Download Getting the message across: how do plant cells exchange

Review
TRENDS in Plant Science
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Vol.9 No.1 January 2004
Getting the message across: how do
plant cells exchange macromolecular
complexes?
Karl J. Oparka
Cell-to-Cell Communication Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK DD2 5DA
A major pathway for macromolecular exchange in plants
involves plasmodesmata (PD), the small pores that connect adjoining cells. This article considers the nature of
macromolecular complexes (MCs) that pass through PD
and the pathways and mechanisms that guide them to
the PD pore. Recent cell-biological studies have identified proteins involved in the directional trafficking of
MCs to PD, and yeast two-hybrid studies have isolated
novel host proteins that interact with viral movement
proteins. Collectively, these studies are yielding important clues in the search for components that compose
the plant intercellular MC trafficking pathway. Here,
they are placed in the context of a functional model that
links the cytoskeleton, chaperones and secretory pathway in the intercellular trafficking of MCs.
Plasmodesmata (PD) form a major pathway for information exchange in plants, and have been the focus of
several recent studies. PD have a unique architecture that
permits the passage of both small solutes and certain
macromolecules that, at first glance, seem too large to pass
through the PD pore. For recent reviews describing the
role of PD in short-and long-distance transport in plants,
see Refs [1 – 4].
Non-cell-autonomous proteins
Proteins that can move between plant cells have been
termed non-cell autonomous proteins (NCAPs) [5]. Some
proteins, such as green fluorescent protein (GFP), can
pass through PD by non-selective movement (i.e. without
requiring a specific interaction with components of the PD
pore) [2]. Recently, evidence has begun to accumulate that
some plant transcription factors might also pass between
meristematic cells by simple diffusion [6], suggesting that,
for some proteins, cell-to-cell movement might occur by
default unless the protein is retained at a specific subcellular location [6]. However, of the many NCAPs identified to date, many appear to show selective transport
through PD and also increase the size exclusion limit of the
PD pore (Table 1). It is difficult to identify common motifs
in these proteins that interact specifically with PD and at
least some NCAPs probably interact with other cellular
proteins to mediate their intercellular passage.
In a recent study [5], the NCAP CmPP16 (a protein first
detected in Cucurbita phloem sap [7]) was used as bait for
the affinity purification of interacting proteins present
within a PD-enriched cell wall fraction. A 40 kDa protein
termed NCAPP1 (non-cell-autonomous pathway protein 1)
was detected that was immunolocalized to the cortical
Table 1. Non-cell-autonomous proteins and modifiers of plasmodesmata
Protein
Plasmodesmata modification
Function
Refs
Virus
Several viral MPs and CPs
Groundnut rosette virus (ORF3)
SEL regulation
–
RNA trafficking
Long-distance RNA trafficking
[2,4,10,11,35,36,49,56]
[61]
Plant
Assorted transcription factors
CmPP16
CmPP36
–
SEL regulation
SEL regulation
[62,63]
[7]
[48]
CmNACP
SEL regulation
HSP70 (from phloem exudate)
PP1, PP2 (from phloem)
SUT1
Thioredoxin h
Assorted phloem proteins
ISE 1
SEL regulation
SEL regulation
–
SEL regulation
SEL regulation
SEL regulation
–
Long-distance RNA trafficking
Phloem movement (when
proteolytically cleaved)
Long-distance developmental
signaling, shoot meristem function
Protein trafficking
RNA interaction
Protein trafficking (CC-SE only)
–
Protein trafficking
–
[63]
[9]
[64,65]
[66]
[67]
[65]
[68]
Abbreviations: CC-SE, companion cell-sieve element; CP, coat protein; ISE, increased size exclusion; MP, movement protein; NCAP, non-cell-autonomous protein; ORF, open
reading frame; SEL, size exclusion limit, ‘ –‘ indicates unknown or unclear.
Corresponding author: Karl J. Oparka ([email protected]).
