Update on Plant Endocytosis
Molecular Dissection of Endosomal Compartments
in Plants1
Jens Müller, Ursula Mettbach, Diedrik Menzel, and Jozef Šamaj*
Institute of Cellular and Molecular Botany, University of Bonn, D–53115 Bonn, Germany (J.M., U.M., D.M.,
J.Š.); and Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, SK–95007 Nitra,
Slovak Republic (J.Š.)
The first indications for the existence of endocytosis
in plants were already obtained two decades ago. For
instance, electron-dense tracers were shown to incorporate into membrane-bound structures within the
cell (Hillmer et al., 1986, 1988; Tanchak and Fowke,
1987; Galway et al., 1993). The identification of clathrincoated pits, associated with the plasma membrane
(PM), was another indication of endocytic events in
plants (Robinson and Hillmer, 1990). Nevertheless,
unambiguous and conclusive evidence for endocytosis
and its vital role for plant cells was missing for a long
time. In the last 5 years, however, a broad range of
molecular markers was developed (for example, Ueda
et al., 2004; Uemura et al., 2004; Jaillais et al., 2006;
Ortiz-Zapater et al., 2006; Lam et al., 2007) and, together with lipid marker dyes such as FM1-43 and
FM4-64 (Vida and Emr, 1995; Emans et al., 2002; Bolte
et al., 2004), widely used to analyze PM vesicular
recycling and endocytosis as well as to identify and
characterize the corresponding endomembrane compartments. Several recent findings suggest that diverse
PM and extracellular cargoes are internalized by endocytosis in plants. For example, endosomal internalization of auxin carriers and pathogen receptors are
highly relevant with respect to physiological responses
of plant cells, such as auxin-regulated polar growth or
pathogen defense. Thus, endocytosis is regarded essential for plant development and for plant adaptation
to the environment. Quite a number of reviews have
appeared recently reflecting the growing attention that
is given to the endocytosis in the field of cell and developmental plant biology (for example, Šamaj et al.,
2004, 2005, 2006; Baluška et al., 2006; Geldner and
Jürgens, 2006; Mo et al., 2006; Robatzek, 2007). In this
Update, we highlight the most important results related to plant endocytosis, especially the most recent
findings published in the last 2 years. We discuss them
with particular focus on molecular components found
1
This work was supported by the Deutsche Forschunggemeinschaft (grant no. SA 1564/2–1 to J.Š.).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jozef Šamaj ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.107.102863
on the putative early and late endosomes. Additionally, we take a closer look at the structure and dynamics of endosomal compartments and on the approved
and/or putative functions of associated molecules.
Finally, we draw attention to the experimental tools,
which turned out to be instrumental in the research
efforts addressing endocytosis and endosome functions in plants.
THE ROLE OF ENDOCYTOSIS IN PLANTS
In the past years, a significant body of evidence accumulated suggesting a role of regulated endocytosis
in plant development. For example, it was shown in
tip growing cells such as root hairs and pollen tubes
that the strong secretory machinery operating at the
apex is directly accompanied by endocytosis throughout the area of the clear zone (Šamaj et al., 2006 and
refs. therein). Other examples indicate a pivotal role
of endocytosis in the control of cell wall metabolism
(Robert et al., 2005; Johansen et al., 2006). In these
studies it is suggested that the activity of the cellulosesynthase complex may be regulated by way of directed
cycling between the PM and endomembrane compartments. Another type of endocytic cargoes are PM
anchored arabinogalactan proteins that are internalized, and by passing through multivesicular bodies
(MVBs) may end up in the vacuoles (Herman and
Lamb, 1992; Šamaj et al., 2000). Additionally, pectins
were also proposed to be internalized via endocytosis
and subsequently rerouted during cytokinesis to provide the developing cell plate with building material
(Baluška et al., 2002, 2005; Dhonukshe et al., 2006).
Further ultrastructural experiments clarifying whether
cell wall pectins are directly accumulated in endosomal compartments (as it was shown for arabinogalactan proteins) might further strengthen this idea.
It may generally be assumed that one crucial requirement for metabolism, development, and signaling is the controlled uptake of biopolymeric substances
such as pectin but also small metabolites from the cell
surface and its distribution in the cell. In many cases,
uptake of small metabolites is accomplished by carrier
proteins residing in the PM. Additionally, there is
growing evidence that spatial distribution and activity
of auxin carriers depend on endocytosis-mediated
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Müller et al.
regulatory mechanisms, proposed to be controlled by
cycling between the PM and endosomal compartments (Geldner et al., 2001). One extensively studied
example is the strictly regulated polar transport of
auxin by pin-forming (PIN) efflux carriers (Gälweiler
et al., 1998). According to Geldner and coworkers
(Geldner et al., 2001, 2003) PIN proteins are continuously cycling between the PM and the endosomal
compartment. Recently it was shown that auxin promotes its own efflux by regulating endocytic activity
and probably degradation of carrier proteins like PIN1
(Paciorek et al., 2005) and PIN2 (Abas et al., 2006).
However, the identity of the endosomal compartment
in which PIN carriers are recycled is not clarified yet
because PIN proteins were not unambiguously localized on the ultrastructural level. The internalization of
PIN1 is known to be mediated by GNOM, an adenosine ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF), which itself acts on an endosome
that seems to be associated with the small GTPase called
Ara7 in Arabidopsis (Arabidopsis thaliana; Geldner et al.,
2003). Ara7 in turn is thought to be associated with the
late endosomes, termed prevacuolar compartment
(PVC) and/or MVB (Sohn et al., 2003; Kotzer et al.,
2004; Lee et al., 2004; Haas et al., 2007), and is known to
be involved in the endocytic pathway (Ueda et al., 2004).
