Vacuolar protein sorting mechanisms in plants



W. Van den Ende, Laboratory of Molecular Plant Biology, KU Leuven, Kasteelpark Arenberg 31, Leuven, Belgium

Fax: +32 16321967

Tel: +32 16321952



Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant-specific entities and sorting determinants appear to exist. The alternative post-Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi-independent route involving plant-specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions.


adaptor protein


ADP ribosylation factor


clathrin-coated vesicle


coat protein complex


dense vesicle


endoplasmic reticulum


lytic vacuole


mannose 6-phosphate receptor


protein storage vacuole


receptor membrane ring-H2


secretion-associated RAS-related protein


soluble nethylmaleimide sensitive factor attachment protein receptor


trans-Golgi network


tonoplast intrinsic protein


vacuolar sorting determinant


vacuolar sorting receptor


A typical plant or animal cell contains up to 10 000 different types of proteins, whereas a yeast cell contains approximately 5000. For proper functioning, each of these numerous proteins must be localized to a precise intracellular compartment, cellular membrane or organelle, or directed to the exterior of the cell [1]. In plants, the endomembrane system is a complex network of organelles specialized in the synthesis, transport, modification and secretion of proteins and other macromolecules. This system is composed of several functionally distinct membrane compartments: the endoplasmic reticulum (ER), the Golgi apparatus including the trans-Golgi network (TGN), secretory vesicles, the vacuole and endosomes [2]. The membranes of mitochondria and chloroplasts do not belong to the endomembrane system [1-3]. Connection between the various endosomal compartments is achieved through tightly controlled, constant budding and fusion of vesicle shuttles [4]. The endomembrane system integrates several dynamic routes: the secretory pathway (including biosynthesis and sorting) and the endocytic pathway. As a result of their overlapping routes and cargo distribution centres, it is difficult, if not impractical, to separate these pathways from each other. Proteins and other cargos are synthesized and programmed to follow a certain sorting pathway to reach their final destination. Although the dynamic activities of endomembrane systems are highly conserved among all eukaryotes, higher plants have developed some unique mechanisms [5].

In plants, the secretory and biosynthetic trafficking pathways are involved in a series of vital mechanisms, such as gravitropism, autophagy, hormone transport, cytokinesis and abiotic/biotic stress responses [6, 7], as well as in ion secretion by salt glands, nectar production, and the secretion of viscin, which is the elastic, mucilaginous and sticky tissue that attaches falling parasitic mistletoe seeds to branches [8]. Over recent years, remarkable progress has been achieved in the understanding of plant protein and membrane trafficking by the use of different systems and approaches [4, 9]. The path followed by a protein depends on the interactions between sorting motifs present in the protein and the motif-recognizing machinery. Many of these motifs are universally conserved among eukaryotes (yeast and humans) [9]. However, sorting motif studies in plants are still in their infancy compared to animal and yeast systems. In plants, studies mainly address tissue-specific and organelle-specific trafficking processes [9]. Generally, the protein secretory pathway begins in the ER, passes through the Golgi complex, and finally reaches the vacuole, other compartments or the cell surface [4, 9]. This review mainly focuses on the mechanisms involved in protein sorting to the central vacuole as a unique, multifunctional plant-specific organelle.

Sorting of proteins to plant vacuoles

Vacuolar proteins reach the different types of vacuoles through a vesicle-mediated biosynthetic trafficking pathway that includes the ER, the Golgi apparatus, the TGN and the endosomes/prevacuoles (Fig. 1). In plant cells, two different types of membrane receptors are known: the vacuolar sorting receptor (VSR) family [9] and the receptor membrane ring-H2 (RMR) family [10].

Figure 1.