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Review
TRENDS in Plant Science
Vol.9 No.1 January 2004
endoplasmic reticulum. N-terminal deletion to the transmembrane domain of this protein produced a dominantnegative mutant that blocked the trafficking of specific
NCAPs such as CmPP16 and the movement protein (MP)
of tobacco mosaic virus (TMV). Transgenic tobacco plants
expressing the mutant form of NCAPP1, or in which the
gene encoding NCAPP1 was silenced, were compromised
in their ability to regulate leaf and flower development,
consistent with NCAPP1 having a role in the selective
transport of key developmental proteins. NCAPP1 is probably one of several proteins used on the NCAP pathway,
possibly shuttling NCAPs to the PD pore [5]. Other putative NCAP pathway proteins remain to be identified,
although some of these are likely to be resident within the
PD pore itself, playing a role in trafficking NCAPs through,
rather than to, PD.
New insights from protein –protein interactions
Several recent studies have examined the interaction
between viral MPs and plant proteins in an attempt to
identify host factors involved in the trafficking of macromolecular complexes (MCs) to PD (Table 2). With few
exceptions, plant proteins that interact with MPs can be
grouped into distinct categories.
Chaperones
Several viral MPs have now been shown to interact with
DNAJ-like chaperones, a group of small proteins belonging
to the HSP40 subclass (Table 2). DNAJ proteins have a
range of functions including protein import into organelles
and in regulating HSP70 chaperone activity [8]. Collectively, HSP-like chaperones might play a role in the partial
unfolding of proteins before their translocation through
the PD pore [1].
Recently, HSP70 has emerged as a possible chaperone
for trafficking endogenous MCs to PD [9]. HSP cognate 70
(HSPc70) chaperones isolated from PD-rich wall fractions
and from Cucurbita phloem exudates were found to interact with PD and to modify the PD size exclusion limit.
Interestingly, a cytosolic, non-phloem, HSP70 protein did
not possess the necessary motif for modifying the size
exclusion limit, indicating that the HSP70 identified
in phloem might have a crucial role in mediating the
non-cell-autonomous movement of MCs into or out of
the phloem system. In a gain-of-movement function assay,
the same motif, when transferred to a human HSP70
protein, allowed the human homologue to modify the PD
size exclusion limit. These data provide compelling evidence for a role of HSP-like chaperones in trafficking MCs
to and through PD. Interestingly, beet yellows virus (BYV)
encodes in its genome a HSP70 homologue that associates
with PD, and is essential for viral cell – cell movement [10].
However, the viral HSP70 does not contain a plant-like
PD-interacting domain [9], suggesting that this virus
might use a different pathway to interact with and traffic
through PD. This example illustrates an interesting case
Table 2. Plant proteins that interact with viral movement proteins
Virus
Host protein
Putative function
Refs
TSWV
TSWV
PMTV (TGB 2)
Chaperones
DNAJ-like
DNAJ-like
DNAJ-like
Chaperone (regulator of HSP70)
Chaperone
Chaperone
[69]
[18]
A. Ziegler and
L. Torrance
(unpublished)
ToMV
ToMV
TBSV
GRV (ORF 3)
Nucleus
KELP
MBF1
HFi22
Fibrillarin
Transcriptional co-activator of pathogenesis-related protein
Transcriptional co-activator
Homeodomain protein (leucine zipper)
Unknown
TBSV (19K)
REF
Transcriptional co-activator, RNA export factor
[14]
[70]
[13]
M. Taliansky
(unpublished)
J. Uhrig et al.
(unpublished)
TMV
TMV
TMV
TMV
TSWV
GFLV
GRV (ORF 3)
CaMV
TMV
TVCV
CaMV
TCV
PVX (triple-gene-block-interacting
proteins)
Cytoskeleton
Actin
Tubulin
Tubulin
MPB2C
At4/1
Vesicle trafficking
KNOLLE
Unknown
MP 17
PD and cell periphery
PME
PME
PME
AtP8
TIPs
vRNA movement
vRNA movement
vRNA degradation
Negative regulator of MP function
Limited homology to myosin or kinesin
[71]
[72]
[17]
[91]
[18]
t-SNARE (syntaxin)
RNA and vesicle trafficking (N-terminal Rab sequence)
Rab receptor (homology to rat PRA1)
[32]
M.Taliansky
(unpublished)
[73]
MP –PD interaction (pectin esterification)
MP –PD interaction
MP –PD interaction
Unknown (contains RGD motifs)
Interact with b-1,3-glucanase
[33,34]
[33]
[33]
[74]
[37]
Abbreviations: CaMV, cauliflower mosaic virus; GFLV, grapevine fan leaf virus; GRV, groundnut rosette virus; MP, movement protein; PD, plasmodesmata; PMTV, potato mop
top virus; PVX, potato virus X; TBSV, tomato bushy stunt virus; TCV, turnip crinkle virus; TMV, tobacco mosaic virus; ToMV, tomato mosaic virus; TSWV, tomato spotted wilt
virus; TVCV, turnip vein clearing virus; vRNA, viral RNA; 19K, 19 kDA.
