Transport Between the Endoplasmic Reticulum and Golgi Apparatus ...

Review

Crossing the Divide – Transport Between theEndoplasmic Reticulum and Golgi Apparatus in Plants

Sally L. Hanton*, Lauren E. Bortolotti, LucianaRenna, Giovanni Stefano and Federica Brandizzi

Department of Biology, 112 Science Place, University ofSaskatchewan, Saskatoon, Saskatchewan S7N 5E2,Canada*Corresponding author: Sally L. Hanton, [email protected]

The transport of proteins between the endoplasmicreticulum (ER) and the Golgi apparatus in plants is anexciting and constantly expanding topic, which hasattracted much attention in recent years. The study ofprotein transport within the secretory pathway is a rela-tively new field, dating back to the 1970s for mammaliancells and considerably later for plants. This may explainwhy COPI- and COPII-mediated transport between the ERand the Golgi in plants is only now becoming clear, whilethe existence of these pathways in other organisms isrelatively well documented. We summarize current know-ledge of these protein transport routes, as well as high-lighting key differences between those of plant systemsand those of mammals and yeast. These differences havenecessitated the study of plant-specific aspects of proteintransport in the early secretory pathway, and this reviewdiscusses recent developments in this area. Advances inlive-cell-imaging technology have allowed the observationof protein movement in vivo, giving a new insight intomany of the processes involved in vesicle formation andprotein trafficking. The use of these new technologies hasbeen combined with more traditional methods, such asprotein biochemistry and electron microscopy, to increaseour understanding of the transport routes in the cell.

Key words: COPI, COPII, endoplasmic reticulum, ER exportsite, Golgi, protein transport

Received 9 December 2004, revised and accepted forpublication 24 January 2005

The secretory pathway is complex system of organelles

specialized for the synthesis, transport, modification and

secretion of proteins and other macromolecules (1). As

such, this system plays a vital role in the life of a cell. It

is generally assumed that protein transport through the

secretory pathway is controlled by vesicular transport

intermediates that carry cargo molecules from one orga-

nelle to the next. Vesicular transport can occur in the

forward (anterograde) direction, from the endoplasmic reti-

culum (ER) towards the plasma membrane, or in reverse

(retrograde). The entire secretory pathway exists in equili-

brium between anterograde and retrograde transport, and

any disruption of a part of this balance can result in

dramatic changes in the biology of a cell.

Vesicle budding and fusion mechanisms at the ER–Golgi

interface are highly conserved between species (2–4). The

formation of vesicles is induced by the action of cytoplas-

mic coat protein complexes (COPs) that polymerize on the

membrane surface, capturing both cargo molecules and

those that function in vesicle direction, such as soluble

N-ethylmaleimide-sensitive factor attachment protein

receptors (SNAREs) in the process. The membrane

becomes deformed during the polymerization of the coat,

resulting in the formation of a nascent vesicle. Small

proteins with GTPase activity regulate the assembly and

disassembly of the vesicle coat by cycling between GTP-

bound (activated) forms and GDP-bound (inactivated)

forms. The activated form initiates the recruitment of

coat proteins to the membrane, whereas hydrolysis of

GTP to GDP alters the conformation of the GTPase and

triggers uncoating of the vesicle. The GTPase activity is

tightly regulated by guanine-nucleotide exchange factors

(GEFs) and GTPase-activating proteins (GAPs), which pre-

vent unproductive cycles of membrane coating and

uncoating.

The Secretory Pathways of Plants and Animalsare not Identical

The organization of the organelles that make up the secre-

tory pathway of plants differs greatly from that of mammals

and yeast. Despite the identification of plant homologues of

proteins that are known to be involved in vesicular transport

in other systems, the mechanisms in plants have not yet

been fully characterized. Given the differences in the fea-

tures of the secretory pathway of plants compared with

those of other organisms, it seems likely that plants have

evolved unique characteristics for achieving efficient protein

transport between organelles. We discuss current know-

ledge of these characteristics, although further study will be

necessary to elucidate many of the processes involved in

protein transport in plant cells.

