Download Protein Secretion in Plants: from the trans

Traffic 2002; 3: 605–613
Blackwell Munksgaard
Copyright C Blackwell Munksgaard 2002
ISSN 1398-9219
Review
Protein Secretion in Plants: from the trans-Golgi
Network to the Outer Space
Gerd Jürgens* and Niko Geldner
ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der
Morgenstelle 3, D-72076 Tübingen, Federal Republic of
Germany
* Corresponding author: Gerd Jürgens,
[email protected]
Functional analysis of exocytosis in yeast and animal
cells has led to the identification of conserved elements and mechanisms of the trafficking machinery
over the last decade. Although functional studies of
protein secretion in plants are still fairly limited, the
Arabidopsis genome sequence provides an opportunity
to identify key players of vesicle trafficking that are
conserved across the eukaryotic kingdoms. Here, we
review and add to recent genome analyses of trafficking components and highlight some plant-specific
modifications of the common eukaryotic machinery.
Furthermore, we discuss the evidence for targeted,
polarised secretion in plant cells, and speculate about
possible underlying cargo sorting processes at the
trans-Golgi network and endosomes, based on what is
known in animals and yeast.
Key words: Arabidopsis genome, ARF, endosomes, intracellular trafficking, plasma membrane, polarised secretion, rab, SNARE complex, trans-Golgi network,
vesicle budding, vesicle fusion
Received 3 June 2002, revised and accepted for publication 14 June 2002
Protein delivery to the cell surface, via the endomembrane
system, is a common feature of eukaryotic cells. Integral proteins of the plasma membrane as well as secreted proteins
are synthesised at the endoplasmic reticulum (ER) and are
inserted into or translocated across the ER membrane. All
subsequent steps of protein trafficking involve transport vesicles that bud from a donor membrane and fuse with a target
membrane. Most vesicles that leave the ER are trafficked to
the Golgi complex, although some vesicles bypass the Golgi
on their way to the vacuole (1). The Golgi complex is a major
sorting station where trafficking routes diverge to the plasma
membrane, the endosome, the vacuole or the cell plate (2)
(Figure 1). Post-Golgi sorting occurs in the endosome which
is also involved in recycling of vesicles to the plasma membrane.
Extensive analysis of protein secretion in yeast and animal
cells suggests that the basic machinery was invented by a
unicellular eukaryote and subsequently adapted to the needs
of multicellular life. For example, targeted secretion to the
apical vs. baso-lateral surface of polarised epithelial cells requires sorting of proteins destined to specific subdomains
within the plasma membrane and uses modified subsets of
trafficking components already present in yeast. Compared
to yeast and animals, much less is known about mechanisms
of protein secretion in plants (2). Considering that plants and
animals display different cellular organisations and also have
achieved multicellularity independently during evolution, it
seems likely that protein secretion has undergone specific
modifications in the plant lineage.
A few examples from plant development may illustrate requirements that have to be met by the protein secretory system. Vegetative pollen cells secrete a peptide ligand (SCR)
into their cell wall that interacts with a Ser/Thr kinase receptor
complex (SRK-SLG) in the plasma membrane of stigmatic
cells, ensuring recognition of matching partners in the selfincompatibility response (3). Similarly, signaling between the
stem cells and the organising centre of the shoot meristem
requires interaction between a secreted peptide ligand
(CLV3) and a Ser/Thr kinase receptor complex (CLV1-CLV2)
for continual reassessment of cell fate and maintenance of
shoot meristem organisation (4). Whereas secretion of the
ligand into the apoplast has been demonstrated, internalisation of the ligand-receptor complex and potential recycling
of the receptor complex to the plasma membrane have not
been studied. Root hairs and pollen tubes grow by delivery
of transport vesicles to their tips (5). Moreover, pollen tubes
grow directionally towards the ovules for fertilisation, which
requires continual changes of vesicle targeting to the plasma
membrane in response to as yet unknown chemotactic signals (6). Plant cells are able to elongate directionally up to
200 times their original length (7), which requires differential
insertion of membrane material into subdomains of the
plasma membrane. Some cell types such as trichomes and
root hairs undergo localised outgrowth from specific sites
within the plasma membrane (8,9). Finally, the cells of the
endodermis partition their surface into two domains, with an
inner surface separated from an outer surface by the Casparian strip (10). This is formally similar to the zonula adherens
border between apical and baso-lateral domains of animal
epithelial cells.