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TRENDS in Plant Science
of molecular mimicry, in which a host protein appears to
have been usurped by a plant virus to fulfil its requirement
for cell – cell transport. However, to date, few viruses have
been shown to encode a direct homologue of a plant
chaperone and it is likely that some viral groups have
instead developed the ability to ‘recruit’ essential host
chaperones during the infection process.
Nuclear proteins
Protein – protein interaction studies have revealed that
some viral MPs interact with nuclear proteins to achieve
successful cell-to-cell movement. In the case of DNA-based
geminiviruses, the virus must enter the nucleus for
replication [11] and recent studies have identified nuclear
components that interact with geminivirus MPs [12].
However, the MPs of some RNA-based viruses also interact
with nuclear components (Table 2), particularly transcription factors, suggesting that one mechanism by which viral
MPs might achieve selective transport through PD is to
commandeer host proteins that themselves have the capacity to move through PD. For example, the MP (P22) of
tobacco bushy stunt virus (TBSV) interacts with a homeodomain leucine-zipper transcription factor, HFi22 [13].
The binding of HFi22 to P22 might enable directional
transport of P22– RNA complexes through PD [13]. However, the binding of P22 to HFi22 might also prevent the
host transcription factor from activating expression of one
or more defence genes, a view that agrees with a recent
suggestion that some viral MPs interact with transcriptional co-activator proteins to influence host gene expression [13,14]. Collectively, these data raise the possibility
that at least some viral MPs must interact with nuclear
proteins to mediate cell-to-cell virus movement. A challenge for the future will be to distinguish between those
viral proteins that enter the nucleus to influence host
defence genes from those that do so to interact with a
nuclear MC destined for export.
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Vol.9 No.1 January 2004
Cytoskeletal proteins
The plant cytoskeleton provides an attractive route by
which MCs might reach PD [15,16] and several viral MPs
have been shown to interact with elements of the cytoskeleton (Table 2). However, such an association might
not indicate the direction of viral MP trafficking, nor
imply that the cytoskeleton directs MCs specifically to PD.
Indeed, in the case of TMV, recent studies suggest that
microtubules might transport MPs as part of a targeted
degradation mechanism [17]. Protein – protein interaction
assays have revealed a direct association of some viral MPs
with myosin- or kinesin-like proteins [18], suggesting that
actin –myosin-based motility might be required for trafficking some MCs to PD. In the future, it will be essential
to determine the direction of MC trafficking along cytoskeletal components. In this respect, fusions of viral MPs
to recently developed photo-activatable fluorescent proteins [19,20] might provide a useful approach to study MC
trafficking along the cytoskeleton, as well as determining
the kinetics of transport.
