compound that preferentially blocks the transportof dense vesicles relative to that of clathrin-coated vesicles.The carboxy-terminal 13 amino acids of PT20 also targetedsporamin to the vacuole. This transport was sensitive towortmanin and greatly reduced by the addition of one ormore glycine residues at the carboxyl terminus. Furtherexperiments showed that the carboxy-terminal sequenceSFKQVQ functions as a vacuolar sorting signal. Whetherthe amino- or carboxy-terminal vacuolar sorting signal isused preferentially in planta remains unknown.

SNARES are involved in transport to vacuolesVesicle transport of proteins requires the docking andfusion of transport vesicles with target organelles. In theSNARE hypothesis, docking is accomplished through aspecific interaction between an integral membrane pro-tein on the vesicle (v-SNARE) and an integral membraneprotein on the target organelle (t-SNARE). Each kind ofvacuole or intermediate compartment must have one or

more t-SNAREs, and each kind of vacuole is likely tohave a unique v-SNARE (for review see [44]). A fewv- and t-SNAREs have been identified in plants, butmany more remain undiscovered.

Two v-SNAREs, AtVTI1a and AtVTI1b, were recentlycloned from Arabidopsis because of their homology to theyeast v-SNARE Vti1p [45•]. Vti1p interacts with the yeastprevacuolar t-SNARE PEP12p, and AtVTI1a was immuno-precipitated with the putative Arabidopsis t-SNAREAtPEP12p. AtVTI1a could also substitute for Vti1p inGolgi-to-prevacuole transport in yeast. When visualized byimmuno-electron microscopy, an AtVTI1a-containing con-struct that was overexpressed in transgenic Arabidopsis wasfound in the Golgi and electron-dense vesicles near theGolgi with equal frequency. AtVTI1b substituted for Vti1pin yeast vacuolar import pathways other than the Golgi-to-prevacuole pathway. In yeast, therefore, AtVTI1a andAtVTI1b 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, wasone of the first t-SNAREs cloned from plants, and recentattempts have been made to demonstrate its function [46].α-SNAP (i.e. α-soluble N-ethylmaleimide-sensitive factorattachment protein) binds to the SNARE complex andfacilitates the formation of a 20S complex that dissociatesin the presence of ATP. AtPEP12p was shown to bindrecombinant mammalian α-SNAP. When detergent-solubi-lized Arabidopsis root proteins were separated on a glyceroldensity gradient, AtPEP12p was present in complexes ofless than 4S to more than 20S. In the presence of ATP, thesize distribution of these complexes shifted to less than11S. These data suggest functional parallels betweenAtPEP12p and t-SNARES such as PEP12p.

A homolog of AtPEP12p, AtVAM3p, has also been charac-terized [47•]. AtVAM3p interacts with AtVTI1a inimmunoprecipitation experiments, suggesting that it has arole in post-Golgi trafficking. On sucrose gradients,AtVAM3p was not separated from AtPEP12p, leaving openthe question of whether these two proteins play redundantor separate roles in vesicle trafficking.

The uncertain nature of prevacuolarcompartmentsAlthough there is agreement that plant cells might containa prevacuolar compartment, the nature of this organelleremains unclear. Prevacuoles are defined as organellesthat receive cargo from transport vesicles and subsequentlydeliver that cargo to the vacuole by fusion with the tono-plast. Alternatively, a prevacuole can be defined as anorganelle that contains t-SNAREs and v-SNAREs, whichbind to v-SNAREs on transport vesicles and t-SNAREson vacuoles, respectively. To date, a prevacuolar compart-ment that fits either of these definitions has not beenunequivocally identified [48•].

472 Cell biology

The multi-vesicular body in developing pea cotyledonshas been proposed to be a prevacuolar compartment, largelyon the basis of results from immuno-electron microscopy.In this tissue, multi-vesicular bodies are strongly labelledwith antibodies to legumin and α-TIP, both vacuolar pro-teins. Multi-vesicular bodies also accumulate tracers forendocytosis. Interestingly, monensin prevents dense-vesi-cle traffic in pea cotyledons and results in swelling ofmulti-vesicular bodies and Golgi. These are tantalizingobservations, but further support is needed to define thefunction of the multi-vesicular body and to assign to it therole of prevacuolar compartment.