The cellular organisation of plants may entail specific modifications of protein secretion. Not only does an ER network
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Figure 1: Model of the trafficking routes in the late secretory system. Schematic of an Arabidopsis root meristem cell expressing
plasma membrane markers located to distinct subdomains (marker names are coloured and in italic). Coloured arrows mark the respective
possible pathways to different plasma membrane compartments from the TGN and/or the endosome. The size of the arrows indicates the
possible differences in relative contributions to the total transport of these plasma membrane markers. Names of membrane compartments
are in bold. Reported locations of proteins of the transport machinery mentioned are in plain text. The circle of dotted lines represents the
ill-defined plant endosome, whose structure and subcompartments (early endosomes, sorting endosomes, etc.) will have to be defined in
the future.
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occur at the cell periphery, but also the Golgi complex is dispersed into many stacks whose numbers vary from about 20
to 400 in plant cells (11). Furthermore, the endomembrane
organisation is highly dynamic, due to extensive cytoplasmic
streaming, which moves organelles about the cell and may
bring Golgi stacks and ER in close proximity (12,13). As plant
cells enlarge, they often contain a large central vacuole that
leaves a thin layer of cytoplasm underneath the plasma membrane. Specific cell types contain two different kinds of vacuole: a lytic vacuole akin to those in yeast or the lysosome of
animal cells and a storage vacuole from which reserve materials can be mobilised (14). The plant cytoskeleton also has
unique features. Actin filaments mediate cell growth, cytoplasmic streaming, ER–Golgi association and organelle
movement. Microtubules are not nucleated from localised
MTOCs (centrosomes) and form particular arrays such as interphase cortical hoops that maintain cell shape, and preprophase band and phragmoplast that mediate oriented cell division and the execution of cytokinesis, respectively (15). However, MTs do not seem to be necessary for basic cell growth,
as protein trafficking to the plasma membrane occurs in MTdeficient cells (16). Moreover, the extracellular matrix (ECM)
of plant cells is a largely polysaccharidic cell wall, whereas
the animal ECM is mainly proteinaceous. Finally, due to the
absence of cell movement, plant organs are shaped by
oriented cell division and regulated cell expansion, with the
latter resulting from targeted protein secretion.
This review serves two purposes. First, we will exploit the
Arabidopsis genome sequence to provide an overview of
some central players of the plant secretion machinery by
comparison with the components that have been functionally
characterised in animals and yeast. Second, we will summarise recent findings that may shed light on protein trafficking
between the trans-Golgi network (TGN) and the plasma
membrane, discussing the role of the endosomal system in
protein sorting. Trafficking from the TGN to the vacuole has
been reviewed recently (17) and will not be covered here.
Plant Proteins Involved in Vesicle Trafficking
Studies in yeast and animals have characterised components
of an ever-growing molecular machinery that ensures the
budding of vesicles with defined sets of cargo proteins from
the donor membrane as well as their recognition by, and fu-
Table 1: Overview of Arabidopsis thaliana protein families involved in vesicle trafficking. Asterisks indicate searches done by the authors
Protein group
Subgroup/class
Proteins/
protein numbers
AGI numbers
Ref.
ARF
G-proteins
class I
ARF1 – ARF6
*
plant class A
plant class B
Gea/GNOM/GBF
Sec7p/BIG class
ARF7 – ARF8
ARF9
GNOM, GNL1, GNL2
AtBIG1-5
ARF-GAP1 class
Age2p class
Age2p-like class
Gcs1p class
12 subfamilies
exocyst
3 members
2 members
3 members
2 members
57 members
8 of 8 subunits
VFT
3 of 3 subunits
HOPS
6 of 6 subunits
Sec34/35p
SYP (syntaxins)
VAMP
SNAP25 hom.