Vesicle trafficking proteins
Rabs
In all eukaryotes, key regulatory proteins play a central
role in determining vesicle transport specificity [21]. These
include the Rab GTPases, which determine key membrane
fusion events between donor and acceptor membranes
[22,23]. In the case of plant viruses, one means of achieving
selective transport to PD would be to ‘grab a Rab’ that is
travelling to the correct subcellular location. This could
occur in either of two ways: by binding directly to a Rab
that traffics to PD as part of its internal cycling mechanism, or by becoming attached to a vesicular cargo that is
being delivered to PD by an appropriate Rab. The large
number of unique proteins in and around PD (Table 3)
suggests that many of these are targeted to PD by vesiclemediated pathways. By attaching to a cargo that is being
trafficked to PD, a viral MC could be delivered to the
Table 3. Macromolecules found in and around plasmodesmata
Macromolecule
Function
Comments
Refs
Callose
Pectin
Pectin methylesterase
Actin
Myosin
Myosin VIII
Centrin
Calreticulin
Ca-dependent protein kinase
Ca-independent protein kinase
41 kDa protein
45 kDa protein
PRms
RTM proteins
Ubiquitin
PD 08
PD 09
PD 10
PD 11
Wound sealing SEL regulation
Wall architecture
Pectin de-esterification MP interaction
SEL regulation
SEL regulation
SEL regulation
SEL regulation
SEL regulation
Protein phosphorylation signaling
Protein phosphorylation signaling
Unknown
Unknown
Unknown
Restrict phloem entry of tobacco etch virus
Turnover of PD proteins
Unknown
Unknown
Unknown
Unknown
–
Enriched around PD
Putative PD receptor
Cytoskeletal PD component
Might link desmotubule to plasma membrane
Unconventional plant myosin enriched at PD
Ca-binding cytoskeletal protein
Ca-sequestering protein
Possible involvement in MP phosphorylation
MP phosphorylation
Isolated from PD-enriched wall fractions
Isolated from nodal walls of Chara
Pathogenesis-related protein
RTM1 localized to punctate spots (possibly PD) in SEs
Localized in suspension cultures
Unknown (contains WD-40 repeat)
Homology to monodehydroascorbate reductase
Homology to Rab11
Unknown protein (TIN15.3; At)
[39,75,76]
[77]
[33,34,78]
[51,79–81]
[75,81,82]
[53,54]
[83]
[84]
[85]
[86]
[87]
[81]
[88]
[89]
[90]
[28]
[28]
[28]
[28]
Abbreviations: MP, viral movement protein; PD, plasmodesmata; RTM protein, restricted tobacco etch virus movement; SEL, size exclusion limit.
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TRENDS in Plant Science
‘correct’ subcellular address. This suggests that PD might
represent a specific site for vesicle recycling events.
Recent studies have begun to implicate Rabs and their
related proteins in MC trafficking to PD (Tables 2 and 3).
Although Rab proteins are generally associated with the
regulation of vesicle-mediated transport [22], they might
also play a role in trafficking large ribonucleoprotein complexes to the cell periphery. For example, in the Drosophila
oocyte, Rab11 has been shown to be involved in highly
polarized mRNA trafficking [24]. Unlike yeast and human
genomes, which have only two and three genes encoding
Rab11 proteins, respectively, Arabidopsis has more than
25 such related genes [25], each with a possible different
localization and function. Recently, transgenic tomato
plants were produced that express an antisense Rab11
sequence [26]. The plants showed abnormal development,
mimicking that seen when the expression of homeobox
genes is disrupted [27], suggesting that Rab11 might be
connected with the trafficking of transcription factors
through PD. Recently, we have shown that the N-terminal
moiety of Rab11, along with other plant cDNAs, associates
strongly with PD when expressed as a cDNA– GFP fusion
(Figure 1) [28]. It would be interesting in the future to
determine whether Rab proteins do indeed play a role in
regulating the trafficking of MCs to PD.
KNOLLE
The KNOLLE protein is a target-soluble N-ethylmaleimide-sensitive-factor attachment protein receptor
(t-SNARE) that belongs to the syntaxin family [29].
Specific t-SNAREs located on target membranes form
part of the integral membrane trafficking machinery of
eukaryotic cells and interact directly with vesicle SNARE
(v-SNARE) complexes to allow specific vesicle– membrane
fusions to occur [30]. During the formation of the
phragmoplast, Golgi-derived vesicles are delivered to the
Figure 1. The N-terminal moiety of a Rab11 protein localizes to plasmodesmata
(PD) when expressed as a green fluorescent protein (GFP) fusion. The figure
shows an optical stack of a single spongy mesophyll cell. PD are labelled at the end
walls connecting cells (arrows). Scale bar ¼ 10 mm. Reproduced, with permission,
from Ref. [28].