The ER in plants is pushed to the cortex of the cell by the

large central vacuole, whereas the mammalian ER has no

such constraints and can exist throughout the cell, radiating

Traffic 2005; 6: 267–277Copyright # Blackwell Munksgaard 2005

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2005.00278.x

267

out from the nuclear envelope. The domains of the ER

from which proteins are exported to the Golgi apparatus,

termed ER export sites (ERES), are relatively immobile in

mammals (5), while in plants, they are highly motile (6).

Similarly, the mammalian Golgi apparatus remains rela-

tively stationary in the perinuclear region of the cell and is

much larger than the plant Golgi. It has been reported that in

various plant cellular systems the Golgi apparatus is present

as multiple stacks that are distributed throughout the

cytosol and are capable of rapid movement (7–9)

(Figure1). The mammalian secretory pathway contains an

additional organelle known as the ER-Golgi intermediate

compartment (ERGIC), which does not exist in plants,

although the cis-Golgi may play a similar role. In contrast,

the plant cell contains one or more vacuolar compartments

(10), which can perform different functions such as protein

degradation, turgor maintenance and protein storage,

depending on the tissue and species. Animal cells contain

lysosomes, which perform a similar degradative function

to that of the lytic vacuole in plants, but are much smaller

and are distributed throughout the cell. The presence of

different types of vacuoles in plants necessitates the exist-

ence of additional transport pathways that allow cargo

molecules to travel to these organelles. Finally, the most

distal location in the plant cell is the cell wall, which requires

the secretion of a variety of molecules for its generation and

maintenance. This transport is one of the driving forces

behind the secretory pathway.

Despite these differences in the secretory pathways, pro-

teins in both plants and animals are generally transported

in the anterograde direction from the ER to the Golgi

apparatus, at which point they are sorted for further trans-

port either forward, in the direction of the cell surface and

organelles in the later secretory pathway, or back towards

the ER.

The Complexity of Protein Transport in Plants

Figure 2 shows a schematic representation of the secre-

tory pathway in plants, summarizing the main transport

pathways that have been established and proposed.

Export from the ER to the Golgi occurs via a COPII-

mediated mechanism (arrow 1) (6,11,12), but evidence

0.0 9.4 18.9

47.237.828.3

Figure 1: The endoplasmic reticulum and Golgi apparatus in plants are motile. Confocal laser scanning microscope images showing

an epidermal cell from tobacco leaves, coexpressing a GFP-tagged Golgi marker protein and a YFP-tagged endoplasmic reticulum (ER)

marker protein. Images were taken at different time-points (seconds; shown at top left of each image) to demonstrate the movement of

both ER and Golgi. The white arrowhead indicates a single Golgi body throughout the time series, while the empty arrowhead indicates

movement within the ER. Size bar¼2 mm.

Hanton et al.

268 Traffic 2005; 6: 267–277

for a second anterograde pathway that is not controlled by

COPII has also been postulated (arrow 2) (13). To balance

these forward transport pathways, at least one retrograde

pathway must exist to carry cargo molecules back from

the Golgi to the ER. Retrieval of proteins from Golgi to ER

is required for a variety of reasons. Some proteins are

intended to be resident within the ER, sometimes with a

role in the folding and modification of newly synthesized

proteins. These proteins can escape from the ER through

accidental incorporation into the lumen of export carriers,

and then need to be retrieved from the Golgi (12,14). It

seems likely that other proteins that are involved in the

export machinery itself may also be salvaged so that they

can be re-used in a subsequent round of vesicle formation

and transport. The retrieval mechanism is based on the

presence of one or more signals in the sequence of the

protein (15), which can be recognized by a receptor or

receptor-like protein. The binding of ligand to receptor is

thought to induce the formation of a retrograde vesicle to

transport ER-resident proteins back to their destination, as

has been shown in mammals (16,17). These vesicles are

known as COPI vesicles (arrow 3) (18,19), although COPI-

independent retrograde transport pathways have been

proposed in mammalian cells (20–22). One or more of

these alternative routes may also exist in plants to balance

the effects of the COPII-independent anterograde route

suggested by Tormakangas et al. (13).