NPSN
VTI, Gos1,
Bet1, Membrin
Sec1 group
Vps45 group
Vps33 group
Sly1 group
5 of 8 subunits
24 members
14 members
3 members
3 members
3, 2, 2, 2 members,
respectively
KEULE, Sec1a, Sec1b
VPS45
VPS33
Sly1
At3g62290; At2g47170; At1g23490;
At1g70490; At1g10630; At5g14670
At5g17060; At3g03120
At2g15310
At1g13980; At5g39500; At5g19610
At3g43300; At1g01960; At3g60860;
At4g35380; At4g38200
At2g35210; At4g17890; At5g46750
At3g17660; At5g54310
At3g07940; At4g05330; At4g21160
At2g37550; At3g53710
See reference
At1g47550, At3g10380, At1g71820
(At1g21170/At1g76850), At5g03540
(At1g10385/At5g49830), At5g12370
(At3g56640/At4g02350)
(At1g71270/At1g71300), At1g50500,
At4g19490
At2g05170, At2g38020 (VCL1),
At1g12470, At1g08190, At4g36630,
At3g54860
See reference
See reference
ARF-GEFs
ARF-GAPs
Rab GTPases
Rab effectors
SNAREs
Sec1 family
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See reference
*
*
*
*
*
*
*
*
(44)
*
*
*
(52)
(57)
(57)
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Jürgens and Geldner
sion with, the correct target membrane. Screening of the Arabidopsis genome for related sequences to key players of intracellular trafficking in nonplant organisms gives some idea
of conserved elements of the machinery between yeast,
plants and animals. For the sake of brevity, we will limit our
discussion to two processes: vesicle budding from the donor
membrane and vesicle interaction with the target membrane.
Vesicle Budding
Vesicle budding requires small GTPases of the ARF family,
their guanine-nucleotide exchange factors (ARF-GEFs) and
GTPase-activating proteins (ARF-GAPs) for coat recruitment
and cargo selection. ARFs not only act to recruit COPI and
clathrin coats to membranes but also play a role in the control
of membrane lipid composition, actin remodeling and related
events (18). Structurally related ARF-like proteins (ARLs) are
members of the same subgroup of the ras superfamily, but
have different functions. For example, Arabidopsis ARL2 is
involved in microtubule formation (16). The six mammalian
ARFs have been grouped into three classes based on their
structure and function. Class I consists of three members
(ARF1–3), class II is represented by ARF4 and ARF5,
whereas ARF6 constitutes the most divergent class III (19).
By contrast, there are only three ARFs in yeast. Class I ARF1
and ARF2 are functionally redundant (20). Yeast ARF3 is divergent from both yeast class I ARFs and human ARF6 (21).
In Arabidopsis, six of nine putative ARF genes encode class
I ARF proteins with 98–100% amino acid identity (Table 1).
Four of them can be grouped into two pairs derived from
segmental genome duplication events. Additionally, there are
three more divergent ARFs, including one pair of duplicates,
with about 60% amino acid identity to human ARF1. The
third divergent ARF seems to represent a subclass of its own
(Table 1). The less conserved Arabidopsis ARFs are also divergent from human ARF6 or yeast ARF3, arguing for an independent evolution of divergent ARF classes from class I
ancestors in the three kingdoms.
The ARF GDP/GTP exchange factors (ARF-GEFs) also underwent diversification during the separate evolution to multicellularity. Yeast have four such regulators of ARFs: the largely
redundant couple Gea1p/Gea2p, and Sec7p and Syt1p (18).
ARF-GEFs are defined by the catalytic Sec7 domain. The
mammalian cell displays a much greater diversity of Sec7
domain proteins than yeast. There are to date five different
classes of exchange factors described, accounting for about
10 different ARF-GEFs three of which show a completely
new arrangement of domains that are not present in yeast
ARF-GEFs (22,23). These new ARF-GEF classes appear to
have evolved for specialised transport functions in a multicellular context, such as matrix adhesion or receptor down-regulation (24,25).
Plant ARF-GEFs appear to show a different pattern of diversification. Arabidopsis has only two classes of exchange factors which can be distinguished by the presence or absence
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of an N-terminal dimerisation domain and are related to yeast
Gea1/2p and Sec7p large ARF-GEFs, respectively (22,26).