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Vol.9 No.1 January 2004
equatorial plane, where they fuse to form a membrane
network that matures centrifugally into a disc-shaped cell
plate [31]. In a recent study, the MP of grapevine fanleaf
virus, which forms PD-associated tubules, was found to
interact with KNOLLE in co-immunoprecipitation studies,
and also to co-localize with KNOLLE at the developing cell
plate [32]. The authors proposed a model in which the
grapevine fanleaf virus MP is transported to specific sites
in the cell, possibly by co-packaging with KNOLLE in the
same Golgi-derived vesicles. The increasing number of
vesicle-trafficking proteins found to interact with viral
MPs (Table 2) suggests that the targeted delivery of some
components to PD might occur via post-Golgi compartments. Protein mediators of vesicle trafficking and fusion
(e.g. Rabs and SNAREs) would provide attractive ‘targets’
for viral MPs, allowing the directed translocation of the
viral genome to PD.
Peripheral proteins
Pectin methylesterase
Several viral MPs appear to interact with putative PD
components. However, direct in situ interactions between
PD proteins and viral MPs are lacking in many studies and
it is probably more appropriate to refer to ‘peripheral’
proteins than to PD proteins. The MP of TMV, along with
other viral MPs (Table 2), has been shown to interact with
pectin methylesterase (PME) [33,34], an enzyme involved
in modifying pectin-rich regions of the wall (Table 3).
Conceivably, MPs only interact with PME once the MP has
been transported to the PD [35]. However, another possibility is that MPs interact with PME before its delivery to
the plasma membrane [1]. In this scenario, PME might
function as a hijacked cargo rather than a bona fide PD
receptor for MP. Provided that this interaction occurred
early enough in the infection cycle, MPs could ‘piggy back’
on the host MC trafficking machinery to reach their
required location.
TGB12K-interacting proteins
Potato virus X requires gene products of the triple-gene
block (TGB), along with the viral coat protein, to mediate
cell-to-cell movement [35]. Using the 12 kDa (12K) TGB
protein (which increases the PD size exclusion limit [36])
as bait in a yeast two-hybrid assay, three host TGB12Kinteracting proteins (TIPs) have been identified [37]. All
three TIPs interacted with b-1,3-glucanase, an enzyme
involved in callose degradation. Callose is an integral
component of the neck region of PD [2,3] and previous
studies have shown a direct link between callose degradation and virus movement [38,39]. Thus, regulation of
b-1,3-glucanase activity by viral MPs might be a means by
which some viruses overcome host deposition of callose
during infection [39]. Significantly, TIPs are detected in
non-virus-infected material and virus infection does not
affect their expression [37]. Therefore, TIP proteins might
normally function to regulate callose deposition around
PD. Not all viral MPs interact with TIPs, indicating that
MP – TIP binding is yet another piece of a jigsaw in which
specific virus – host interactions control the movement
of MCs to PD.
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Vol.9 No.1 January 2004
37
How is specific molecular complex trafficking achieved?
The ability of viral MCs to bind cargos and/or host
chaperones does not in itself guarantee successful delivery
of the MC to the PD pore. Indeed, random diffusion of MCs
would appear to be an inaccurate way to interact with PD,
even if the chaperone– MC has an integral PD-interaction
motif [40]. If many MCs do use components of the host
cytoskeleton to access PD [15,16], what factor(s) bind the
MC to the cytoskeleton and how is successful PD targeting
achieved? Important clues have been obtained from recent
studies of MC trafficking in yeast and Drosophila.
or they might be spatially separated on different proteins
within the pore. In the context of the putative PD docking
protein, it is significant that both transcription factors and
viral MPs might interact with a common PD receptor
protein [48], consistent with the notion that plant viruses
acquired the capacity for cell – cell movement from the
plant genome [49]. The putative PD docking protein(s)
awaits isolation. However, given its apparent ability to
interact with a wide range of protein cargos, it might
have multiple binding domains capable of forming stable
protein – protein interaction platforms.
Chaperone –motor associations
In many animal cells, the destination of mRNAs is determined by specific sequences in their 30 untranslated
regions (UTRs) that are often referred to as ‘zip codes’
[41]. Zip codes are recognized in turn by transacting
proteins that determine the correct subcellular address of
the mRNA [41]. Of these transacting proteins, linkers
between RNA and cytoskeletal motors are key determinants of mRNA destination [41 – 43]. Many chaperonerelated proteins bind directly to a molecular motor,
ensuring the delivery of the MC to the cytoskeleton.