On the other hand, because PIN1 cycling takes place
very rapidly (Geldner et al., 2001), it seems to be more
likely that GNOM acts on an early endosomal compartment. Moreover, a recent study demonstrated the
association of Arabidopsis SORTING-NEXIN1 (AtSNX1)
with an endosomal compartment, which is distinct
from that containing GNOM (Jaillais et al., 2006). SNXs
belong to a protein family known to be involved in the
regulation of intracellular trafficking (Carlton et al.,
2005). Jaillais et al. (2006) have shown that AtSNX1 not
only resides on compartments that are carrying PIN2
proteins, but is also involved in their proper distribution, because snx1 null mutants exhibit multiple auxinrelated defects. Future work on SNX1 might give more
insights into the regulated endocytic cycling of PIN2
carriers and the identity of endosomal structures involved in this process. Another example for regulated
uptake and distribution through endocytic cycling is
the Arabidopsis boron transporter AtBOR1, which was
shown to accumulate in the PM under low boron conditions, whereas it gets internalized and degraded if
the external boron concentration rises to toxic levels
(Takano et al., 2005). One of the most striking discoveries of the last year was the unambiguous demonstration of the existence of receptor-mediated endocytosis
(RME) in plants. Although it has long been speculated
about the existence of RME (Horn et al., 1990), and a
set of candidate receptors have been proposed to reside in the PM and in endosomal structures (Russinova
and de Vries, 2006 and refs. therein), clear evidence for
RME was provided by Robatzek et al. (2006) only
recently. These authors showed that the Arabidopsis
leucin-rich repeat receptor FLAGELLIN SENSITIVE2
(FLS2) resides at the PM and gets internalized into
endosomal compartments upon specific binding of its
ligand, flg22, a small bacterial flagellin peptide that is
known to elicit basal defense responses (see GomezGomez et al., 2001; Gomez-Gomez and Boller, 2002).
There are more candidates possibly involved in induced endo- and exocytotic events mediating plant
immune responses (Robatzek, 2007) and future work
will show whether RME is, like in animals, a key
regulator in plants specifically recognizing putative
pathogens.
ENDOSOMAL COMPARTMENTS IN ANIMALS
In animal cells several types of endocytic processes such
as phagocytosis (uptake of particles), pinocytosis (uptake
of fluid), clathrin-mediated endocytosis, cavaeolaemediated endocytosis, and clathrin- and caveolaeindependent endocytosis occur. Clathrin-dependent,
receptor-mediated internalization of extracellular
components/ligands is clearly the best characterized
endocytic process. It involves multiple endosomal
organelles, through which endocytic cargo is passed
in a stringent order. The first step in this pathway is
the formation of clathrin-coated pits and vesicles serving for internalization of receptor-ligand complexes at
the PM (Mellman, 1996). Internalized vesicles then fuse
with the sorting endosome, representing one type of
early endosome. This is the first endosomal compartment on the route to recycling and degradation. Within
this organelle, because of its acidic lumen, ligands
start to get dissociated from their receptors (Johnson
et al., 1993) and are sorted according to their destination (Mo et al., 2006 and refs. therein). Proteins
determined for recycling are either directly delivered
back to the PM or they are first passed on to another
early endosomal compartment called the endosomal
recycling compartment (Sheff et al., 1999; Hao and
Maxfield, 2000). Furthermore, they can be transported
from the endosomal recycling compartment to the
trans-Golgi network (TGN; Wilcke et al., 2000; Lin
et al., 2001). Proteins that are destined for degradation
are packed and delivered to the late endosome, the
so-called MVBs, before they finally reach the lysosome (Goldstein et al., 1985; Mukherjee et al., 1997).
In some cases, cargo gets recycled from the MVB to
the TGN with the help of retromer complexes. For
example, this was shown for the cation-independent
Man 6-phosphate receptor, a receptor maintaining the
transport of lysosomal hydrolases from the TGN to
MVB (Arighi et al., 2004). MVBs received their name
because they possess inner luminal vesicles derived
from membranous invaginations. It is believed that
formation of such inner luminal structures serves a
double purpose. First, it enables the degradation of
membrane-integral proteins/receptors and second it
terminates their signaling function (Katzmann et al.,
2002). Internalization of transmembrane proteins is
mediated through so-called ESCRT complexes, which
recognize monoubiquitinylated proteins and enable
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Endosomes in Plants
their delivery into the inner luminal vesicles of MVB
and to the lysosome. Therefore, monoubiquitinylation
seems to serve as a signal attached to proteins that
are determined for degradation within the vacuole/
lysosome (Katzmann et al., 2001; Babst et al., 2002a,
2002b).
ENDOSOMAL MARKERS IN PLANT CELLS
The straightforward approach to analyze the organization of the endosomal system within plants is to
identify the intracellular compartments that are involved in this trafficking route. A relatively well established way to identify and characterize these structures
is to use specific molecular markers, which are known
to act predominantly at one specific endomembrane
compartment. Eukaryotic cells possess a broad range of
highly conserved protein families known to be essential
for proper protein sorting, directed vesicular trafficking, and fusion of endosomal/PM-derived vesicles
with the target membranes. All of these protein families
can be divided into subclasses, which often are associated specifically with only one endosomal compartment. The most common protein families used in this
context are t-SNAREs/syntaxins, small GTPases, and a
set of diverse endosomal sorting receptors. T-SNAREs
belong to the syntaxin family and are responsible for
the fusion of vesicles with its specific target membrane
(Jahn and Scheller, 2006). The Arabidopsis genome
encodes at least 54 t-SNARE proteins. Some of them are
already characterized as specifically involved in fusion
events on distinct endosomal compartments and therefore are potentially useful molecular markers. For example, the Arabidopsis SNARE SYP41 is associated
with the TGN, while SYP21 is thought to be associated
with a late endosomal compartment (Uemura et al.,
2004). A comprehensive report about the localization
and the important role of SNARE’s in plants is given in
a topical review by Surpin and Raikhel (2004).