Model of protein sorting pathways to the plant vacuole. (A) Protein sorting to the PSV. Proteins destined for the PSV initiate their life in the ER and can be sorted to the PSV through Golgi-dependent or -independent pathways. In the Golgi-dependent pathway, proteins are transported to the Golgi via COPII vesicles, and then aggregate and become the cargo of DVs, being transported into multivesicular bodies and then fusing into the PSV. Notably, some storage proteins that need processing in the Golgi reach the PSV via precursor-accumulating vesicles. In the Golgi-independent pathway, PSV residents are packed into precursor-accumulating vesicles and are transported to the PSV. MVB, multivesicular body; PAC, precursor-accumulating vesicle; PV72/RMR, vacuole sorting receptor. (B) Protein sorting to the LV. Proteins destined for the LV initiate their life in the ER and can be sorted to the LV through Golgi-dependent or -independent pathways. In the Golgi-dependent pathway, the protein can be (a) recognized by BP80/VSR and becoming the cargo of CCV, after which it is transported to the prevacuolar compartment and, finally, to the LV; or (b) transported via a putative AP3-mediated pathway to the LV. In the Golgi-independent pathway, the LV residents are transported through the ER bodies. A PSV (α-TIP) might be transformed into an LV (γ-TIP). Fusions of LVs or PSVs make up the central vacuole. PVC, prevacuolar compartment; BP80/VSR, vacuole sorting receptor.

The plant vacuole

The number and the size of plant vacuoles depend on cell type and developmental stage. A single central vacuole may occupy as much as 80% of the volume of a cell. Plant vacuoles are essential multifunctional organelles distinct from similar organelles in other eukaryotes. They serve both physical and metabolic functions, and are crucial to processes involved in the cellular responses to environmental and biotic factors, as well as to general cell homeostasis [11, 12]. Vacuoles typically store water, ions, secondary metabolites and nutrients. Similar to animal lysosomes, they also act as a repository for waste products, excess solutes and toxic substances [13-16], and play key roles in (programmed) cell death [17-20].

Two types of vacuoles can be found in plant cells, the protein storage vacuole (PSV) and the lytic vacuole (LV) [21]. Typically, vacuolar storage proteins accumulate in PSVs. PSVs have higher pHs and lower hydrolytic activities than LVs, and predominate in storage tissues (e.g. cotyledons and endosperm in seeds, tubers), as well as in vegetative tissues of adult plants (e.g. bark, leaves, pods) [22, 23]. Proteins stored in PSVs are mainly used as nitrogen or carbon sources during seed germination and plant development [24]. However, PSVs can also contain large amounts of toxic proteins (e.g. lectins, protease inhibitors and ribosome inactivating proteins), which can be considered as the result of a cytosolic detoxification process or as a defence against predators [25-27]. PSV proteins could be processed through the action of vacuolar processing enzymes or proteases [28-30]. By contrast, LVs are usually found in vegetative tissues. They have an acidic pH and contain an abundance of hydrolytic enzymes [31, 32]. LVs are used for storage and as a depository of unwanted materials in plant cells. They receive extracellular components via endocytosis and phagocytosis, and intracellular material via autophagy, as well as via the biosynthetic trafficking pathway and membrane-bound transport systems. LVs modulate the degradation of a multitude of macromolecules and other compounds. As such, they are considered key regulators in cellular homeostasis [33, 34].

Tonoplast intrinsic proteins (TIPs) have been used as intracellular markers for vacuolar biogenesis and identity. The expression of TIPs, although tissue-specific, varies greatly throughout development. TIPs are classified into five categories: α, β, γ, δ and ε-TIPs [35]. α and β-TIPs are seed-specific. γ-TIPs associate with the LV, whereas α-TIP and δ-TIP associate with the PSV [36, 37]. ε-TIPs are primarily found in root and floral organs. Different TIPs can coexist in the same cell, suggesting the presence of both LVs and PSVs [33]. Evidence that LVs and PSVs can occur together has been found in the root tip cells of barley and pea seedlings [31, 38], as well as in protoplasts of barley aleurone and tobacco [32]. Interestingly, it was reported that, during the germination of Arabidopsis seeds, the LV is primarily embedded in the PSV and then derives from it, instead of being generated de novo [21, 31, 39]. Moreover, depending on physiological conditions, LVs can be transformed into PSVs and vice versa [40-42]. In addition to PSVs and LVs, a third type of vacuole has been suggested in Arabidopsis [43, 44]. During leaf senescence, senescence-associated vacuoles, with a smaller size and containing aggregates, are formed de novo [45], and are characterized by a higher cysteine-protease activity and a lower pH than LVs [46].

The existence of two types of plant vacuoles with distinct contents and functions implies that separate trafficking pathways must exist for their respective cargo [5], with a need for correct separation in the Golgi/TGN apparatus (Fig. 1). Furthermore, it can be hypothesized that the concurrent existence of LVs and PSVs provides plants with extra flexibilities to deal with changing environmental conditions.