Several molecular motors, including those from the myosin, kinesin and dynein families, have been shown to
interact with cellular proteins that determine transport
specificity [43]. The dual function of Rab GTPases, namely
their specificity for their cargo and their ability to link
their cargo to the cytoskeleton, make these proteins
attractive candidates for mediators of MC trafficking
[41,44]. In yeast, specialized ‘adaptor proteins’ perform
the function of linking motor proteins to a specific cargo
[45]. In yeast, a class V myosin (myo4) has been shown
to transport ASH1 RNA in a highly polarized manner
(localization of ASH1 at the bud tip is required for matingtype switching [46]) via interactions with adaptor proteins
known as She proteins [45].
It is estimated that several hundred different transport
complexes can travel along the cytoskeleton in a single
eukaryotic cell [44]. Plants possess up to 17 myosins [47],
although the functions of these have yet to be determined.
In the same way that yeast-two hybrid approaches have
begun to identify plant proteins that interact with viral
MPs, using the newly discovered motor proteins from the
Arabidopsis database as bait in yeast-two hybrid studies
should allow identification of many of the interacting
transport complexes that are shuttled by different molecular motors in plants.
Search for plasmodesmal components
Several proteins have now been localized to PD (Table 3),
although the list is not extensive. The paucity of known PD
components is largely because it is difficult to isolate intact
PD from the cell wall fraction for proteomic analyses.
In our search for proteins that interact specifically with
PD, we have exploited a virus-based vector expressing
random cDNA– GFP fusions [28]. One of the unknown
PD proteins we have isolated encodes a WD-40-repeat
sequence (Table 3), a regulatory protein domain involved
in protein – protein interactions [50]. The interaction of
these PD-associated proteins with other plant proteins
and viral proteins will be an interesting future subject area.
Getting through the plasmodesmal pore
Assuming that a MC has recruited the necessary machinery to target it to PD, the next major challenge is successfully to negotiate passage through the PD pore. Several
criteria must be met for successful transport of the MC
through PD. The first is the docking of the MC with a
putative PD receptor at or within the orifice of the PD pore,
the second is the successful initiation of ‘gating’ (transient
increase in the size exclusion limit) of the pore, and the
third is the trafficking of the MC through the pore from one
cell to the next [48]. The PD components responsible for
each of these functions might be present on a single protein
Role for protein kinases?
If components of the cytoskeleton are an integral component of PD, how are the individual elements regulated
to achieve specific trafficking of MCs? Regulation of
cell signalling in both plants and animals involves
signal cascades that require protein phosphorylation –
dephosphorylation cycles, and it is tempting to invoke a
role for specific protein kinases in regulating PD function.
It is known that the MPs of several viruses are phosphorylated during infection [56]. However, it is not known
whether the endogenous MCs that are trafficked through
PD require phosphorylation to mediate their passage.
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Myosin within plasmodesmata
In the structural model proposed by Robyn Overall and
Leila Blackman [51], actin is depicted as running through
the PD pore, closely associated with the central desmotubule. Putative myosin ‘spokes’ radiate out from the actin,
physically linking it to the plasma membrane. Such a
structural arrangement could create tension between
the plasma membrane and the desmotubule, regulating
pore aperture. Myosin has a role in generating tension
between adjacent membranes in many mammalian cells
[52]. Recently, myosin VIII, a unique plant unconventional
myosin, has been localized to PD [53] and has been
implicated in the regulation of PD function [54,55]. This
myosin might be bound to the plasma membrane within
PD, possibly by its C-terminal globular region [54]. However, myosin VIII also has a characteristic motor-domain
region, common to all myosin motors, as well as four IQ
motifs that are predicted to bind calmodulin [54]. It is
therefore possible that myosin VIII functions as a Ca2þregulated molecular motor that is capable of trafficking
cargo along the actin filaments that traverse the PD
pore (see below).