Unlike mammals with only one Gea1/2p class (GBF) and two
Sec7p class (BIG1/2) ARF-GEFs, the Arabidopsis genome
encodes 3 Gea1/2p and 5 Sec7p-like exchange factors. The
sequence divergence between family members (20–60%
identity) suggests at least partially non-redundant functions.
Only the Gea1/2p class ARF-GEF GNOM has been functionally characterised. Although gnom embryos have severe patterning defects, the mutant cells are viable and can be grown
in culture (27–29). GNOM might act in signal-dependent recycling of plasma membrane proteins from endosomal compartments to the cell surface (29) (Figure 1 and see below).
Neither yeast nor mammalian ARF-GEFs of the Gea/GBF/
GNOM class appear to act in endosomal recycling, which in
mammals rather involves mammal-specific ARF-GEFs, such
as EFA6, ARNO or ARF-GEP100 (22,23). Thus, plants seem
to have evolved new members of an existing ARF-GEF class
to regulate recycling events, whereas ARF-GEFs with newly
assembled domain structures have evolved for the same process in animals.
Yeast ARF1-GAP mediates interaction between ER-Golgi vSNAREs and COPI coat proteins (30). Three other yeast ARFGAPs act at the TGN, whereas mammals also have structurally divergent ARF-GAPs functioning in the periphery of the
cell (18,31). In Arabidopsis, ten genes encode ARF-GAPs of
which three groups are related to the yeast ARF-GAPs acting
at the TGN, Age2p, Gcs1p and Glo3p, respectively, while the
members of the fourth group show similarities to human
ARF-GAP1 (31,32) (Table 1).
Transport vesicles can be distinguished by their coats which
are formed by a limited set of proteins, including COPI, COPII
and clathrin. Coat proteins are captured by specific adaptor
proteins and aid in cargo recruitment and vesicle budding
(33). Whereas COPI- and COPII-coated vesicles traffic between ER and cis-Golgi or between Golgi cisternae, clathrincoated vesicles bud from the TGN or the plasma membrane
and are thus involved in post-Golgi trafficking.
Clathrin-coated vesicles have been detected at the TGN and
plasma membrane, and at the cell plate in dividing cells of
Arabidopsis (34–36). Arabidopsis has two genes encoding
slightly divergent putative clathrin heavy chains, and clathrin
has been immunolocalised to budding vesicles (35). In addition, one of three putative clathrin light chains was shown
to interact with light chain-free mammalian clathrin hubs in
vitro (37).
Clathrin is recruited by adaptor (AP) complexes that consist
of four subunits: two large subunits (b and one of a, g, d or
e), the medium subunit m and the small subunit s. Like mammals, and in contrast to Drosophila or yeast, Arabidopsis has
four adaptor complexes, AP-1 to AP-4 (38). AP-2 mediates
clathrin recruitment in endocytosis from the plasma membrane, whereas AP-1, AP-3 and AP-4 are associated with the
trans-Golgi network and/or endosomes in mammals.
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Whereas AP-1 and AP-2 function with clathrin, AP-4 is most
likely part of a nonclathrin coat. Interestingly, the Arabidopsis
genome encodes three putative g subunits of AP-1 as opposed to two in mammals and only in one each in Drosophila
and yeast, and three b subunits that cannot be clearly assigned to either AP-1 or AP-2 (38,39). The reason for this
potential diversity of AP-1 and, possibly, AP-2 complexes remains to be determined as no functional data are available in
Arabidopsis.
Clathrin adaptors recruit cargo proteins via interaction with
their cytosolic tails, e.g. m2-adaptin of AP-2 recognises a tyrosine-based endocytic sorting motif (40). In plants, the only
evidence for cargo recruitment is the in vitro interaction of
the TGN-localised vacuolar cargo receptor AtELP with the
mammalian TGN-specific AP-1 clathrin-adaptor complex
(41,42).
Interaction of Vesicles with the Target
Membrane
The fusion of transport vesicles with their target membrane
is preceded by tethering and docking, both of which contribute to the target specificity of vesicle fusion. Initially, rab-GTP
on the vesicle membrane interacts with effectors that tether
the vesicle to the target membrane (43). This is followed by
the formation of trans-SNARE complexes that dock vesicles
to target membranes, facilitating subsequent fusion.
