Annals of Botany 92: 167±180, 2003
doi:10.1093/aob/mcg134, available online at www.aob.oupjournals.org
INVITED REVIEW
Protein Transport in Plant Cells: In and Out of the Golgi²
U L L A N E U M A N N , F E D E R I C A B R A N D I Z Z I and C H R I S H A W E S *
Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane Campus,
Oxford OX3 0BP, UK
Received: 3 March 2003 Returned for revision: 8 April 2003 Accepted: 6 May 2003
In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glycosylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticulum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occurs
via bulk ¯ow. In this Botanical Brie®ng, we review the latest data on ER/Golgi inter-relations and the models
for transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER±
Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass through
the Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternae
from the cis- to the trans-face, a number of membrane proteins reside in the different Golgi cisternae.
Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for the
retention of proteins within the Golgi. In non-dividing cells, protein transport out of the Golgi is either directed
towards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lytic
vacuole and protein storage vacuoles. In general, transport to either of these from the Golgi depends on different
sorting signals and receptors and is mediated by clathrin-coated and dense vesicles, respectively. Being at the
heart of the secretory pathway, the Golgi (transiently) accommodates regulatory proteins of secretion (e.g.
SNAREs and small GTPases), of which many have been cloned in plants over the last decade. In this context,
we present a list of regulatory proteins, along with structural and processing proteins, that have been located to
the Golgi and the `trans-Golgi network' by microscopy.
ã 2003 Annals of Botany Company
Key words: Review, Golgi, endoplasmic reticulum, prevacuolar compartment, vacuole, plasma membrane, protein
transport, protein sorting, vesicles, SNAREs, small GTPases.
INTRODUCTION
In plants, the Golgi apparatus is central to the synthesis of
complex cell wall polysaccharides and of glycolipids for the
plasma membrane, as well as the addition of oligosaccharides to proteins destined to reach the cell wall, plasma
membrane or storage vacuoles. The Golgi apparatus is also
the key organelle in sorting proteins, sending them to their
various destinations within the cell. The majority of these
proteins are imported into the Golgi from the endoplasmic
reticulum (ER), a major organelle of the endomembrane
system involved in the folding, processing, assembly and
storage of proteins, as well as in lipid biosynthesis and
storage (Vitale and Denecke, 1999). The relative importance of the two major Golgi functions in a plant cell, the
assembly and processing of oligo- and polysaccharides on
the one hand and protein sorting on the other, depends on the
cell type and its developmental and physiological state
(Juniper et al., 1982). Nevertheless, the two functions
cannot be regarded as completely unrelated processes;
newly synthesized cell wall polysaccharides have to reach
the correct target destination and must therefore be appropriately sorted.
* For correspondence. Fax +44 1865 483955, e-mail chawes@brookes.
ac.uk
² In memorium of Jean-Claude Roland who, as an expert in plant cell
walls, would always have appreciated the importance of the Golgi
apparatus.
The plant Golgi apparatus shares many features with its
animal counterpart, but also has unique characteristics. The
most important difference concerns its structure. Whereas in
animal cells the Golgi apparatus occupies a rather stationary
perinuclear position, in plant cells the Golgi is divided into
individual Golgi stacks, which are generally considered to
be functionally independent (Staehelin and Moore, 1995)
(Fig. 1). The number of Golgi stacks per cell and the number
of cisternae per stack vary with the species and cell type, but
also re¯ect the physiological conditions, the developmental
stage and the functional requirement of a cell (reviewed by
Staehelin and Moore, 1995; Andreeva et al., 1998b).
Despite these variations, each individual Golgi stack can
be described as a polarized structure with its cisternal
morphology and its enzymatic activities changing gradually
from the ER-adjacent cis-face to the trans-face (Fitchette
et al., 1999). Proteins destined for secretion enter the Golgi
at the cis-face and subsequently move towards the transface where the majority of proteins exit the stack en route to
the plasma membrane or vacuolar system (for exceptions
see below). In between the cisternae of the Golgi stack,
intercisternal elements can be observed, mainly towards the
trans-face (Ritzenthaler et al., 2002) (Fig. 1A). Although no
matrix proteins surrounding the plant Golgi stack have yet
been identi®ed, the existence of a matrix has been predicted
from the appearance on micrographs from ultra-rapidly
frozen root cells of a clear zone, excluding ribosomes,
around each Golgi (Staehelin et al., 1990). The Golgi matrix
Annals of Botany 92/2, ã Annals of Botany Company 2003; all rights reserved
168
Neumann et al. Ð Protein Transport in Plant Cells
F I G . 1. Transmission electron micrographs of Golgi stacks in tobacco (A) and maize roots (B). A, Cross-section of a Golgi stack in a tobacco root cap
cell. High-pressure freezing and freeze-substitution improves the ultrastructural preservation of intercisternal ®laments towards the trans-face of the
Golgi stack (arrows). B, Face view of a Golgi cisternum in a maize root meristematic cell. Zinc iodide and osmium tetroxide impregnation selectively
stains the ER and the Golgi and clearly shows the fenestrated margins of the Golgi cisternum. ER, Endoplasmic reticulum; M, mitochondrion; V,
vesicle. Bars = 200 nm.