Review
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TRENDS in Plant Science
Vol.9 No.1 January 2004
To date, several protein kinases have been implicated in
PD function and in MP trafficking (Table 3). Protein
kinases present within PD might be involved in the
phosphorylation of MCs directly, or alternatively might
play a role in the phosphorylation of the chaperones and/or
cytoskeletal motors that deliver them. Phosphorylation of
the myosin bound to the plasma membrane within PD
would provide an attractive mechanism for modulating the
size exclusion limit. In many animal cells, phosphorylation
by myosin-specific kinases results in the dissociation of
the myosin motor from its attached membrane [43].
Interestingly, the unique C-terminus of myosin VIII, a
PD-localizing myosin, contains several predicted phosphorylation sites for protein kinases A and C [54].
However, the role of phosphorylation in the regulation
of this unconventional plant myosin has yet to be
demonstrated.
Linking structure to function
Here, I propose a model that couples known structural
components of PD to a potential regulatory mechanism
in which protein kinases regulate the activity of key
components within PD (Figure 2). It should be stressed
that this model borrows heavily from recent developments
in animal and yeast biology, and that other mechanisms,
including passive transport for some MCs [6], remain a
possibility. However, the model is intended to generate
debate about how selective MC trafficking through PD
P
K
Cell wall
P
D
Plasmodesma
Nucleus
Ribonucleoprotein
complex
Cytoplasmic chaperone
(putative Rab)
Nuclear export factor
Actin
D
PD-docking protein
Golgi-derived cargo
K
PD-kinase
Endosomal
compartment
Myosin
TRENDS in Plant Science
Figure 2. Potential trafficking pathways to and through plasmodesmata (PD). (Left) A ribonucleoprotein complex traffics from the nucleus to the cytoplasm, assisted by a
nuclear export factor. A cytoplasmic chaperone then interacts with the macromolecular complex (MC) and binds it to a specific myosin motor. These interactions determine
transport specificity to the PD pore. At the neck region of the pore, the MC binds to a putative docking protein, which in turn activates a myosin-specific kinase that
phosphorylates the C-terminus of the myosin motor, resulting in its release from the membrane. The MC is then free to traffic via the motor domain of the myosin along
actin filaments associated with the desmotubule that spans the central part of the pore. (Right) Protein cargos destined for secretion at or around PD are packaged at the
trans face of the Golgi apparatus. A cytoplasmic chaperone (putative Rab protein) associates with the vesicle membrane and binds it to myosin. As above, these specific
interactions determine vesicle destination. At the neck region of the PD pore, the vesicle contents are discharged by regulated exocytosis. The plasma membrane in this
region, along with essential PD components, is subsequently recycled by an endocytic step and delivered to an endosomal sorting compartment.
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Vol.9 No.1 January 2004
might be achieved. In this particular model, the cytoskeletal motor, not the cargo, is phosphorylated to permit
MC trafficking through the pore (although it is appreciated
that other protein kinases within PD might phosphorylate
MCs directly). The model is based on the requirement
for chaperone– motor interactions to traffic MCs in plants,
a feature that has already become established for MC
trafficking in yeast and mammals.
In the case of MCs originating inside the nucleus
(Figure 2, left), a chaperone protein (nuclear export factor)
is required to transport the MC into the cytoplasm. In the
cytoplasm, the MC associates with a second chaperone
(putative Rab protein) that in turn binds the cargo to an
appropriate motor protein. The specific interaction between
chaperone and motor protein determines transport specificity and targets the cargo to PD. One of the plant-specific
myosins is envisaged to link the MC – chaperone complex
to the actin cytoskeleton via its motor domain. The continuity of the actin cytoskeleton from cytoplasm to PD
provides a pathway for directional traffic of the MC to the
PD orifice. At the PD, a docking protein binds the MC,
either by attaching directly to the cargo or, alternatively, to
the myosin motor protein at its C-terminus. The gating
motif on the MC activates a myosin-specific kinase that
phosphorylates the C-terminus of the myosin motor, resulting in its release from the membrane and a resultant
increase in the size exclusion limit of the pore. The MC is
then free to traffic to another cell via the motor domain of
the myosin, along actin filaments that span the central
pore. In this model, phosphorylation – dephosphorylation
cycles of the molecular motor regulate the detachment and
attachment of the MC from the plasma membrane lining
the PD pore, providing a generic mechanism for PD regulation that is based on the motor protein, not the cargo.
