Download Vacuolar protein sorting mechanisms in plants

REVIEW ARTICLE
Vacuolar protein sorting mechanisms in plants
Li Xiang1, Ed Etxeberria2 and Wim Van den Ende1
1 Laboratory of Molecular Plant Biology, KU Leuven, Belgium
2 Horticulture Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA
Keywords
adaptor protein complex; BP80;
endomembrane; ER body; Golgi; protein
storage vacuole; signal peptide; sorting;
trafficking
Correspondence
W. Van den Ende, Laboratory of Molecular
Plant Biology, KU Leuven, Kasteelpark
Arenberg 31, Leuven, Belgium
Fax: +32 16321967
Tel: +32 16321952
E-mail: [email protected]
(Received 14 July 2012, revised 8
November 2012, accepted 11 December
2012)
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 postGolgi 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.
doi:10.1111/febs.12092
Introduction
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 mem-
branes 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
Abbreviations
AP, adaptor protein; Arf, ADP ribosylation factor; CCV, clathrin-coated vesicle; COP, coat protein complex; DV, dense vesicle;
ER, endoplasmic reticulum; LV, lytic vacuole; MPR, mannose 6-phosphate receptor; PSV, protein storage vacuole; RMR, receptor
membrane ring-H2; Sar, secretion-associated RAS-related protein; SNARE, soluble nethylmaleimide sensitive factor attachment protein
receptor; TGN, trans-Golgi network; TIP, tonoplast intrinsic protein; VSD, vacuolar sorting determinant; VSR, vacuolar sorting receptor.
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Vacuolar protein sorting in plant cells
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].
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
980
A
B
Fig. 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 Golgidependent 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 Golgiindependent pathway, PSV residents are packed into precursoraccumulating 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 (a-TIP) might be
transformed into an LV (c-TIP). Fusions of LVs or PSVs make up
the central vacuole. PVC, prevacuolar compartment; BP80/VSR,
vacuole sorting receptor.
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,
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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: a, b, c, d and e-TIPs
[35]. a and b-TIPs are seed-specific. c-TIPs associate
with the LV, whereas a-TIP and d-TIP associate with
the PSV [36,37]. e-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.
Vacuolar protein sorting in plant cells
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,
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Vacuolar protein sorting in plant cells
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 RASrelated 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 targetSNARE 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 highlyregulated 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
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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 transGolgi to reach their final destination [65,81]. The
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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 GTPbinding protein, Arf). The clathrin unit is a threelimbed 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
Vacuolar protein sorting in plant cells
[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 b-conglycinin a 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
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Vacuolar protein sorting in plant cells
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].
VSRs
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 l1 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 VSRcargo 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 precursoraccumulating 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
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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
a/b1, a/b2, d/b2, e/b4 and ξ/b5, respectively), a medium subunit (l1–l5) and a small subunit (r1–r5) [141
–144]. The c/b1 and a/b2 subunits of AP1 and AP2
bind clathrin via clathrin-binding sites within their
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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 l
subunits, although the AP subunits that recognize
dileucine-based sorting signals remain unidentified.
There is evidence indicating that AP1 binds via its c
and r1 subunits [157], whereas AP3 binds via its b
unit [145,158].
Protein sorting to the vacuole
Protein sorting to the PSV
Storage proteins are transported to the PSV via Golgidependent 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
Vacuolar protein sorting in plant cells
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. Precursoraccumulating 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
FEBS Journal 280 (2013) 979–993 ª 2012 The Authors Journal compilation ª 2012 FEBS
985
L. Xiang et al.
Vacuolar protein sorting in plant cells
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, c adaptin earcontaining, 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 lm 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
986
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 postGolgi, 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.
Acknowledgements
Li Xiang and Wim Van den Ende are supported by
grants from FWO Vlaanderen.
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