In plants, small GTPases are divided at least into four
major groups, the Rac/Rop (Rho of plant), the Ran, the
Arf/Sar, and the Rab families. Although Ran GTPases
were identified in a variety of plant species their roles
are not well characterized. From animal systems they
are known to regulate the nuclear trafficking of RNA
and proteins and in plants they are speculated to be
involved in the regulation of the cell cycle (for review,
see Meier, 2007). While Rop proteins seem to be mainly
involved in signaling events and cytoskeletal organization, Rabs and Arfs are more important for the correct
protein sorting as well as for regulated vesicular trafficking and fusion with the target organelle (Rutherford
and Moore, 2002; Molendijk et al., 2004; Šamaj et al.,
2006). These latter small GTPases have already been
used to define plant-specific membrane compartments,
including those of the endocytic machinery (for example, Ueda et al., 2001, 2004; Sohn et al., 2003; Kotzer
et al., 2004; Lee et al., 2004; Voigt et al., 2005). Endosomal
sorting receptors, useful as molecular markers to define
plant endosomal compartments are, for example, the
vacuolar sorting receptor (VSR) BP-80 from pea (Pisum
sativum) and its Arabidopsis homolog AtELP1, which
are known to predominantly associate with PVCs/
MVB (Li et al., 2002; Tse et al., 2004). This organelle
mediates the transport of proteins to both lytic vacuoles
(Li et al., 2002) as well as to protein storage vacuoles
(Otegui et al., 2006). Meanwhile, there is a larger number of additional proteins used as molecular markers.
Some of them do not even occur in plants, but are used
as heterologous markers because they are known to
define specific compartments or trafficking pathways
in other eukaryotic cells. One actual example is the
human transferrin receptor (hTfR) which, if heterologously expressed in Arabidopsis protoplasts, binds its
ligand, transferrin, at the PM and internalizes it to
endosomal compartments (Ortiz-Zapater et al., 2006).
Thus, hTfR appears to be a useful tool for studying the
mechanism of RME in plants. In most cases, however, it
seems that it is not possible to transfer the animal or
yeast (Saccharomyces cerevisiae) models of endosomal
organization directly to the situation in plants, even
though there often is a high partial homology between
the identified protein families within the eukaryotes.
For example, in Arabidopsis there are three known
homologs of the mammalian Rab5 family of small G
proteins, namely Ara7 (RabF2b), Rha1 (RabF2a), and
plant-specific Ara6 (RabF1). While in mammals members of this family are clearly associated with early
endosomes (Gorvel et al., 1991; Bucci et al., 1992),
several studies have shown that they can be associated
with a late endosomal compartment in plants (Sohn
et al., 2003; Kotzer et al., 2004; Lee et al., 2004; Dettmer
et al., 2006; Haas et al., 2007). Furthermore, plants
seem to lack some of the protein subclasses known to
be essential regulators of protein sorting, vesicle trafficking, and fusion in animals. For example, there
are no homologs of the mammalian Rab4 and Rab9
subclasses of small Rab-GTPases, which are both
involved in the regulation of endosomal trafficking
(Nielsen, 2005).
Another possibility to better characterize endosomal
compartments and their spatial and functional organization is to use membrane-impermeable fluorescent
tracers, which make it possible to directly visualize the
endocytic trafficking routes. One well established tracer
is the red styryl marker dye FM4-64 which, if exogenously applied, incorporates into the PM and passes
through the diverse endosomal compartments on its
way to the vacuole (Vida and Emr, 1995; Bolte et al.,
2004). Additionally, the GFP technology, visualizing proteins within living cells by expression of proteins tagged
to GFP or its spectral variants in transgenic plants or
cell lines, is of great advantage. The combined application of marker dyes like FM4-64 with molecular
markers tagged to GFP is widely used and helps substantially to define and characterize intracellular endosomal compartments in plants (for example, Ueda et al.,
2001, 2004; Tse et al., 2004; Voigt et al., 2005; Dettmer et al.,
2006). Importantly, immunogold electron microscopy
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Müller et al.
using specific antibodies against resident proteoglycans and proteins provides an important tool for unambiguous identification of endosomal compartments
(Herman and Lamb, 1992; Šamaj et al., 2000; Tse et al.,
2004; Dettmer et al., 2006; Preuss et al., 2006; Lam et al.,
2007).
PHARMACOLOGICAL AGENTS
The use of drugs that affect vesicular trafficking
between endomembrane compartments by modifying their dynamic behavior as well as structure and
organization turned out to be very useful tools in determining the origin, assignment, and functional interconnections of distinct endosomal compartments
within the endocytic network. In addition to diverse
drugs that are disturbing the dynamics of cytoskeletal
elements and therefore affect the vesicular/organellar
motility and organization, there are mainly three drugs,
which directly interfere with vesicular trafficking and
fusion events, namely Brefeldin A (BFA), Wortmannin,
and Tyrphostin A 23.
BFA is a fungal toxin that targets an endoplasmic
reticulum (ER) or endosome-located GEF. This GEF is
essential for the activation of the small GTPase Arf1
(Jackson and Casanova, 2000; Nebenfuhr et al., 2002).