Rab proteins are thought to determine the fusion competence of membranes and to specify target membranes by
acting as molecular scaffolds and regulators of membrane
composition. As many as 57 rabs have been identified in
Arabidopsis, nearly matching the number of rabs in mammals and by far exceeding those of yeast, Drosophila or Caenorhabditis elegans (44). Rab functions are conserved
across eukaryotes, such that their subcellular localisation can
be inferred from known localisations of members of the same
subfamily in other species. The large number of Arabidopsis
rab proteins results from diversification within 12 subfamilies.
The rab11 subfamily, for example, contains 26 members,
which may reflect differential tissue expression or functional
redundancy of late-endosomal rabs. Alternatively, this diversification might suggest a complex subdomain structure of recycling endosomes in plants [for review, see (43)]. The rab11
homologue PRA2 has been reported to localise to the ER and
to regulate brassinosteroid biosynthesis in response to light,
an unprecedented case of a rab function possibly unrelated
to membrane trafficking (45). However, the same protein
was recently localised to the Golgi and endosomes rather
than the ER (46) (Figure 1). Regardless of this discrepancy,
both reports agree on differences in localisation or function
between PRA2 and its closest homologue PRA3, supporting
the notion of functional diversity among rab11 subfamily
members in plants.
Another example of a plant-specific rab variant is Ara6, a
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member of the rab5 subfamily, which lacks the strictly conserved C-terminal isoprenylation (47). Instead, N-myristoylation and palmitoylation of an N-terminal extension is necessary for proper localisation of Ara6 to endosomal compartments (Figure 1). Since only a subset of early endosomes
appears to be Ara6-positive, plants may have functionally distinct early endosome populations.
Rab effectors are specific to the target membrane and often
consist of protein complexes. For example, the exocyst complex of eight proteins conserved between yeast and mammals mediates tethering of Sec4p/rab3 vesicles to the
plasma membrane (48). In yeast, the exocyst targets vesicles
to a specific site of the bud plasma membrane via the Sec3p
protein, although t-SNAREs appear more broadly distributed
in the target membrane (49). The mammalian exocyst targets
vesicles to the baso-lateral surface of epithelial cells (50). A
Ypt6p effector complex named VFT (for Vps 53) mediates
tethering of putatively endosome-derived vesicles to the
trans-Golgi network (51). A Ypt1p effector complex named
Sec34/35 complex appears to tether vesicles to the cis-Golgi
stack (52). Interestingly, several components of these three
complexes show similarities that suggest a common evolutionary origin (53). The Arabidopsis genome encodes
homologues of all eight exocyst, three VFT and five of eight
Sec34/35 complex proteins (Table 1). However, no functional
analyses have been performed. Vesicles trafficking to the
vacuole are tethered to the target membrane via the Ypt7p
effector complex named HOPS, which is also conserved between yeast and mammals (54). Again, homologues of all
six components of the HOPS complex are encoded in the
Arabidopsis genome (Table 1). In this case, mutations in the
VACUOLELESS1 (VCL1) gene encoding the Vps16p homologue were shown to be embryonic lethal and to lead to defects in vacuole biogenesis (55).
Vesicle docking is brought about by the pairing of complementary SNAREs on opposite membranes (56). SNARE
complexes consist of pathway-specific synaptobrevins (vSNAREs) and syntaxins (t-SNAREs), whereas more promiscuous t-SNARE light chains or SNAP25s contribute the
remaining two coiled-coil domains of the four-helical bundle
in yeast and mammals. Again, the Arabidopsis genome encodes a larger number of syntaxins (called SYP, syntaxins of
plant) than does the human genome (57). Especially, there
are nine SYP1 proteins related to the plasma-membrane syntaxins Sso1/2p and syntaxin1. They include KNOLLE, a cytokinesis-specific syntaxin only found in plants, and the plasma
membrane-located Syr-1 required for protein secretion (58–
60) (Figure 1). It will be interesting to determine the tissue
specificity and the subcellular location of the remaining
members of this group. Members of four other SYP subfamilies form at least five complexes involved in TGN/prevacuolar
compartment (PVC) trafficking (61). Members of the SYP2
and SYP4 syntaxin family are closely related by sequence,
but SYP41 and SYP42 have been shown to localise to different regions of the TGN. In addition, functional analysis by
reverse genetics suggests unique requirements of each, as
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evidenced by pollen lethality (62). As in yeast and animals,
the SNAP25-homologue SNAP33 appears to be more promiscuous, interacting with KNOLLE at the cell plate and also
localising to the plasma membrane (63) (Figure 1).