The model incorporates recent data that implicate
specific vesicle trafficking events in the delivery of cargos
to PD (Figure 2, right). Specific proteins destined for
secretion at or around PD are packaged at the trans face of
the Golgi before their delivery to the PD pore. Notice that
the coordination of this vesicle-mediated transport requires
a similar subset of chaperones and molecular motors to
achieve specific targeting. At the PD pore, the protein
cargo is discharged to the apoplast by regulated exocytosis.
The model also incorporates an endocytic vesicle recycling
step in which key PD components are returned to intracellular compartments for degradation. Figure 3 shows a
putative pathway for MPs to PD. Several viral MPs associate with the endomembrane system early in the viral
infection cycle, and several of these are predicted to have
transmembrane domains [17,35,57,58]. A viral MP is shown
to traffic to PD by becoming incorporated, along with
its host-interacting partner, into Golgi-derived vesicles
destined for secretion at PD (Figure 3). Such vesicles
achieve directed trafficking to PD by binding to an appropriate Rab protein. At the PD pore, specific interactions
between v-SNARE and t-SNARE complexes on the vesicle
and plasma membrane, respectively, allow the vesicle
membrane to fuse with the plasma membrane, leaving
the MP inserted into the plasma membrane within PD.
The MP might then subsequently associate with the PD
trafficking machinery (Figure 3). Conceivably, viral RNA
attached to the MP could be trafficked on the cytosolic face
of such a transport complex.
Future directions
The aim of this article is to highlight areas of future
research that might yield information about the pathways
and mechanisms of selective MC trafficking. Future
studies will gain from further isolation of PD proteins
and determination of the cellular components with which
they interact, particularly the chaperones and molecular
motors responsible for directional trafficking to PD. This
article focuses on targeted MC movement. In the future, it
will be essential to identify those MCs that move between
cells by nonselective movement and to distinguish between
the mechanisms that underlie selective and nonselective
transport through PD. The elusive protein kinases that
regulate the trafficking of specific MCs remain to be
Cell wall
GTP
GTP
Plasmodesma
Ribonucleoprotein
complex
Rab protein
Movement
protein
Plasma membrane
v-SNARE
t-SNARE
Golgi-derived
cargo
TRENDS in Plant Science
Figure 3. Putative mechanism by which some viral movement proteins (MP) might use the endomembrane system to target plasmodesmata (PD). The viral MP interacts
with a specific host protein via an endomembrane-spanning domain before (or during) the packaging of the cargo protein by the Golgi. A specific Rab protein then guides
the vesicle to the vicinity of PD. Specific interactions between v-SNARE and t-SNARE proteins on the vesicle membrane and plasma membrane, respectively, permit the
vesicle to fuse with the plasma membrane, discharging the protein cargo to the apoplast. The MP is then left associated with the plasma membrane at the neck of the PD
pore. The figure also shows how a viral ribonucleoprotein complex could be trafficked to PD by its attachment to the cytosolic face of the MP. Such a ribonucleoprotein
complex might subsequently interact with the PD trafficking machinery to permit selective trafficking of the viral genome.
http://plants.trends.com
40
Review
TRENDS in Plant Science
characterized. New evidence is beginning to emerge for a
role of the endomembrane system in delivering viral MPs
to PD, and this important area requires further study.
Determining the vesicle trafficking proteins with which
viral MPs interact will provide clues leading to the dissection of the endogenous MC trafficking pathway to PD.
Once a battery of PD-interacting proteins has been identified, protein imaging and analysis techniques such as
FRAP (fluorescence recovery after photobleaching) [59]
and FRET (fluorescence resonance energy transfer) [60]
will assist in determining PD protein turnover rates, as
well as establishing meaningful in situ protein – protein
interactions within the PD pore. Central to our understanding of MC trafficking in plants will be the acceptance
that PD are not isolated structures embedded in the
cell wall but rather an integral component of an intercellular network that includes nuclear proteins, cytoskeletal proteins, motor proteins, vesicle trafficking proteins,
PD-specific chaperones and protein kinases. The challenge
for the future is to place the pieces of this jigsaw puzzle into
a meaningful order.
Acknowledgements
I thank Trudi Gillespie for drawing the figures and SEERAD and the
Gatsby Foundation for financial support.
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