Sorting of vacuolar proteins is initiated in the ER

The initiation of vacuolar/lysosomal protein sorting in the ER is a very conserved mechanism in yeast, animals and plants [5, 9]. The ER represents the first compartment of the secretory system [47]. The ER membrane connects to the nuclear envelope, and forms a wide network of thin tubules and cisternae in the cortical and inner parts of the cell [47]. Both transmembrane proteins and soluble, secreted proteins contain an ER signal sequence including a hydrophobic 20–25 amino-acid segment. As a result of its hydrophobic nature, the signal sequence is inserted into the ER membrane. The difference between the secreted and transmembrane proteins is that the hydrophobic sequence is removed by the ER signal peptidases from the secreted proteins, although not from the transmembrane proteins [48, 49]. Based on the model of Singer and Nicolson [50], integral membrane proteins can be classified as: (a) type I transmembrane proteins containing a single transmembrane domain with an Nout/Cin orientation (‘in’ means cytoplasmic side, ‘out’ means lumen); (b) type II transmembrane proteins containing a single transmembrane domain with an Nin/Cout orientation; (c) type III membrane proteins (multiple transmembrane domains in a single polypeptide chain); and (d) type IV membrane proteins (multiple transmembrane domains reside on a single or multiple individual polypeptide chain).

The ER is also an important check point of correct protein folding and assembly, known as stringent quality control [51]. Only properly folded and assembled proteins are allowed to exit the ER. Misfolded proteins are recognized by molecular chaperones (e.g. BiP, calnexin) and retained in the lumen of the ER in an attempt to re-fold them to their correct, native structure [52-54]. Persistent misfolded proteins are transferred to the cytosol and degraded by the proteasome system [55, 56]. Proteins that have erroneously reached the Golgi can also return to the ER when ER retention signals are present [57].

Vesicular trafficking

Plant cells, similar to all eukaryotes, are structurally and metabolically subdivided into different compartments, with communication between organelles being accomplished mainly by vesicular trafficking. This is a highly-regulated and directional/targeted process. In general, the vesicle trafficking process involves budding, vesicle release, targeted transport, tether, and membrane recognition and fusion [58]. Vesicle trafficking is also involved in secretion into the apoplast and, during endocytosis, recycling proteins from the plasma membrane to the endosome, the TGN and the lysosome/vacuole [5, 47]. In all eukaryotes, clathrin-coated vesicle (CCV), caveolin, coat protein complex I (COPI) and coat protein complex II (COPII) vesicles carry out these functions and have an ubiquitous presence and budding machineries.

Vesicle formation is not a default, passive event, but instead, requires a specific driving force to carry out a series of highly-regulated events including recognition and binding between the receptor at the donor membrane and an activated GTP-ADP ribosylation factor (Arf) complexed with the secretion-associated RAS-related protein (Sar)1 complex, recruiting coatomer, membrane distortion and dissociation under the assistance of fatty acyl CoA, and the formation and release of a vesicle. Similar to animals and yeast, in plants, most of the Sar/Arf1 GTPases are well conserved. The Arf1 family is necessary for COPI vesicle and CCV formation, whereas the Sar1 family is mainly involved in COPII vesicle formation [59, 60]. After formation, the vesicle is released and travels towards the target compartment assisted by actin filaments, where it disassembles with the help of a cytosolic Hsc70 chaperone. After the uncoating process [61, 63], specific vesicle soluble nethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, exposed on the vesicle's membrane, bind to cognate target-SNARE proteins complexed with synaptosomal-associated protein 25. Next, nethylmaleimide sensitive factor and soluble nethylmaleimide sensitive factor attachment proteins bind to this complex, which dissociates after vesicle fusion. Sec1p and Rab proteins serve as effectors for SNARE complex regulation during vesicle targeting and fusion [62-64].

Trafficking between the ER and the Golgi apparatus

Although the sorting of plant vacuolar proteins commences at the ER [64], the Golgi apparatus is the major sorting station in the plant cell [65]. The ER and Golgi communicate with each other via a highly-regulated traffic system. Transfer from the ER to the Golgi apparatus is the first step in protein sorting via the biosynthetic trafficking pathway. This is mediated by COPII vesicles. The retrograde transport from the Golgi to the ER is accomplished via COPI vesicles, which are morphologically and biochemically different from the COPII vesicles of the retrograde system [66, 67].