An AtPEP12p-containing compartment in Arabidopsis hasalso been called a prevacuolar compartment [45•,46].Electron micrographs show this putative prevacuolar com-partment to be a vesicular/tubular compartment of less than100 nm in diameter. Cell fractionation and biochemical datashowed that AtPEP12p was located in a post-Golgi compart-ment [49], and this compartment has subsequently beencalled a prevacuolar compartment [45•,46,50]. Unambiguousinterpretation of these biochemical data is difficult, however,because the AtPEP12p-containing compartment was notcleanly resolved on density gradients. Likewise, the electronmicrographs are difficult to interpret because the subcellularstructures were poorly preserved. Additional data are neededto confirm that the AtPEP12p-containing compartment is aprevacuolar compartment.

ConclusionsPlant vacuoles and prevacuolar compartments are part of acontinuum of endomembrane compartments. All of thesecompartments are specialized for individual functions. Mostof them are dynamic and can change morphologically andfunctionally to suit the needs of the cell. Although ourunderstanding of plant vacuoles remains rudimentary, weare beginning to appreciate the plasticity of this organelle.Rapid progress is being made in the areas of tonoplast trans-port and the regulation and import of vacuolar proteins. Thesame cannot be said for other, equally fundamental aspectsof vacuole biology. The mechanisms that control vacuoleidentity are unknown, so too are the means by which onevacuole fuses with another or fragments into many. Thechallenge for plant biologists is to merge views of vacuolefunction, morphology and biochemistry into a clear, unifiedpicture that encompasses the dynamic nature of the vacuole.

AcknowledgementsThe authors thank Masayoshi Maeshima and Christophe Maurel for criticalreview of this manuscript. Eleanor Crump assisted in editing the manuscriptand her help is gratefully acknowledged.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

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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 thehypocotyl and tobacco cells. Eur J Biochem 2000, 267:3090-3098.

VCAX1p is shown to be a functional Ca2+/H+ antiporter that is predomi-nantly located in the tonoplast. The possibility that VCAX1p also functions inthe 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 tosuffer 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 potentialmechanism for engineering salt tolerance into plants.

25. Rea PA, Li Z-S, Lu Y-P, Drozdowicz YM: From vacuolar GS-X pumpsto multispecific ABC transporters. Annu Rev Plant Physiol PlantMol 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 ofthese transporters in plants. The structural and functional characteristics of thesetransporters in yeast and plants are discussed in this highly detailed review.

27. Sidler M, Hassa P, Hasan S, Ringli C, Dudler R: Involvement of anABC transporter in a developmental pathway regulating hypocotylcell 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 ofglucuronide uptake by glutathione and its conjugates. Plant J2000, 21:289-304.

The energy-dependent uptake of two flavones by an MRP-like transporterwith broad specificity was characterized. In experiments with isolated tono-plast vesicles, application of valinomycin doubled the uptake rate of oneflavone 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 isdependent on membrane potential. This finding raises the possibility thatthe activity of some MRP-like transporters may be coordinated with theactivity of other tonoplast transporters, especially the proton pumps.

29. Jiang L, Rogers JC: Sorting of membrane proteins to vacuoles inplant cells. Plant Sci 1999, 146:55-67.

30. Hawes CR, Brandizzi F, Andreeva AV: Endomembranes and vesicletrafficking. 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 vacuolarprotein 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 transportvesicles. Plant Cell 1999, 11:1509-1524.

Localization studies using cell fractionation and immuno-electron microscopyshow that Golgi-derived dense vesicles contain storage proteins and thatclatherin-coated vesicles contain BP-80. These data lend support to a modelfor protein import in which proteins destined for lytic vacuoles are sorted intoseparate transport vesicles from proteins destined for storage vacuoles.