Later transport in the secretory pathway is mediated by

other vesicle types such as clathrin-coated vesicles (arrow

4) (23), which have been proposed to mediate transport

from the Golgi apparatus to the lytic vacuole, and dense

vesicles (arrow 5) (24,25), which convey cargo proteins to

the storage vacuole. In addition to these routes that carry

cargo from the Golgi to the vacuoles, a variety of vesicle

types that appear to transport storage proteins directly

from the ER to the protein storage vacuole (PSV) have

been identified in seeds (arrow 6) (26–28). It has been

shown in developing wheat grains that aggregates of sto-

rage proteins form within the ER bud off to form vesicular

structures surrounded by rough ER (27), which are incor-

porated into the vacuole through a mechanism similar to

autophagy. Comparable structures have been identified in

germinating seeds, designated KDEL-tailed cysteine pro-

teinase-accumulating vesicles (KVs) (26), and in maturing

seeds, termed precursor-accumulating (PAC) vesicles (28).

The contents of these vesicles are mainly unglycosylated

storage proteins, which do not require Golgi-mediated

modifications. However, other storage proteins that carry

complex glycan chains have been found on the periphery

of the PAC vesicles (28). This may indicate that Golgi-

derived vesicles carrying these glycoproteins have fused

with the PAC vesicles en route to the PSV. Alternatively,

these proteins may be transported to the Golgi for glycan

processing to take place, followed by recycling to the ER

for packaging into PAC vesicles and transport to the PSV.

Further investigation into the precise mechanisms by

which proteins are transported to the PSV is required to

clarify these ambiguities.

The multitude of transport pathways present in plant cells

means that the trafficking of proteins in vesicular inter-

mediates is a vast subject that cannot be properly dis-

cussed in a single review. This review therefore focuses

specifically on the complex area of transport between the

ER and the Golgi in plant cells. We discuss the formation

PSV

5

6 Golgi

TGN4 PVC

PVC

Lyticvacuole

3

ER

1 2

Figure 2: Overview of the

secretory pathway in plants.

Schematic representation of

organelles and their connecting

protein transport routes in the

plant-secretory pathway. Routes

are numbered as follows: 1 –

COPII-mediated endoplasmic

reticulum (ER)–Golgi traffic; 2 –

COPII-independent ER–Golgi

traffic; 3 – COPI-mediated Golgi–

ER traffic; 4 – traffic in clathrin-

coated vesicles (CCVs) from the

trans-Golgi network (TGN) to the

prevacuolar compartment (PVC);

5 – traffic via dense vesicles

(DVs) to the protein storage

vacuole (PSV); 6 – direct ER–

PSV traffic.

Protein Traffic at the Plant ER–Golgi Interface

Traffic 2005; 6: 267–277 269

mechanisms and functions of COPI vesicles and review

current knowledge of putative COPII carriers, as well as the

various models for the transfer of proteins between ER

and Golgi in plant cells.

Anterograde Transport from the ER isMediated by COPII

Although COPII-mediated export of proteins from the ER is

not the only anterograde ER–Golgi route that has been

proposed in plants, it has received the most attention and

is therefore the best-characterized pathway between

these organelles at this time. Transport mediated by

COPI and COPII is very similar in many respects. The

main difference between the two is that while COPII

carriers originate in the ER and export newly synthesized

proteins to the Golgi complex, COPI vesicles bud from the

cis-Golgi and travel to the ER. In mammalian cells, COPII

vesicles transport proteins as far as the ERGIC. After this,

it has been suggested that the proteins are repackaged

into vesicles that mediate transport to the Golgi apparatus

and may be involved in forward transport through the Golgi

(29,30). A recent study has shown that protein transport

occurs from the ERGIC both forward to the Golgi and back

towards to the ER (30), supporting the argument that the

ERGIC is a discrete stable compartment. However, this does

not preclude the possibility that it eventually develops into

the cis-Golgi in an extension of the cisternal maturation

model (31–34). It appears that the ERGIC in mammals is

similar in function to the cis-Golgi in plants, in that it is a point

fromwhich proteins can be returned to theERor transported

forward to distal locations. It has also been reported that

COPI vesicles may bud directly from the ER in yeast and

mammals (35,36), although no data supporting this

possibility in plants have yet been presented.