The COPII vesicles mediate the anterograde traffic export protein from ER the to the Golgi apparatus in eukaryotic cells [68, 69]. COPII is composed of three components: Sar1, Sec23/24 and Sec13/31. After budding off from the ER, COPII vesicles can either directly fuse with cis-Golgi, or first fuse with each other and then to the cis-Golgi [68, 70, 71].

The role of COPI vesicles for retrograde traffic from the Golgi to the ER or within the Golgi apparatus from the trans- towards the cis-Golgi in plants was first demonstrated in transgenic tobacco [72]. COPI consists of coatomer (F-COP and B-COP subunit) and Arf G-protein. Two classes can be discerned: COPIa vesicles are derived from cis-cisternae, and COPIb vesicles are derived from medial and trans-cisternae [73]. Different classes of COPI vesicles are caused by multiple isoforms of COP subunits [73]. The distribution of proteins between ER and Golgi is maintained by the balanced cooperation of COPI and COPII transport routes. Inhibition of COPI function results in impaired trafficking between the Golgi and the ER and disruption of the ER export sites [72, 74, 75].

For exit of proteins from the ER, different types of motifs have been identified that are recognized by COPII. Di-acidic (e.g. DXE/EXE), di-basic (e.g. RKXRK) and di-aromatic motifs in the cytosolic tail of transmembrane proteins were reported to be important for the export of proteins from the ER in yeast and animals [68]. The first evidence for a di-acidic motif (DAE) in protein export from the ER in plants was obtained for a sugar transporter in tobacco [76]. Next, Schoberer et al. [77] demonstrated the importance of a di-basic motif (RKR) in tobacco as well.

Retrograde Golgi-ER transport of soluble proteins is achieved by C-terminal H/KDEL motifs that are recognized by the receptor ER retention defective 2 protein embedded in the membrane. Thus, H/KDEL motifs are considered to be responsible for the retention of soluble proteins in the ER lumen, and are named ER retention signals. H/KDEL containing proteins never undergo the typical modifications observed in Golgi-derived enzymes [78, 79].

Intra-Golgi and post-Golgi transport

The plant Golgi apparatus appears to be more complicated than its animal counterpart. It is divided into functionally independent individual Golgi stacks and has a polarized structure, whereas the animal Golgi apparatus is perinuclear and stationary [75]. After receiving newly-synthesized proteins from the ER, the Golgi apparatus performs covalent modifications, and then further distributes the proteins to various final destinations [80].The cis-Golgi constitutes the entrance to the apparatus, whereas vesicles leave at the trans-Golgi to reach their final destination [65, 81]. The Golgi is also implicated in protein trafficking to nonsecretory organelles such as peroxisomes and chloroplasts. The Golgi apparatus plays an important role in post-translational modification mainly through transmembrane processing enzymes. Proteins enter the cis-Golgi, move through the med-Golgi and reach the trans-Golgi where the modification process is completed, such as glycosylation, sulfation and phosphorylation [82]. Two models of intra-Golgi transport have been proposed: the vesicle shuttle and the cisternal maturation models. Notably, multiple Golgi-independent protein transport pathways exist for delivering cargo molecules from the ER to a variety of destinations, such as celery mannitol dehydrogenase delivery to the extracellular space [83] and barley aspartic protease transport to the vacuole [84].

Post-Golgi vesicle transport: the CCV

The CCVs were the first type of coated vesicles to be described in eukaryotes. Typically CCVs are 50–100 nm in diameter, formed at the plasma membrane and TGN/endosomes and are involved in the trafficking of protein cargo between these organelles [85]. There is evidence for their formation at the lysosomes of animals [86] and the vacuoles of yeast [87], yet there are no confirmed reports of CCV formation in plant vacuoles. They are also implicated to play a role in other processes such as defence responses and cytokinesis. The formation of a clathrin coat requires various adaptor proteins (APs), clathrin and dynamin (a GTP-binding protein, Arf). The clathrin unit is a three-limbed shaped triskeleton, each limb containing one clathrin heavy chain and one light chain [88]. Two other proteins, amphiphysin and synaptojanin, are involved in CCV formation as well. The clathrin coat can be depolymerized by cytosolic Hsc70, and re-used [89-91]. A distinctive characteristic of plant CCVs is their larger size. Capacitance measurements of endocytic events during fluid phase endocytosis in tobacco BY-2 protoplasts estimated the size of vesicles to be a mean of 133 nm [92]. These estimates are similar to those obtained by Gall et al. [93] in turgid guard cells. By contrast, estimates of CCVs in nonplant systems appear to be between 70 and 100 nm [94].