34. Jiang L, Rogers JC: The role of BP-80 and homologs in sortingproteins to vacuoles. Plant Cell 1999, 11:2069-2071.

35. Miller E, Anderson M: The role of BP-80 and homologs in sortingproteins 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 ismediated by large precursor-accumulating vesicles. Plant Cell1998, 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 Physiol1999, 40:263-272.

A chimeric gene was constructed that contained a truncated pumpkin 2Salbumin linked to a selectable marker. When expressed in vegetative cells ofArabidopsis, the protein product accumulated in novel vesicles that hadcharacteristics of precursor-accumulating vesicles. These data suggest thatthe formation of precursor-accumulating vesicles is driven by the synthesisof specific proteins. They also highlight the risk of using transgenic plants tostudy 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 vac-uolar sorting signals. These are discussed in the context of protein-sortingreceptors and putative transport pathways.

39. Kirsch T, Paris N, Butler JM, Beevers L, Rogers JC: Purification andinitial characterization of a potential plant vacuolar targetingreceptor. Proc Natl Acad Sci USA 1994, 91:3403-3407.

40. Shimada T, Kuroyanagi M, Nishimura M, Hara-Nishimura I: A pumpkin72-kDa membrane protein of precursor-accumulating vesicleshas characteristics of a vacuolar sorting receptor. Plant CellPhysiol 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 domainsrequired for binding to a synthetic proaleurain peptide were identified. A modelfor the interaction of BP-80 with proaleurain is presented that may indicatehow 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 sortingsignal receptor BP-80 and the precursor form of Na-PI. The suggestion ismade that BP-80 may bind to a carboxy-terminal vacuolar sorting signal. Thisis a novel finding that challenges prevailing views of how vacuolar proteinsare 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 vacuolarsorting signals. Plant Cell Physiol 1999, 40:1152-1159.

Putative targeting signals from the amino and carboxyl terminus of the 20-kDapotato tuber protein were linked to sweet potato sporamin in order to assesstheir effectiveness in targeting sporamin to the vacuole. Both a canonical amino-terminal vacuolar sorting signal and a carboxy-terminal sorting signal functioned

474 Cell biology

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 fusionand 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 prevacuolarcompartment. 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 func-tioned in yeast Golgi-to-prevacuole transport and AtVTI1b in two alternativepathways. AtVTI1a co-localized with the putative vacuolar sorting signalreceptor AtELP and the t-SNARE AtPEP12p. A model is presented in whichproteins recognized by AtELP are sorted into vesicles containing AtVTI1a,which are subsequently targeted to a post-Golgi compartment through aninteraction with AtPEP12p.

46. Bassham DC, Raikhel NV: The pre-vacuolar t-SNARE AtPEP12pforms 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 thev-SNARE AtVTI1a. AtVAM3p was not separated from AtPEP12p on

sucrose density gradients, and antibodies to both t-SNAREs labeled thesame compartments as seen by immuno-electron microscopy. Theauthors propose that plant endomembrane compartments may havemore than one t-SNARE. Whether AtVAM3p and AtPEP12p are redun-dant, or act in cell-specific or pathway-specific transport requires furtherexperimentation.

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 ofGolgi-mediated transport of seed storage proteins, that is illustrated withexamples from the authors’ research. Particular emphasis is given tovesicular transport and the sorting of storage proteins from proteins des-tined for lytic vacuoles. The evidence for prevacuolar compartments isbriefly reviewed. The authors suggest that multivesicular bodies may beprevcuolar compartments.

49. Conceicao ADS, Marty-Mazars D, Bassham DC, Sanderfoot AA,Marty F, Raikhel NV: The syntaxin homolog AtPEP12p resides ona 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 cargoreceptor partially colocalizes with AtPEP12p on a prevacuolarcompartment in Arabidopsis roots. Proc Natl Acad Sci USA 1998,95:9920-9925.

Vacuoles and prevacuolar compartments Bethke and Jones 475