Its inactivation at the interface between ER and cisGolgi leads to the disruption of the formation of COPIcoated vesicles, thereby stopping the membrane
replenishment flow from the ER to the Golgi. Eventually,
Golgi stacks disappear and secretion is stopped, whereas
endocytosis may continue and produce a surplus of
internalized membranes, which aggregate and form the
so-called BFA compartments in plants. There is also
one ARF/GEF member called GNOM that is sensitive
to BFA, which is localized on the endosomal compartment and was proposed to be involved in trafficking
from endosome to the PM (Geldner et al., 2003). At
lower concentration it is possible to observe different
types of membrane aggregates, depending on the cell
type and plant used in the experiment. In dividing
tobacco (Nicotiana tabacum) BY-2 suspension cells a
hybrid ER-Golgi compartment is formed as well as a
BFA compartment, possessing a mixture of the TGN
vesicles and presumptive endosomal vesicles. Additionally, it has been shown that also MVBs/PVCs can
form aggregates at higher concentrations of BFA, which
are distinct from preferentially TGN-derived aggregates (Tse et al., 2006). In Arabidopsis root cells, the
core of the BFA compartment consists of TGN vesicles
surrounded by the rest of the Golgi at the periphery
(Grebe et al., 2003; Šamaj et al., 2004) and in epidermal
cells of the root apex in maize (Zea mays), BFA compartments are shown to consist of homotypically fused
TGN (Hause et al., 2006). Because it was clearly demonstrated for diverse plant species that the effect of
BFA is reversible (e.g. Driouich et al., 1997; Geldner
et al., 2001; Ritzenthaler et al., 2002) this drug can be
successfully used for physiological recovery studies.
Therefore, with regard to this point, the use of BFA can
have some advantages in comparison to genetic approaches such as RNAi technology or T-DNA insertion mutants and their revertants.
Wortmannin is the second drug extensively used in
studies on endosomal organization and trafficking. It
is known to target phosphoinositide 3 kinase (Ui et al.,
1995) inhibiting vacuolar/lysosomal trafficking at the
level of late endosomes in both mammalian (Bright
et al., 2001) and plant cells (Matsuoka et al., 1995) in a
dose-dependent manner. On the subcellular level, it
has been shown that Wortmannin causes MVB/PVC
to form vacuolated structures but it does not affect
the Golgi (Tse et al., 2004). However, results from
Wortmannin treatments in plants always have to be
interpreted with some caution because it is known to
inhibit also phosphoinositide 4 kinase at higher concentrations (Matsuoka et al., 1995).
Unlike BFA and Wortmannin, which are extensively
used as inhibitors of vesicular trafficking in plants,
tyrphostins represent a relatively new class of drugs.
They have been primarily used as inhibitors of Tyr
kinases in animal cells. From these it is known that
tyrphostin A23 is able to inhibit the interaction of the m2
subunit of the adaptor complex AP-2 with the YXXF
internalization motif of transmembrane proteins such
as hTfR. As a consequence, the internalization of these
receptor proteins within clathrin-coated vesicles is
blocked (Aniento and Robinson, 2005 and refs. therein).
In a recent study, Ortiz-Zapater et al. (2006) demonstrated that internalization of heterologously overexpressed hTfR is effectively inhibited by tyrphostin A23
in Arabidopsis protoplasts. These results suggest that
tyrphostin A23 is able to inhibit endocytosis of putative
membrane proteins bearing the YXXF motif also in
plants. Nevertheless, since the direct target of tyrphostin A23 is not known and the final proof for the existence of clathrin-mediated endocytosis in plant cells is
still missing, results from experiments using tyrphostins as endocytotic inhibitors always have to be interpreted with caution.
In summary, if used carefully BFA and Wortmannin
as well as tyrphostin A23 can be used as valuable
pharmacological tools for the study of secretory and
endocytic pathways because they affect different endomembrane structures and trafficking routes in a cell
type, plant species, and dose-dependent manner.
IDENTIFICATION OF ENDOSOMAL
COMPARTMENTS IN PLANTS
Using ultrastructural analysis as well as different
molecular markers, a couple of endosomal compartments have been described during the past years in
plants that partially resemble those known from animals and yeast. Initially, it seemed to be difficult to
integrate these structures into a functional endocytic
pathway. Nevertheless, recent studies provide first,
important insights into their functions, organization,
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Endosomes in Plants
and interconnections. A model of the plant endosomal
system based on the most recent findings is shown in
Figure 1.
MOLECULAR AND GENETIC DISSECTION OF
CLATHRIN-DEPENDENT TRAFFICKING IN PLANTS
Like in animals, more than one form of endocytosis
might exist in plants. Different reports suggested the
existence of several types of endocytosis such as fluid
phase uptake (for example, Baluška et al., 2004), phagocytosis of bacteria (for example Son et al., 2003), and
lipid raft-mediated endocytosis (for example, Grebe
et al., 2003). In relation to the latter, it was previously
speculated that the recycling of PIN auxin efflux carriers is dependent on their sequestration within PM
lipid raft microdomains in Arabidopsis (Grebe et al.,
2003; Willemsen et al., 2003).