Vacuolar sorting machinery

Plant vacuolar sorting determinants (VSDs)

VSDs are necessary for correct post-Golgi sorting to the vacuole. Plant cells contain three different categories of VSDs: (a) sequence-specific VSDs; (b) C-terminal VSDs; and (c) protein structure-dependent VSDs [80, 95]. Without these motifs, vacuolar proteins follow the default pathway and are secreted to the surface of the cell, whereas their introduction into a secreted protein could redirect it to the vacuole [96]. In general, N-terminal sequence-specific VSDs and C-terminal VSDs are removed during protein maturation after vacuolar sorting by the action of vacuolar proteases [97].

Sequence-specific VSDs are generally considered to be recognized by VSRs for sorting to the LV, as in the case of barley aleurain and sporamin [98-101]. Functional sequence-specific VSDs (NPIXL/NPIR) were also described in storage protein sorting to the PSV, such as castor 2S albumin and ricin [102-104]. Sequence-specific VSDs function independently of their molecular position, although they are most often situated at the N-terminus of a protein. For example, sporamin type sequence-specific VSDs are still able to direct protein to the vacuole after translocation from the N-terminus to the C-terminus [99, 105]. There are some examples of C-terminal and even internal sequence-specific VSDs [106].

In plants, C-terminal VSDs were first discovered in barley lectin and tobacco chitinase [107, 108]. The minimal length is four amino acids [103]. Many random C-terminal peptides are sufficient to target a reporter protein to the vacuole. For example, the C-terminal VSD of tobacco chitinase A (GLLVDTM), and the FAEAI and LVAE motifs of barley lectin, are necessary and sufficient for vacuolar targeting [107-109]. Moreover, the IAGF motif from 2S albumin of Passifloraceae, the PLSSILRAFY motif of the β-conglycinin α unit of soybean and the KISIA motif from the 11S albumin of Amaranthus are all functional C-terminal VSDs [106, 110, 111]. C-terminal VSDs must strictly localize to the C-terminal part of the protein. Moreover, the introduction of C-terminal glycosylation sites or addition of extra alanine stretches at the C-terminus led to cell surface secretion [112, 113]. Some proteins even combine a sequence-specific VSD and a C-terminal VSD for dual targeting to the PSV matrix and to globoids [114]. Importantly, random C-terminal VSDs appear to be rather unique to plants. Despite more extensive research on animal systems, only a few cases have been reported in animals [115], where tyrosine motifs (YXXΦ) are more commonly used [116, 117].

The physical and structural VSDs are based on protein structure, and can be subdivided in two types. The first type may be composed of multiple internal domains forming a higher-order structure to function as a VSD. This is the case for the 11S globulin legumin from field bean. The second type is formed by the aggregation of seed storage proteins, presumably occurring in the Golgi apparatus. Proproteins are often more hydrophobic than their mature counterparts and therefore they form aggregates. This nonreceptor-mediated sorting mechanism was reported for seed storage proteins reaching the PSV through dense vesicles (DVs), which are unique vesicles only occurring in plants [106, 113, 118, 119].