The formation of both COPII and COPI vesicles requires

the activation of a specific small GTPase, which causes the

recruitment of structural components of the vesicle coat to

the membrane, resulting in the formation of a vesicle that

can then bud from the membrane and travel to its destina-

tion. COPII vesicles have not yet been isolated from

plants, but homologues of several of the components of

the COPII coat have been identified (11,37,38) and have

been used to study the pathway (6,12). The small, cytoso-

lic GTPase Sar1p mediates COPII vesicle formation in

yeast and mammals. Three isoforms of Sar1p have been

identified in Arabidopsis (39), at least one of which

(AtSar1a) is capable of complementing Saccharomyces

cerevisiae mutants (38). This suggests similarities

between the mechanisms of vesicle budding in the two

systems. The activation of Sar1p is mediated by an

ER-localized integral membrane protein called Sec12p, a

functional homologue of which has been identified in

Arabidopsis (37,38). Sec12p is a GEF, recruiting Sar1p to

the ER membrane in order to become activated through

the exchange of GDP for GTP, which initiates vesicle for-

mation. Sec12p is not incorporated into COPII carriers (6,

40), meaning that activated Sar1p can maintain its mem-

brane association independently of Sec12p. It has been

shown in crystallization studies that mammalian Sar1p

possesses an amphipathic N-terminal domain that is

exposed on binding to GTP and allows direct interaction

of Sar1p with the membrane (41). The homology between

mammalian and plant Sar1p suggests that this may also be

the case in plants, although this has yet to be confirmed

experimentally.

It has been demonstrated through in vitro studies of non-

plant systems that the coat proteins of COPII vesicles

form two heterodimeric complexes that are sequentially

recruited to the membrane by activated Sar1p (40,42).

Sec23/24p binds to Sar1p, after which the Sec13/31p

complex completes the coat. The assembly of the coat

proteins on the membrane causes membrane curvature

and eventually vesicle budding. After this, the protein coat

must be released in order for the vesicle to fuse with the

target membrane (in this case the Golgi apparatus). This

step is induced by the hydrolysis of GTP by Sar1p, causing

a conformational change that allows Sar1p to dissociate

from the membrane (41). The hydrolysis of GTP is stimu-

lated by the presence of a GAP, Sec23p (43). The fact that

the GAP is a functional part of the COPII coat means that it

is in close proximity to Sar1p, which may cause rapid GTP

hydrolysis and increase the instability of the vesicular

structure. The release of Sar1p from the membrane

causes the dissociation of the coat proteins, leaving a

membranous vesicle that can fuse with the Golgi appara-

tus and release its contents. Proteins are then sorted for

further anterograde transport or retrograde transport back

towards the ER. The instability of the COPII vesicle may

account for some of the difficulties faced when attempting

to isolate them from plants. However, daSilva et al. (6)

demonstrated that recruitment of Sar1p to ERES can be

enhanced by overexpressing certain membrane cargo pro-

teins. It may therefore be possible to isolate COPII vesi-

cles using an analogous experimental system to increase

the number of vesicles produced.

Disruption of COPII-mediated transport has a dramatic

effect on protein transport within the cell (6,9,12,44–47).

This demonstrates the importance of the secretory path-

way to the normal functioning of the cell. The mutation of a

single residue in Sar1p can prevent it from becoming acti-

vated, effectively restricting it to the GDP-bound form and

preventing the budding of vesicles from the ER in mam-

mals, yeast and plants (45,46,48). Similarly, mutation of

another amino acid reduces the GTPase activity of Sar1p,

causing it to be confined to the GTP-bound form. This

mutant GTPase allows vesicles to bud from the ER, but

prevents them from uncoating and fusing with the Golgi

apparatus in yeast (46,49). Although it has been shown that

the GTP-restricted mutant of Sar1p inhibits the export of

proteins from the ER in plants (9,12,45,50), the accumulation

Hanton et al.

270 Traffic 2005; 6: 267–277

of coated vesicles has yet to be confirmed in this system.

However, it has been shown that in conditions of low expres-

sion, a yellow fluorescent protein fusion to Sar1p (GTP) accu-

mulates at ERES in tobacco leaf epidermal cells (6). This may

be taken to mean that the mutant labels a population of

transport intermediates that are not capable of fusion with

the target membrane.