Concerning the question of clathrin-dependent endocytosis in plants, several essential components such
as clathrin and adaptor protein subunits were identi-
fied during the last years (Holstein, 2005; Murphy
et al., 2005; Mo et al., 2006). Furthermore, a recent work
suggests that the plant TGN plays a role not only as a
secretory organelle but also as an early endosomal compartment (Dettmer et al., 2006; Lam et al., 2007) and it
possesses clathrin coats (Lam et al., 2007). The function
of clathrin in endocytosis and vesicular trafficking was
investigated using genetic or pharmacological approaches in two very recent studies (Dhonukshe et al.,
2007; Tahara et al., 2007). Both were using the dominant
negative effect of overexpression of the C-terminal
part of clathrin heavy chain (the so-called clathrin hub
fragment) preventing proper triskelion assembly (Liu
et al., 1995). Tahara et al. (2007) used this approach to
inhibit clathrin-dependent endocytosis in stably transformed tobacco BY-2 cells and they have found suppression of endocytosis as indicated by the inhibition
of FM4-64 dye uptake and the formation of aberrant
spindle and phragmoplast structures, finally resulting
in cytokinetic defects and multinucleate cells. Even
though, these results have to be interpreted with caution, because it was not shown that the inhibition of
Figure 1. Endocytic routes and compartments in plant cells. Internalized components from clathrin-coated pits (CCP) or lipid
rafts (LR) are delivered to an early endosome (EE) that was proposed to be identical to the TGN (Dettmer et al., 2006; Lam et al.,
2007). Internalized cargo is either recycled/secreted back to the PM or delivered to the MVBs/PVC that are assumed to function
as late endosomes (LE). From there, vacuolar proteins as well as proteins determined for degradation are transported to the
vacuole while cargo receptors may be recycled back to the TGN (Oliviusson et al., 2006). Additionally, there is at least one
endosomal compartment that is not identified yet. This compartment is labeled by SNX1 and might be a late endosome since
structural homologs of AtSNX1 in yeast are known to be part of a retromer residing at MVB/PVC (Oliviusson et al., 2006). Both
TGN and MVBs are labeled by FM4-64 and therefore clearly of endosomal nature. Molecular markers are depicted in different
colors. Rab-GTPases are blue, SNAREs are green, VSRs are black, and other molecules are yellow. Inhibitors with their inhibitory
effects are shown in purple. Black arrows indicate established endocytic routes, while punctuated arrows indicate putative ones.
Green colored compartment is not identified at the ultrastructural level but it is labeled by endosomal markers visualized by
confocal laser scanning microscopy.
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Müller et al.
endocytosis is a direct effect of the mutation and
because it is likely that defects in proper triskelion
assembly would not only affect endocytosis but also
the secretory pathway, this study gives indications for
clathrin-dependent trafficking at the forming cell plate
as an essential final step in plant cell division. Dhonukshe
et al. (2007) could demonstrate that the constitutive endocytosis of PIN auxin efflux carriers is clathrin dependent in Arabidopsis protoplasts and roots using genetic
and pharmacological approaches. They showed that
transiently transformed protoplasts overexpressing the
clathrin Hub fragment construct were not able to internalize FM4-64 as well as membrane proteins such as
PINs and the aquaporin PIP2. Pharmacological interference with the clathrin-mediated endocytic machinery
using tyrphostin A23, an inhibitor preventing the recruitment of endocytic cargo, inhibited internalization
of several PM proteins such as PINs, Lti6b, H-ATPase,
and PIP2 but not the general endocytic uptake of FM
dye (Dhonukshe et al., 2007). Although, as mentioned
above, the effects of tyrphostins in plant cells have to
be taken with caution, these data seem to support
the existence of constitutive clathrin-mediated endocytosis of some membrane proteins. Altogether, these
two reports suggest that clathrin-dependent trafficking
might represent a major endocytic route in plant cells.
On the other hand, other types of endocytosis such as
fluid phase uptake of sugars/nutrients and phagocytosislike uptake of Rhizobia might be restricted to specialized organs and tissues (Son et al., 2003; Baluška et al.,
2004; Etxeberria et al., 2005; Baroja-Fernandez et al.,
2006).
MOLECULAR DISSECTION OF TGN AS POTENTIAL
EARLY ENDOSOMAL COMPARTMENT IN PLANTS
In plants, TGN could be defined as an assembly of
electron translucent round and pear-shaped vesicles
reaching their size up to 100 to 150 nm and forming
budding profiles (Dettmer et al., 2006; Hause et al.,
2006; Preuss et al., 2006; Lam et al., 2007). These vesicles show electron-dense clathrin coat at their surface
and often are interconnected via distinct stalk-like connections and partial bridge-like fusions (Fig. 2; Hause
et al., 2006). TGN could be found either as loosely
associated with trans-Golgi side or more independent
from Golgi stacks (Uemura et al., 2004; Hause et al.,
2006; Lam et al., 2007) that likely depends on cell type
and physiological situation (Akihiko Nakano, personal communication). It is not clear yet whether these
two populations of TGN are functionally identical.
In animals, the TGN is described as a structure tightly
associated with the trans-side of the Golgi apparatus and Golgi accumulates around the microtubuleorganizing center in most interphase cells (Kupfer et al.,
1982). In contrast to animals, plants seem to lack typical
microtubule-organizing centers, and the Golgi stacks as
well as the TGNs are not restricted to the proximity of
the nucleus but rather they are distributed over the
entire cell. According to the classical definition from
animal cell biology, early endosomes are the first organelles that are receiving and sorting endocytozed
cargo. Since two recent reports described the plant TGN
as an early endosome it remains to be shown convincingly which endocytic cargo is internalized from PM to
TGN, and subsequently which is the further fate of this
cargo in the plant endocytic pathway.
One of the first molecular markers described as specific for the TGN was the Arabidopsis SNARE protein
SYP41 (Uemura et al., 2004). It has been shown that a
GFP-SYP41 fusion construct, transiently overexpressed
in Arabidopsis suspension-cultured cells, did not colocalize with Golgi or PVC markers but aggregated
under the influence of BFA. Recently it was shown that
the Arabidopsis membrane-integral V-ATPase subunit
VHA-a1 is colocalized with SYP41 and resides in the
Figure 2. Ultrastructure of endosomal compartments
in plants. Typical examples of TGNs (A–C) either
loosely associated with Golgi (A and B) or independent from Golgi (B and C), and of MVBs containing
internal microvesicles (D and E) in root cells prepared
by high-pressure freezing/freeze substitution. Budding profiles and connections between TGN vesicles
are indicated by arrowheads, MVBs are indicated by
arrows, and Golgi is indicated by G. Bar represents
0.35 mm for A, 0.5 mm for B, 0.25 mm for C, 0.2 mm
for D, and 0.3 mm for E. Figures A to C were
reproduced from Hause et al. (2006) with permission
granted by Landes Bioscience.