In 1994, the first VSR was identified from a pea cotyledon CCV preparation. The protein was termed BP80 (binding protein 80 kDa) by its ability to bind in vitro the VSD of barley aleurain [120]. VSRs are type I membrane proteins with a cytosolic motif and a large lumenal domain. They are not related to TGN sorting receptors such as the mannose 6-phosphate receptor (MPR) in animals or the vacuolar carboxypeptidase sorting receptor VPS10 in yeast [121, 122]. VSRs generally recognize NPIRL-(like) consensus motifs in cargo proteins (mostly present at the N-terminus in cargo), whereas, in turn, VSRs themselves are recognized by AP1 via its YMPL consensus motif at the C-terminus. VSRs are generally considered to mediate protein sorting to LV, as supported by several pieces of evidence. First, VSRs recognize and bind the sequence-specific VSDs present in LV proteins, such as barley aleurain and sporamin [123]. Second, VSRs contain a tyrosine motif for interaction with the AP1 complex μ1 subunit and subsequent packing into the CCV [5, 124]. Third, Arabidopsis thaliana vacuolar sorting receptor 1, a BP80 homologue from Arabidopsis, interacts with AtEpsinR1, with its animal homologue being involved in CCV-mediated trafficking to lysosomes [125, 126]. Fourth, the A. thaliana vacuolar sorting receptor 1 C-terminal cytosolic tail interacts with AtVPS35, a prevacuolar compartment localized retromer from Arabidopsis [127]. Fifth, despite the fact that VSR-cargo ligand interaction might be initiated in the ER [64], VSRs are accumulating to a high extent in the prevacuolar compartment and to a lower extent in the TGN [128]. Finally, the pH-dependent ligand binding indicates that the receptor-cargo complex needs to end up in an acidic environment to release its cargo [129].

However, several studies also report interactions between VSRs and VSDs of storage proteins such as 2S albumins in castor bean [10, 102]. PV72, a pumpkin homologue of VSR, was identified in the precursor-accumulating compartments that are proposed as intermediates in the transport of storage proteins to the PSV [130, 131]. Similar results have been found in sunflower [132] and castor bean [112]. By contrast to the vacuolar sorting receptor BP80, ligand binding to PV72 is calcium-dependent because it is released at a low calcium concentration. Additionally, A. thaliana vacuolar sorting receptor 1 (also termed AtELP1) was shown to play a role in sorting storage proteins in seeds, which also shows calcium-dependence [133]. These results indicate that different isoforms may be involved in different sorting pathways. The recent finding that VSRs could be localized at the plasma membrane suggested an additional role for VSR proteins in mediating protein transport towards the plasma membrane and endocytosis in germinating pollen tubes of lily and tobacco [122].

The RMR protein family

The RMR protein family RingH2 was originally discovered as a result of their homology to protease-associated domains in VSRs, indicating their role in binding vacuolar proteins [10]. In vitro experiments showed the capability of RMR to bind to the C-terminal VSD of barley lectin, bean phaseolin and tobacco chitinase, which are then transported to the PSV [134, 135]. The RMRs are type I transmembrane proteins containing a typical N-terminal signal peptide, followed by a protease-associated domain and a single transmembrane domain [136]. By contrast to the short cytoplasmic tail of VSRs, plant RMRs contain a long cytoplasmic tail with a typical C3H2C3 RING-H2 domain [10]. One isoform of Arabidopsis, AtRMR1, has been mainly localized in the late Golgi apparatus, DVs and in the PSV of Arabidopsis embryos by the use of immunogold electron microscopy [137]. AtRMR2 was localized in the PSV [138], whereas other RMRs were also found in the PSV of tomatoes [13] and in members of the Brassicaceae [139]. These findings are compatible with the hypothesis of its role as a receptor in protein sorting to the PSV [122]. Recently, it was demonstrated that rice RMR1 associated with an intermediate vacuolar-like compartment related to the PSV [140].

AP complexes

AP complexes are heterotetramers mediating the formation of transport vesicles, as well as cargo sorting in all eukaryotes. Most of the research on AP complexes has been devoted to animals and yeast. According to Hirst et al. [141], five distinct AP complexes (AP1–AP5) have been identified in eukaryotes to date. AP complexes are composed of two large subunits (termed α/β1, α/β2, δ/β2, ε/β4 and ξ/β5, respectively), a medium subunit (μ1–μ5) and a small subunit (σ1–σ5) [141-144]. The γ/β1 and α/β2 subunits of AP1 and AP2 bind clathrin via clathrin-binding sites within their hinge domains [145]. Each AP complex operates in distinct organelle localization and performs similar functions. The AP1 complex is involved in CCV formation at the TGN and endosomes, mediating the trafficking between these organelles [146, 147]. The AP2 complex contributes to the formation of CCV from the plasma membrane and facilitates clathrin-mediated endocytosis [148, 149]. The AP3 complex is involved in the formation of vesicles from TGN/endosomes, and mediates transport to lysosomes/vacuoles [147, 150]. The clathrin binding of AP3 is under debate. In Arabidopsis, AP3 subunit loss-of-function mutants implicated AP3 in biogenesis and function of the plant LV [151-153]. The AP4 complex was recently defined as a mediator of the transport of the amyloid precursor protein from the TGN to the endosome [154]. It was suggested to be involved in vesicle formation with or without clathrin [155, 156]. The AP5 complex does not associate with clathrin, localizes in late endosomal compartments, and mediates endosomal sorting [144].