High expression of these mutant GTPases often results in

cell death. COPII-mediated transport can also be inhibited

by the overexpression of Sec12p in both plants and yeast

(6,12,44), which causes the continual recruitment of Sar1p

to the ER membrane and prevents vesicle budding. This

titration effect can be reversed by overexpressing Sar1p,

in order that some activated Sar1p molecules are present

and can form functional vesicles (12,44).

COPI – the Retrograde Counterpart of COPII

The existence of COPI vesicles has been demonstrated in

mammalian (51), yeast (52) and plant cells (11,18,19).

COPI vesicles bud from the cis-cisternae of the Golgi (18)

and mediate traffic from the cis-Golgi back to the ER

(53,54). This is an essential pathway that continually

recycles proteins and lipids from the Golgi to the ER in

order to maintain an equilibrium with COPII transport,

sustaining the balance between the anterograde and the

retrograde transport pathways.

The minimal machinery for the budding of COPI-coated

vesicles consists of coatomer, a stable cytosolic complex

comprising seven subunits a-, b-, b0-, g-, d-, e- and z-COP

(51), as well as the small GTPase ARF1p (55,56). Homo-

logues of a-, b-, b0- and g-COP have been isolated from rice

(19), while g-, d- and e-COP have been identified in cauli-

flower, maize and Arabidopsis (11,18). In addition, ARF1p

homologues from Arabidopsis have also been identified

(39,57). These findings suggested that COPI vesicles

exist in plants, a hypothesis which was confirmed by the

in vitro induction of COPI-coated vesicles from cauliflower

cytosol (18).

COPI coat assembly is initiated by the exchange of GDP

for GTP by ARF1p in a similar manner to that of Sar1p. The

GDP-bound form of ARF1p interacts with p23, a Golgi

membrane protein, through the myristoylated N-terminus

of the GTPase (58). GDP is then exchanged for GTP

through interaction with the GEF (56,59), causing a con-

formational shift that allows direct interaction of ARF1p

with the membrane. It has been shown that myristoylation

is not required for this direct interaction (60), but rather that

the 17 N-terminal amino acids of mammalian ARF1p form

an amphipathic structure similar to that of Sar1p that can

be inserted between phospholipids in the Golgi membrane

(61,62). The activated, membrane-associated ARF1p

recruits preassembled coatomer from the cytosol (63).

This induces curvature of the membrane, resulting in the

formation of a nascent vesicle that can then bud from the

membrane.

ARF1p has multiple functions within the cell in addition to

its regulatory role in COPI-mediated transport. These func-

tions appear to be defined by different GEFs that can all

activate ARF1p (64). For example, it has been shown that

ARF1p is involved in the regulation of the BP80-mediated

route to the vacuole in plants (50), probably due to the

interaction of ARF1p with constituents of the clathrin

coat (65–67). The identification of GNOM, an Arabidopsis

ARF-GEF that is localized to the endosomes (68), in con-

junction with the data presented by Pimpl et al. (50), sup-

ports the hypothesis that the nature of the GEF

determines the action of ARF1p. It is not clear which

GEF is responsible for initiating COPI vesicle formation,

but the most likely candidates are thought to be Gea1p,

Gea2p (69) and ARNO (70). Of these, only the Gea GEFs

have been identified in Arabidopsis (71), suggesting that in

plants at least, Gea may be responsible for the activation of

ARF1p, leading to COPI vesicle formation. Further investi-

gation is required to confirm this, as the existence of an

alternative, plant-specific GEF cannot be ruled out.

Uncoating of COPI vesicles requires GTP hydrolysis to

allow ARF1p to dissociate from the vesicle membrane. A

GAP is involved in this process, and both plants and

animals possess several ARF-GAPs, all of which contain

a conserved catalytic domain (39,72). It is not clear which

of these is responsible for COPI uncoating. Fifteen ARF-

GAPs have been identified in Arabidopsis, some containing

plant-specific regions. All 15 have homology to one

another, but also exhibit considerable diversity within this

homology (39). This suggests that the different ARF-GAPs

may perform a variety of functions within the cell, which

might be expected given that 12 ARF GTPases as well as

numerous ARF-related GTPases have been identified in

Arabidopsis (39), although their respective functions are

not yet clear. It is also possible that different GAPs can

act on the same ARF and regulate its function in a similar

manner to that of the GEFs.