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Endosomes in Plants
TGN (Dettmer et al., 2006). Interestingly, the endocytic
marker FM4-64 rapidly colocalizes with VHA-a1 in
Arabidopsis root cells. This observation led to the
conclusion that the TGN indeed serves as an early
endosomal compartment. Moreover, it could be shown
that the V-ATPase inhibitor concanamycin A inhibits
the endocytic trafficking of FM4-64 from TGN as a
putative early endosome to the late endosome and to
the vacuole. Upon concanamycin A treatment FM4-64
was restricted, together with VHA-a1, to the TGN. This
might indicate that the TGN serves a function in both
the secretory/vacuolar and the endocytic pathways.
Dettmer et al. (2006) also showed that BFA causes the
VHA-a1 positive structures to aggregate within BFA
compartments and that the inhibition of VHA-a1 by
concanamycin A prevents this aggregation. Therefore,
they came to the conclusion that the TGN is clearly a
part of the BFA compartments and that VHA-a1 function is important for their formation.
A recently published work that described the localization of SECRETORY CARRIER MEMBRANE PROTEIN1
(SCAMP1) to the TGN (Lam et al., 2007) further strengthened and supported previous conclusions. SCAMPs are
endocytosis-mediating proteins in animals. Plant homologs have already been found in rice (Oryza sativa), Arabidopsis, and pea. Lam et al. (2007) localized the rice
SCAMP1 in transgenic tobacco BY-2 cells both at the
PM and within highly mobile, punctuated, cytoplasmatic structures. Colocalization experiments as well
as treatments with BFA and Wortmannin showed that
these structures are neither Golgi nor MVB/PVC. Moreover, there was a clear colocalization with the organelle
labeled by antibodies against V-ATPase, suggesting that
this structure constitutes the TGN. Indeed, immunogold electron microscopy experiments revealed that
SCAMP1 localized to vesicular structures of the TGN
possessing clathrin, which was usually found close to
the trans-face of Golgi stacks. Furthermore, it was
shown that FM4-64 labels rapidly SCAMP1-positive
organelles, and prior to the MVB/PVC compartment.
Although, this study did not present direct evidence for
the functional role of SCAMPs in plant endocytosis, the
data have shown that SCAMP1 is localized to the TGN,
an organelle, which is potentially serving as an early
endosome in plant cells. Moreover, these authors also
speculated that the partially coated reticulum, a tubuloreticular structure bearing clathrin coats (Pesacreta
and Lucas, 1985) that was previously described as a
putative early endosomal compartment (Tanchak et al.,
1988), is identical to the TGN.
Additionally, RabA4b, an Arabidopsis homolog of
the Rab11 family of mammalian small Rab-GTPases
was recently shown to, at least partially, localize to the
TGN compartment in growing tips of root hairs (Preuss
et al., 2004, 2006). Rab11 proteins in mammals are
known to mediate recycling processes from early endosomal compartments to the PM by acting on recycling
endosomes (Ullrich et al., 1996; Mohrmann and van der
Sluijs, 1999). In animals, recycling endosomes are intermediate compartments, which are receiving inter-
nalized PM proteins from the sorting endosome (the
first endosomal compartment) for further recycling to
the PM (see above). Preuss et al. (2006) identified two
phosphatidylinositol 4-OH kinases (PI-4 K), namely
PI-4Kb1 and PI-4Kb2 as putative effector proteins of
RabA4b. Immunogold labeling experiments in root epidermis cells of high-pressure frozen/freeze-substituted
samples showed that RabA4b is associated with the
TGN in wild-type plants and in double mutants affected in PI-4 kinase b1/b2 function. Interestingly, cell
fractionation and immunoblot experiments using TGNspecific SYP41 antibodies indicated that only the minor
part of RabA4b is associated with TGN, while the major
part labels a yet unidentified compartment (Preuss
et al., 2004). Summarized, these data possibly give the
first indication for the existence of an additional early
endosomal compartment in plants.
MOLECULAR DISSECTION OF MVB AS LATE
ENDOSOMAL COMPARTMENT IN PLANTS
In plants, similarly to other eukaryotic organisms,
vacuolar/lysosomal protein degradation as well as the
delivery of vacuolar proteins is mediated through a
compartment described as either PVC or MVB. Moreover, proteins of the secretory pathway as well as
proteins delivered to protein storage vacuoles apparently have to pass through such a compartment in the
cell. During the last years, several molecular markers
were developed and used to describe late endosomal/
secretory compartments. Markers like VSRs (Li et al.,
2002; Tse et al., 2004), the Arabidopsis syntaxin SYP21/
AtPep12p (da Silva Conceicao et al., 1997; Uemura
et al., 2004), as well as small Rab-GTPases (Sohn et al.,
2003; Kotzer et al., 2004; Lee et al., 2004) contributed
significantly to the study of MVB/PVC functions. MVB
could be defined as round-shaped electron-transparent
vesicular compartment enclosed by limiting outer membrane and containing small internal electron-dense microvesicles (Fig. 2; Tse et al., 2004; Hause et al., 2006; Lam
et al., 2007). Usually, MVBs reach sizes up to 200 to 500
nm. In animal cells, MVBs are defined as intermediate
compartments (between early endosomes and lysosomes) having dual role in the late endocytic/lysosomal
and the secretory pathways, and they are often called
late endosomes. Recent studies on plant MVBs provide a
growing body of evidence that they also play a dual role
in the late endocytic and secretory/vacuolar pathway.