In the biosynthetic and endocytic trafficking pathways, AP complexes selectively recognize sorting signals [145]. A number of such sorting signals have been identified in the last decade, such as tyrosine signals (NPXY and YXXФ signals, where X could be any amino acid and Ф is a bulky hydrophobic amino acid), and dileucine signals ([DE]XXXXL[LI] and DXXLL consensus motifs) [147]. AP complexes are known to interact with tyrosine-based sorting signals via their μ subunits, although the AP subunits that recognize dileucine-based sorting signals remain unidentified. There is evidence indicating that AP1 binds via its γ and σ1 subunits [157], whereas AP3 binds via its β unit [145, 158].

Protein sorting to the vacuole

Protein sorting to the PSV

Storage proteins are transported to the PSV via Golgi-dependent or -independent pathways depending on the cargo protein and plant developmental stage [4, 159].

Unlike LVs, the trafficking of storage proteins from the Golgi apparatus into the PSV is mediated by DVs rather than by CCVs [160, 161]. DVs are small, uniform vesicles (150–200 nm in diameter) containing intrinsic membrane proteins destined for the PSV, and are characterized by their high density electron-opaque lumenal contents [113]. They were first discovered in common bean [160], and later in other plant species, such as wheat [162], pea [163] and Arabidopsis [137]. Within the cis-Golgi, the accumulation and condensation of storage proteins initiates the formation of DVs. Subsequently, these discrete small vesicles are transferred to the TGN concomitant with increased density and, finally, they are released from the TGN [80, 137]. Mature DVs are not protein coated; they can directly fuse with the PSV or first with multivesicular bodies [13, 161, 164, 165]. Multivesicular bodies contain multiple internal vesicles, are present in all eukaryotes, and are involved in various post-Golgi processes of the biosynthetic trafficking pathway. DVs fuse into the multivesicular bodies where they discharge their contents [166]. Multivesicular bodies are then received by the PSV together with their cargos. Therefore, the Golgi-dependent PSV trafficking pathway could be defined as an ER→Golgi→DV→(multivesicular bodies)→PSV pathway (Fig. 1A). Protein transport to the PSV mainly occurs through aggregation sorting, although receptor-mediated sorting might play a role as well [132]. In this case, the involved VSRs are BP80 homologues (such as PV72, AtELP1) and RMRs [80, 112, 113, 167, 168]. Many proteins have been reported that sort to the PSV via DVs, such as legumin, vicilin and sucrose-binding-protein homologue [132, 169].

Transport of storage proteins to the PSV can also take an alternative route from the ER bypassing the Golgi, as shown in Fig. 1A. Globulins, the major vacuolar storage proteins in pumpkins, were suggested to reach the PSV via precursor-accumulating compartments, which are much larger (diameter of 200–400 nm) than DVs, and reach the PSV directly from the ER [130, 170]. Similar results have been obtained for cysteine proteinases [171, 172]. Precursor-accumulating compartments contain unglycosylated precursors of storage proteins, and mediate aggregation sorting. Precursor-accumulating compartments have been found in pumpkin, castor beans and in wheat [130, 173, 174]. Although directly generated from the ER, precursor-accumulating compartments can accept glycosylated proteins derived from the Golgi during their transport to the PSV [112, 130]. The content of precursor-accumulating compartments is incorporated in the lumen of the PSV. The incorporation follows one of two models: (a) fusion between precursor-accumulating compartments and PSV occurs through autophagy [175, 176] or (b) by direct membrane fusion [177].