Disruption of Retrograde Transport InhibitsForward Protein Trafficking

The importance of the COPI pathway in maintaining both

retrograde and anterograde transport has been demon-

strated in several studies. Brefeldin A (BFA) is a drug that

inhibits several ARF1-GEFs including Gea1p and Gea2p

(59,73), preventing the activation of ARF1p and the sub-

sequent formation of COPI vesicles. The inhibitory effect

of BFA on COPI vesicle formation in plants was demon-

strated directly by Pimpl et al. (18) using extracts from

cauliflower. Various other studies have observed the

effects of BFA on the plant-secretory pathway, both in

Protein Traffic at the Plant ER–Golgi Interface

Traffic 2005; 6: 267–277 271

terms of its functionality (6,50,74,75) and in terms of its

morphology (76–79). The morphological effects of BFA on

the plant secretory pathway appear to vary depending on

the system studied, the concentration of the drug used

and the exposure time. In tobacco BY-2 cells, prolonged

treatment with 10 mg/mL of BFA results in the sequential

incorporation of Golgi membranes into the ER and the

formation of a so-called BFA compartment composed of

both organelles (76). In contrast, treatment of maize roots

with 100 mg/mL of BFA has a much less pronounced effect

(77), although the same concentration of BFA in tobacco

leaf cells causes the complete disappearance of Golgi

bodies (80). Much lower concentrations of BFA have a

dramatic effect on the function of the secretory pathway,

0.3 mg/mL being sufficient to reduce the secretion of a

marker protein by almost half in tobacco leaf protoplasts

(50). This indicates that COPI transport is required in order

for anterograde protein trafficking to occur. However, BFA

may affect other pathways within the cell (79), causing

complete disruption of the protein transport machinery

and general collapse of protein trafficking.

A more specific disruption of COPI-mediated transport can

be achieved by using mutant forms of ARF1p that are

restricted to either the GTP or GDP-bound form. The inhi-

bition of vesicle budding (GDP-restricted form) or fusion

(GTP-restricted form) both upset the fine balance between

anterograde and retrograde transport, resulting in an inhibi-

tion of both pathways (50,78,81,82). These studies have

clearly demonstrated the requirement for retrograde trans-

port in order to maintain anterograde transport. When one

of the pathways is inhibited, it is likely that a build-up of

membrane and vesicle proteins occurs within the target

organelle, leaving a depletion of these important vesicle

components in the donor organelle that eventually pre-

vents transport from occurring.

What is the Signal?

Studies in mammalian cells have shown that certain mem-

brane cargo molecules are selected for incorporation into

COPII transport intermediates and that these are concentrated

in specific domainsof theER, termedERES (83–87). Signals in

the cytosolic domains of transmembrane proteins are thought

tomediate the recruitmentof theCOPII coat to themembrane,

thereby inducing vesicle formation. It is not clear whether the

same mechanism operates in plant cells, although the high

degree of homology between theCOPII components in plants

and other systems (38) suggests that the signals involvedmay

be similar. Co-expression of GFP-tagged membrane cargo

proteins and Sar1-YFP has been used to demonstrate the

induction of ERES formation in plants (6), and a recent study

has shown that a plant p24protein can interactwith bothCOPI

andCOPII coat proteins in vitro (88).A similar signalling system

also operates for the assimilation of transmembrane proteins

intoCOPIvesicles for transport back fromtheGolgi to theER in

bothmammalian and plant cells (15, 53, 54). However, several

different types of export signals have been identified in yeast

and mammals (89), and further study is required to demon-

strate whether all of these signals are functional in plants or

whether certain transportmechanismsare specific to different

systems.