The situation in plants, however, might be more complicated because they contain two types of vacuoles.
Nevertheless, not only structurally but also functionally,
plant MVBs seem to resemble, at least partially, their
counterparts described in animal cells.
FUNCTION OF MVB/PVC IN THE
ENDOCYTIC PATHWAY
It is known for a long time that organelles possessing multivesicular structures are involved in endocytic
Plant Physiol. Vol. 145, 2007
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Müller et al.
processes in plants. Already in the 1980s and the early
1990s uptake studies using electron-dense tracers indicated their presence within MVBs (Hillmer et al.,
1986, 1988; Tanchak and Fowke, 1987; Galway et al.,
1993). More recently, the use of molecular markers
localized by immunogold labeling and as GFP-tagged
variants, in combination with fluorescent marker dyes,
made it possible to show that there are cross talks
between the secretory/vacuolar and the endocytic
pathway on the level of MVB/PVCs. There are several
molecular markers known to localize predominantly
to MVB/PVC. In addition to the above mentioned VSR
proteins (Li et al., 2002) and the Arabidopsis syntaxin
SYP21/AtPep12p (da Silva Conceicao et al., 1997;
Uemura et al., 2004) there are some small Rab-GTPases
known to be involved in endocytic vesicular trafficking.
Although, in the past years there were some controversial discussions about the localization of endosomal Rab-GTPases in plants, it seems to be more clear
now that three of them, namely Rha1 (AtRabF2a), Ara7
(AtRabF2b), and Ara6 (RabF1) are associated with the
MVB/PVCs (Sohn et al., 2003; Kotzer et al., 2004; Lee
et al., 2004; Ueda et al., 2004; Haas et al., 2007). Evidence for the assumption that MVBs/PVCs are acting
in both secretory/vacuolar and endocytic pathways
came from studies with Ara7 and Rha1. It was shown
that dominant-negative variants of both GTPases are
blocking the transport of proteins from the Golgi to the
vacuole (Sohn et al., 2003; Kotzer et al., 2004). Later on,
Ueda et al. (2004) could show that Ara6-labeled compartments at least partially colocalize with those labeled by Ara7. Thus, it seems to be likely that at one
point the pathways of Ara6 and Ara7-labeled compartments are converging. Finally, using immunogold
labeling on plastic sections of high-pressure frozen/
freeze-substituted wild-type Arabidopsis roots, Haas
et al. (2007) could recently show that not only Ara7
(AtRabF2b) and Rha1 (AtRabF2a) but also Ara6 (RabF1)
is associated with MVBs.
Because uptake studies with FM4-64 clearly indicated that Ara7, Rha1, and Ara6 are of endosomal
nature (Ueda et al., 2001, 2004), these results strongly
suggested that there is an interconnection between the
late secretory/vacuolar and the endocytic pathway
converging within the MVB/PVC. This was further
supported by Tse et al. (2004) because they showed
that GFP-tagged VSR (BP-80), a receptor which mediates protein transport to the vacuole, was localized to
MVB/PVC and also colocalized with FM4-64 in tobacco BY-2 cells.
Recently, a plant retromer complex was identified in
Arabidopsis (Oliviusson et al., 2006). The retromer is
a protein complex known to be responsible for the
retrograde transport of vacuolar cargo receptors from
the MVB/PVC to the TGN in mammalian and yeast
cells (Arighi et al., 2004; Seaman, 2004, 2005). In yeast,
this complex consists of two subunits. Three proteins,
Vps35p, Vps29p, and Vps26p constitute a large subunit, while two proteins, Vps17p and Vps5p, constitute a small subunit. Oliviusson et al. (2006) cloned the
Arabidopsis homologs of the large subunit (Vps35,
Vps29, and Vps26) and showed that they form a complex, which is associated with MVB/PVC. Moreover,
they colocalized Vps35 with the MVB/PVC markers
Pep12 and the Arabidopsis VSRAt-1. These data suggest the existence of retrograde protein transport from
the MVB/PVC to the TGN that might operate in plants,
resembling that in yeast. In another recent study, the
structural homolog of Vps5p in mammals (Haft et al.,
1998, 2000) named SNX1 was described in Arabidopsis
(Jaillais et al., 2006). The latter authors showed that
AtSNX1 is associated with yet unidentified endosomal
structures, distinct from endosomes, which are regulated by GNOM. These endosomes are involved in
the accumulation and distribution of the auxin efflux
carrier PIN2 (see above). Like Vps5p, SNX1 from mammals is also suggested to be a part of the small
subunit of the retromer (Arighi et al., 2004). Because
AtSNX1 was shown to be involved in endocytic processes, it will be interesting to study if AtSNX1 is also
a part of the retromer complex associated with MVB/
PVC, thus playing a dual role within plant cells. Actually, such dual role was already suggested for SNX1
in mammalian cells because an interaction of SNX1
with the epidermal growth factor receptor was shown
to lead to an enhanced degradation of the epidermal
growth factor receptor in lysosomes (Kurten et al.,
1996).