Protein sorting to the LV

Different pathways for post-Golgi sorting of proteins to lysosomes/vacuoles have been described for yeast and animals. The pathway through CCVs appears to be conserved among all eukaryotes (plants, yeast, animals). Inside the TGN, specific sorting signals are recognized by TGN membrane localized receptors, recruited into CCVs and transported into LVs/lysosomes [165, 178]. In animal cells, the sorting of acid hydrolases to the lysosome is facilitated by the MPR [179]. MPR-ligand complexes are recruited into CCVs at the TGN. This process is mediated by Golgi-localized, γ adaptin ear-containing, Arf-binding proteins and by the AP1 complex through interactions with MPRs tyrosine (YXXФ) and dileucine (LL) motifs at the cytosolic tail [179, 180]. In yeast, the sorting and delivery to the LV is very similar to the MPR pathway of animal cells [181, 182]. This mechanism is assumed to be used for trafficking to plant LVs as well, although LV proteins interact with VSRs, showing no homology to MPRs [85]. Plant VSRs recognize sequence-specific VSDs (e.g. NPIRL-like consensus motifs) in LV targeted proteins, such as barley aleurain [183, 184]. VSRs then interact with AP1 through a tyrosine-based sorting motif (e.g. YMPL) instead of through a dileucine motif as observed in yeast and animals [124]. CCVs, containing cargo-VSR, then bud from the TGN, and discharge their contents after fusion into the prevacuolar compartment [163, 166]. As a result of the lower pH of the prevacuolar compartment, ligands dissociate from their receptor, and the receptor is subsequently recycled back to the Golgi apparatus [185-187]. Importantly, an MPR-independent and a VPS10-independent lysosome sorting pathway were found in animal cells and yeast, respectively [188, 189]. Recent studies on the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts suggest a similar route in plants (Fig. 1B) [190]. AP3, but not AP1, appears to fulfill a central role in this pathway. In animals, AP3 can recognize both dileucine and tyrosine motifs, whereas, in yeast, only dileucine signals can be recognized [145, 191]. Recent reports suggest that AP3 subunits are involved in the biogenesis, morphology and function of the prevacuolar compartment and vacuoles in plants [151-153].

Remarkably, and uniquely in plants, LV resident proteins can be transported directly from the ER to the LV by means of ER bodies as intermediate compartments, bypassing the Golgi apparatus, as shown in Fig. 1B [170]. Thus, one of the emerging differences that appears to distinguish plants from other eukaryotes is the plasticity of the ER to form protein-, oil- or rubber-containing subcellular structures best termed ER bodies [192], which either stably accumulate or are transported to the LV. The ER bodies are in most instances spherical, < 1 μm in diameter, and consist of a dense core of a self-assembling or aggregating protein, oil or rubber, and a membrane of ER origin [193]. However, there is some evidence for nonconventional ER trafficking bypassing the Golgi to the lysosome in animal cells [194]. It was proposed that ER bodies in plants can follow a similar path, bypassing the Golgi and directly fusing with LV, as observed under stress conditions [[195],196].

Conclusions and perspectives

Recent advances in our understanding of the processes involved in the sorting of proteins to the vacuole(s) in plant cells suggest that there are relatively highly-conserved processes among eukaryotes. Most differences are related to the uniqueness of the plant vacuole as a storage compartment, as opposed to the animal lysosome and the yeast vacuole, which function predominantly as hydrolytic compartments. Another distinctive feature is the apparent duplicity and alternative pathways for proteins to reach the lytic and protein storage vacuolar lumen or tonoplast. Furthermore, the variety of chemical substances stored in the vacuole (e.g. protein bodies, resins, gums, latex, sugars, etc.) imposes the acquisition of redundant transport pathways to the vacuole. One aspect of the vesicle-mediated delivery system to the vacuole that awaits clarification is how the vacuoles compensate for the increasing uptake of fluids and added membrane during the vesicle fusion process. At present, there are no indications as to how homeostasis is maintained, although, in yeast, the formation of retrograde CCV has been observed. The alternative post-Golgi, AP3-mediated route has been well described in yeast and animals, although further experimental work is required to determine whether this route is also fully active in plants. Furthermore, the regulatory mechanisms of the AP3-mediated and Golgi-independent routes, as well as the cargos, receptors and possible budding factors involved, still remain elusive and require further exploration. The use of fluorescent probes, transgenic plants and new imaging techniques will likely provide us with a clearer understanding in the near future.


Li Xiang and Wim Van den Ende are supported by grants from FWO Vlaanderen.