It has been shown that soluble proteins are exported from

the ER in plants by a COPII-dependent bulk flow mechanism

(12,14). This means that proteins that are intended to remain

in the ERmay be exported as far as the Golgi apparatus along

with those travelling to distal locations. The escape of pro-

teins from the ER in this manner is combated by the pre-

sence of an H/KDEL motif at the C-terminus of the protein,

which is recognized by a Golgi-localized membrane receptor

(ERD2p) (90). This receptor induces the activation of ARF1p

in mammals by recruiting the GEF (17,91), and thereby

causes the formation of COPI vesicles as described above.

It is not clear whether soluble proteins simply diffuse into

COPII carriers that are created in response to an accumula-

tion of membrane proteins that contain specific signals for

export. It is possible that a receptor for specific soluble

proteins that need to be exported from the ER exists in

plants, and that this could allow a fast-track system of

transport from ER to Golgi. Endoplasmic reticulum-export

receptors (termed Erv14p and Erv29p) for certain soluble

and transmembrane proteins in yeast have been identified

(92–95), although it is not known whether homologues of

these receptors exist in plants.

A di-acidic or di-basic motif in the cytosolic tail of trans-

membrane cargo proteins is thought to be important in

recruiting the COPII coat in mammals (85,86,96), but

other signals that may be involved have also been identi-

fied (89). The type I transmembrane protein ERGIC-53,

which localizes to the ERGIC in mammals, contains a

di-aromatic motif that is reported to be important for its

export from the ER (97). This is similar to the signal identi-

fied in plants by Contreras et al. (88), although the function

of this signal has not yet been analysed in vivo in plants. It

has been shown that the length of the transmembrane

domain plays an important role in regulating the final des-

tination of single-spanning membrane proteins in plants

(98), but export signals may play a role in increasing the

rate of export of proteins from the ER. It may also be that

export signals can override the length of the transmem-

brane domain in determining the destination of the protein.

Much more investigation is evidently needed in plant cells

before any conclusions regarding protein signals required

for ER export can be formed.

Mechanisms of Protein Transfer Between ERand Golgi in Plants

It is generally assumed that the function of ERES is to select

and concentrate cargo molecules into vesicles for export to

the Golgi (6,84,99–101), although non-vesicular carriers

have been observed for some cargoes in mammalian cells

Hanton et al.

272 Traffic 2005; 6: 267–277

(102,103). Golgi stacks in plants are found throughout the

cytosol and travel along actin filaments in leaves (74). The

ER tubules in plants are also dynamic and are constantly

forming and breaking connections (104), giving rise to an

extremely motile system (Figure 1). Protein transport may

be challenged by the relatively high mobility of the ER and

Golgi, which could have led to the evolution of specific

mechanisms in plants in order to permit efficient trafficking.

The mobility of the early plant secretory pathway has sug-

gested three possible mechanisms for protein transport

between the ER and the Golgi (summarized in Figure 3).

The first of these postulates that Golgi bodies move along

the ER to reach vesicles budding from ERES, at which

point the cargo molecules can be transferred from the ER

to the Golgi (7). This model was named the ‘vacuum-

cleaner model’ and was proposed based on the movement

of Golgi bodies along ERmembranes. The second model is

referred to as the stop-and-go model (8), and suggests that

there is an as yet unidentified signal present on fixed ERES

that causes Golgi bodies to become transiently detached

from the actin microfilaments, while they acquire cargo

proteins from the ER, after which they would reattach to

the actin and continue to move. However, neither of these

studies included data demonstrating the occurrence of

cargo transport during the postulated interactions of Golgi

with ERES, meaning that no correlation between cargo

transport and Golgi movement has been experimentally

determined.

A more recent investigation (6) utilized advanced in vivo

imaging techniques to demonstrate that ERES are capable

of movement over the ER, and that they are closely linked

to Golgi bodies (Figure 4). This would allow continual trans-

fer of cargo between ER and Golgi, allowing transport to

go on uninterrupted by Golgi movement. The experimental

evidence provided by daSilva et al. (6) demonstrates that the

models postulating fixed ERES andmotile Golgi do not neces-

sarily reflect the peripatetic nature of the early secretory path-

way in plants. The biological function of the actin-mediated

movement of this system remains to be established, but does

not seem to be related to the transport of cargo molecules

between the ER and the Golgi bodies (6,74). The exact nature

of the connection between the ERES and the Golgi bodies is

also not completely understood. It has been demonstrated

that the activities of the Sar1p-COPII and ARF1p-coatomer

systems jointly serve to form and maintain forward protein

transport to the Golgi bodies, whose components continu-

ously circulate through the ER (6,74). Similarly, ERES are

dynamic structures that exist by virtue of COPI and COPII

A

B

C

Figure 3: Comparison of the

models for protein transport

between endoplasmic reti-

culum export sites (ERES) and

Golgi bodies. Schematic rep-

resentation of the three models

for the transfer of proteins from

the ER to the Golgi apparatus. A)