It has been established for a long time that one of the
main roles of the plant lytic vacuole is the degradation
of proteins, thus closely resembling lysosomes and
vacuoles from animals and yeast, respectively. However, the mechanism of protein targeting for degradation is far from being understood. In eukaryotes, there
are at least two ways of how proteins are getting
degraded. One is mediated by proteasomes, the other
one describes the degradation within lysosomes or
vacuoles. While the targeting of proteins to the proteasomal pathway is well defined (Vierstra and Callis,
1999), much less information is available about the
vacuolar/lysosomal pathway. In animal and yeast systems it was shown that monoubiquitinylation is needed
for proteins to get recognized by ESCRT complexes,
which are first mediating the internalization of monoubiquitinylated proteins into the luminal vesicles of
MVB before they are delivered to the vacuole/lysosome
(Babst, 2005). A recently published study by Spitzer
et al. (2006) opens a new field integrating the MVB/
PVC directly into the degradation pathway, which delivers proteins to the vacuole in plant cells. They show
that all components of the ESCRT complexes are encoded within the genome of Arabidopsis. Moreover,
they could show that the ELC gene, disrupted in the
elch (elc) mutant showing multiple nuclei and trichome
phenotypes, encodes a homolog of the yeast Vps23p
gene. The Vps23p is a part of the ESCRT-I complex
mediating the recognition and delivery of monoubiquitinylated proteins to the MVB. The authors provide
evidence that ELC is actually a part of an ESCRT-I complex in Arabidopsis and that it localizes to endosomal
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Endosomes in Plants
structures. Additionally, they proposed a role of the
ELC protein in regulating the microtubule cytoskeleton by unknown mechanism during cytokinesis. This
may suggest a pivotal role of monoubiquitinylationdependent, ESCRT-mediated protein sorting for degradation of PM-derived signaling receptors within a
yet unknown endosomal compartment. Future work
has to clarify whether ELC is localized to MVBs and
which proteins are degraded in this pathway. In addition to the ESCRT complexes, it is known from animals and yeast that an AAA-ATPase called Vps4p
in yeast and SUPPRESSOR OF K1 TRANSPORT
GROWTH DEFECT1 (SKD1) in mouse is required for
the invagination of the limiting membrane of MVBs,
possibly by supporting the release of the ESCRT complex through its ATPase activity. In a very recent report, Haas et al. (2007) examined an Arabidopsis
homolog of this AAA-ATPase called At-SKD1. Using
immunogold labeling and GFP tagging they could
show that At-SKD1 partially colocalized on MVBs
with the endosomal Rab-GTPases Ara7 (AtRabF2b),
Rha1 (AtRabF2a), and Ara6 (RabF1) as well as with the
endocytic tracer FM4-64, indicating that the labeled
organelles are of endosomal nature. To determine a
potential role of At-SKD1 on the MVB, the authors
created a mutant Arabidopsis line overexpressing an
ethanol inducible version of the GFP-tagged At-SKD1,
which is unable to hydrolyze ATP. These plants possessed enlarged MVBs with fewer internal vesicles,
suggesting a role for the ATPase activity of At-SKD1 in
membrane invagination on MVB. Moreover, when overexpressed in tobacco BY-2 cells, mutated, GFP-tagged
At-SKD1 was solely localized to convoluted membranous structures but not to the cytoplasm, further suggesting the ATPase activity to be essential for proper
localization. Even though this work identified another
component possibly involved in mediating the transport of proteins into the inner luminal vesicles of MVB,
it was not shown that it indeed interacts with the ESCRT
complex. Future experiments have to clarify this open
question.
CONCLUSION AND FUTURE PROSPECTS
In the recent extensive studies employing new tools
such as plant-specific molecular markers, endosomal
marker dyes, advanced imaging techniques, and genetic studies considerably enhanced our knowledge
about endocytosis in plants. The identification and
molecular dissection of early and late endosomal compartments, receptor, and clathrin-mediated vesicular
trafficking along with the finding that plant cells possess more than one type of vacuole, clearly indicate
that the plant endosomal pathways are quite complex.
Furthermore, analysis of the functional organization of
vesicular trafficking routes in plants provided evidence that the secretory and the endocytic machinery
are tightly connected. As mentioned above, the TGN
in animals is primarily responsible for the sorting of
secretory and lysosomal proteins but it takes part also
on the retrograde transport of a set of diverse proteins
including extracellular protein toxins to the TGN that
is mediated by early recycling and late endosomes (for
review, see Bonifacino and Rojas, 2006).
Although there is evidence that some mechanisms
are conserved within all eukaryotes, the recent results
indicate a considerable divergence of endosomal organization in plants with surprising differences between cell types and plant species. As an example,
plant Rab5 homologs seem to be localized to MVBs
representing late endosomes while mammalian Rab5
is localized to early endosomes.
One future challenge will be the identification of the
endosomal compartments, which are involved in RME
of the flagellin receptor FLS2 along with the associated
signaling machinery as well as putative endosomes
possessing DnaJ domain proteins involved in RME
such as KAM2/GRV2 (Tamura et al., 2007). Additionally,
for many PM proteins involved in membrane transport processes, such as PIN, AUX, and BOR carriers,
PIP2 water channel and H-ATPase pump, the identity
of endosomes responsible for their recycling has to be
shown at the subcellular level.
The finding that the TGN and MVB compartments
likely play a dual role, both in the secretory and in the
endocytic pathway, raises several new questions. For
example, it is not clear yet whether one or more subpopulations of diverse TGN and MVB compartments
are operating in plant cells, having either unique or
overlapping functions in the endocytic and/or in the
secretory pathway. Furthermore, it should be clarified
in which cells and under which circumstances is the
TGN with early endosomal characteristics closely associated with the late Golgi cisternae or eventually
represent an independent organelle. The latter possibility was suggested from results on some suspension
and tip-growing cells (Uemura et al., 2004; Preuss
et al., 2006; Šamaj et al., 2006 and refs. therein). The
next interesting topic will be to find out whether vesicular endosomal compartments undergo the process
of maturation as it is known from Golgi cisternae (e.g.
Matsuura-Tokita et al., 2006). Analysis of further protein components and multiprotein complexes of endosomal organelles is certainly necessary to answer these
important questions. Therefore, much more integrated
effort is required in the future to work out the differences and similarities in further detail, before the grand
scheme of the endocytic pathway valid for most eukaryotes emerges.
Received May 25, 2007; accepted July 31, 2007; published October 8, 2007.
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