Arabidopsis encodes 16 putative synaptobrevins, of which
11 form a subfamily related to the endosomal/lysosomal
VAMP7 (57). By contrast, there are no close homologues
to the plasma membrane synaptobrevins Snc1/2p and
VAMP1/2. On the other hand, the Arabidopsis genome encodes 3 ‘novel plant SNAREs’ (NPSNs) that lack close homologues in nonplant organisms. NPSN11 interacts with the
cytokinesis-specific syntaxin KNOLLE and also localises to
the cell plate during cytokinesis (64). It remains to be determined whether NPSNs can substitute for conventional synaptobrevins.
Arabidopsis encodes six putative members of the Sec1 family which in nonplant organisms interact with SNAREs in different ways. Whereas nSec1 stabilises the closed conformation of syntaxin1A (65), Sly1 interaction with the N-terminus of the cis-Golgi SNARE Sed5 may contribute to the
specificity of SNARE complex formation (66,67). In contrast
to yeast, the Arabidopsis VPS45 is located at the TGN and
interacts with the TGN syntaxins of the SYP4 family (68) (Figure 1). The only Arabidopsis Sec1 protein that has been functionally characterised is KEULE which interacts with the cytokinesis-specific syntaxin KNOLLE and is required for vesicle
fusion during cell-plate formation (69,70).
In conclusion, the sequence analysis of the Arabidopsis genome suggests conservation of key regulators of vesicle trafficking. However, in the case of protein families, there has
been differential expansion of subfamilies in plant evolution,
which points to possibly divergent roles of individual members. To clarify this issue, functional analysis needs to be
done by reverse genetics in Arabidopsis.
Post-Golgi Trafficking Pathways in Plants:
Where Does Sorting Take Place?
Protein secretion to the cell surface delivers integral plasma
membrane proteins, such as cellulose synthases, the tSNAREs SYR1 and SNAP33 or Hπ-ATPase, and extracellular
proteins located in the cell wall, such as lipid transfer protein,
endoxyloglucan transferase or endoglucanase, and secreted
signaling peptides, such as CLV3 or SCR. In all these cases,
the entire plasma membrane is the target of vesicle trafficking. However, there are other proteins that accumulate in specific subdomains of the plasma membrane. This is illustrated
by the apical localisation of the auxin uptake carrier AUX1
(71), the basal localisation of the putative auxin efflux carrier
PIN1 (28,72) and the lateral localisation of COBRA, a GPIanchored protein necessary for correct differential cell
elongation (73) (Figure 1). These three proteins can be found
in the same cells of the Arabidopsis seedling root, suggesting that targeted secretion can distinguish between dif610
ferent subdomains of the plasma membrane. Other proteins
localised to specific subdomains include PIN2, which accumulates at the apical end of root epidermal cells (74), and
PIN3, which is predominantly observed at lateral plasma
membranes of root cortical cells (75).
How these differential localisations are achieved by targeted
secretion in plant cells is not known. In mammalian polarised
epithelial cells, syntaxins are differentially localised to either
apical or basolateral plasma membrane domains (76), and
this was shown to be important for correct sorting of marker
proteins (77). It is important to note that epithelial cells have
mechanical diffusion barriers that prevent correctly targeted
proteins from diffusing laterally into the neighbouring plasmamembrane domain. Such barriers are not apparent in the depicted root meristem cell (Figure 1), suggesting that different
mechanisms prevent the mixing of apical, lateral and basal
markers. One possible mechanism would be a self-organising scaffolding machinery. Alternatively or in addition, segregated proteins might be continually resorted by recycling, as
will be discussed below. However, where does sorting happen in the first place?