The vacuum cleaner model. Golgi

bodies move along the ER

surface, picking up cargo. The

entire ER is capable of exporting

proteins. B) The stop-and-go

model. Golgi bodies move along

the ER and stop at fixed ERES,

where protein transport takes

place. After transfer of cargo

from ER to Golgi, the Golgi body

moves to the next site and

collects more cargo. C) The

mobile ERES model. Golgi

bodies and ERES move together,

a l lowing cont inua l prote in

transport between the two

organelles.

Protein Traffic at the Plant ER–Golgi Interface

Traffic 2005; 6: 267–277 273

cycling (6). It is difficult to envisage how Golgi bodies and

ERES can move together, yet not be physically linked. The

movement of the Golgi may be associated with the actin

cytoskeleton via a mechanism that has not yet been investi-

gated, while ERES may be differentiated on the ER, as the

Golgi bodies travel over the ERmembranes due to continuous

cycling of COPI and COPII components. It is unclear why the

movement of Golgi stacks through the cell should be neces-

sary, if cargo transport can occur while they are stationary. It

may be that Golgi movement is required for the efficient

transport of proteins to distal locations. Further investigation

is required to determine the precise nature of ER–Golgi trans-

port.

Future Perspectives

Although our knowledge of the protein transport between

the ER and the Golgi in plants has increased manyfold in

recent years, there are several aspects of this process that

remain elusive. In addition to specific subjects discussed

earlier in this review, such as the unidentified GEF involved

in COPI vesicle formation and the reason for Golgi move-

ment on the ER network, there are wider questions to be

addressed.

The direction of vesicles to their destinations is mediated

by SNAREs, which together with various cytosolic factors

form complexes that allow fusion of vesicles with specific

target organelles. Genes encoding 54 different SNAREs

have been identified in Arabidopsis and are localized on

organelles throughout the secretory pathway (105,106).

Six of these SNAREs are found in the ER membrane and

a further nine in the Golgi membranes (106), but it is not

clear whether these proteins exhibit functional redundancy

or if they each have a specific function, perhaps in different

pathways. Small GTPases called Rabs are also involved in

vesicle trafficking. Plants contain 57 Rab GTPases, some

of which have a high homology to mammalian Rabs,

others apparently being unique to plants (107). Although

a few of the Rabs in plants have been studied in some

detail (108–113), little is known about the localization and

function of most of these proteins. These are just two

examples of areas that remain poorly understood within

the larger topic of protein transport in plant cells, leaving

plenty of scope for further research in the field.

Current research into the subject of ER to Golgi transport

in plants increases our knowledge of the topic on an

almost daily basis. However, the complexity of the inter-

actions between different proteins and between proteins

and membranes requires yet more investigation. The

advances in confocal microscopy that allow us to observe

the relative rates of transport of different proteins in living

cells provide an ideal tool to complement more traditional

biochemical approaches. Together, these techniques can

provide us with accurate, quantitative information to aid in

our understanding of the pathways in the plant cell.

Acknowledgments

This work was supported by grants awarded to FB from the University of

Saskatchewan, CFI and the Canada Research Chair fund. SLH is also

indebted to the Department of Biology, U of S, for a Post-Doctoral Fellow-

ship. GS is supported by a Graduate College Studies Award and CRC, and

LR by a University of Saskatchewan New Faculty Award. Dr JP Taylor

(Chapel Hill, USA) is thanked for critically reading the manuscript. SLH

thanks Dr J Denecke (Leeds, UK) and L Kriek (Oudenaarde, Belgium) for

continued support and inspiration over the last few years.

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