The TGN has been recognised as a major sorting site in the
secretory pathway where proteins destined for the lysosome/
vacuole are segregated away from exocytic cargo. This is also
the case in plants since the vacuolar sorting receptor AtELP
has been localised to the TGN (41,42) (Figure 1). It has also
been shown recently through live imaging that transport vesicles from the TGN directly fuse with the plasma membrane,
without passing through any intermediate compartment (78).
If the TGN acts as a last sorting station before outer space,
differential secretion towards plasma membrane subdomains
would require budding of several distinct vesicle populations
from the TGN. However, there is evidence from both mammalian and yeast cells for ‘indirect’ trafficking to the plasma
membrane, with vesicles first being delivered to the endosomal system (79–82). The endosome is an important sorting
centre in the cell where cargo destined for degradation is
separated from proteins to be recycled back to the surface.
Thus, it is a rather straightforward idea that newly synthesised proteins travel from the TGN to the endosome
where they join proteins from the endocytic pathway, and
both sets of proteins are sorted together for their respective
destinations at this point. The direct pathway from the TGN
to the plasma membrane could be taken by cargo such as
cell-wall material or secreted proteins that would not undergo
recycling through endosomes. In a growing plant cell, this
would probably still constitute the major part of vesicle flow
to the plasma membrane. However, things are likely to be
more complicated, since sorting of some apical or basolateral
markers does clearly also take place in the TGN. This is even
the case in nonpolarised cells, where different vesicle populations are not destined to distinct plasma membrane compartments (78). Currently, data from mammals and yeast only
indicate the existence of two pathways from the TGN to the
plasma membrane, but we are far from understanding why
one protein takes the direct route and another travels via the
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endosome, and what contributions the two pathways make
to overall secretion.
In plants, post-Golgi trafficking to the plasma membrane has
not been analysed and it is thus not clear whether there are
multiple pathways. However, BFA treatment leads to rapid
internalisation of plasma membrane proteins, which is most
likely due to a block of re-secretion to the plasma membrane
(29). BFA-induced internal accumulation appears to be plant
specific, since in animals endocytosis and recycling of
plasma membrane markers are resistant to BFA (83,84).
Thus, BFA is a tool to study post-Golgi secretion in plants.
Several plasma membrane proteins accumulate intracellularly
in response to BFA treatment (28,29,63,85). It has been
shown that PIN1 accumulates in so-called BFA compartments that consist of aggregates of membrane vesicles and
are distinct from adjacent Golgi stacks (29). The BFA compartments involved in PIN1 recycling most likely represent
endosomes. If this is the case, proper targeting of PIN1 and
possibly other plasma membrane proteins may be effected
by sorting in endosomes rather than at the TGN. By comparison, newly synthesised PIN1 accounts only for a minor proportion of transported PIN1 protein in the cell. Thus, regardless of whether this minor fraction is sorted at the TGN or
passed on to the endosome for sorting, the determining step
for the polar localisation of PIN1 should be sorting at the
endosome. How sorting and targeting to the correct plasma
membrane subdomain is brought about is entirely unknown.
For example, none of the several putative plasma membrane
SNAREs of Arabidopsis has been shown to be asymmetrically localised in the plasma membrane, in contrast to animals (76). In the case of the COBRA protein, which is localised to the lateral subdomain of the plasma membrane, its
GPI anchor may be instructive for targeting since, in animals,
segregation into lipid rafts has been postulated to target GPIanchored proteins to the apical end of epithelial cells (86).
Future Perspectives
Both the analysis of the Arabidopsis genome and the still
limited number of functional studies point to a similar complexity of the secretory system in plants as in animals. More
functional studies are required to analyse mechanisms of
plant protein secretion, and the necessary tools are now becoming available. Specifically, reverse-genetics analysis of
Arabidopsis genes has become commonplace with the availability of large collections of insertion lines, and facile transformation makes it easy to express specific variants of regulatory components of vesicle trafficking. In addition, the paucity
of suitable subcellular markers for identifying membrane
compartments will be overcome by the widespread use of
GFP fusions. We can thus expect a dramatic increase in
knowledge about protein secretion in plants, which will
eventually enable us to differentiate between a common eukaryotic heritage and plant-specific ways of sorting out
things.
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