has been suggested to play an important role in the
maintenance of stack organization against the shearing
forces during cytoplasmic streaming (Staehelin and Moore,
1995).
Confocal microscopy of Golgi-targeted proteins or
peptides fused to the green ¯uorescent protein (GFP) has
revealed that individual stacks are highly mobile within the
plant cell, moving over the ER on an actin-network (Boevink
et al., 1998; NebenfuÈhr et al., 1999; Brandizzi et al., 2002b)
(Fig. 2); this has resulted in them being christened `stacks on
tracks' (Boevink et al., 1998) or `mobile factories'
(NebenfuÈhr and Staehelin, 2001). The fact that the plant
Golgi apparatus is divided into highly mobile biosynthetic
subunits certainly poses major problems when trying to
elucidate mechanisms for controlled protein import into and
targeted product export out of the stack.
In this Botanical Brie®ng, we summarize recent ®ndings
regarding protein transport from the ER to the Golgi
apparatus and sorting of proteins and membranes as they
exit the Golgi.
E N T E R I N G TH E G O L G I:
IMPORT FROM THE ER
movement is necessary for ER-to-Golgi transport. The
`vacuum cleaner model' (Boevink et al., 1998) suggested
that Golgi stacks move over the surface of the ER picking up
products, similar to a vacuum cleaner picking up dust whilst
moving over a carpet (Fig. 3A). This implies that the whole
surface of the ER is capable of protein export, and that
continued formation of cargo vectors occurs. In contrast, the
hypothesis underlying the `stop-and-go' or `recruitment
model' (NebenfuÈhr et al., 1999) is that Golgi stacks receive
cargo from the ER at de®ned export sites, which produce a
local stop signal that transiently halts stack movement
(Fig. 3B). This model is supported by the observation that
actin-based Golgi movement is not necessary for ER-toGolgi membrane protein transport (Brandizzi et al., 2002b).
Although more attractive, as protein transport out of the ER
would be restricted to con®ned areas, the recruitment model
suggests a rather stationary image of the ER surface. A third
model might therefore propose that protein export from the
ER is restricted to speci®c export sites, which could be
either highly mobile within the ER membrane or mobile due
to the movement of the ER surface (Fig. 3C). Thus, Golgi
stacks and ER export sites may move together in an actindependent fashion, forming discrete `secretory units'
(Brandizzi et al., 2002b).
Towards a more dynamic model of ER-to-Golgi protein
transport
Receptor-mediated protein transport or bulk ¯ow?
The discovery that Golgi stacks move in close association
with the ER and that this movement is actin-dependent
(Boevink et al., 1998) led to the question of whether Golgi
Regardless of the physical model of ER-to-Golgi transport, export from the ER and import into the Golgi are two
intimately related processes. How proteins are sorted and
Neumann et al. Ð Protein Transport in Plant Cells
169
F I G . 2. Confocal laser scanning micrographs showing the spatial relationship between Golgi stacks and ER (A) and between Golgi stacks and actin
®laments (B). A, 3D-reconstruction (Velocityâ) of serial optical sections through the cortical cytoplasm of a tobacco leaf epidermal cell transiently
transformed with a GFP-fusion targeted to the ER (GFP-HDEL in green) and a YFP-fusion labelling the Golgi (ST-YFP in red). Golgi stacks are in
close association with the ER network. B, Optical section through the cortical cytoplasm of a tobacco BY2 cell stably transformed with ST-GFP (in
green). Af®nity labelling of actin by rhodamine-phalloidine (in red) reveals that Golgi stacks are aligned with actin ®laments (arrows). Bars = 10 mm.
concentrated at sites of ER export is therefore a key
question. Are they actively sorted with the help of speci®c
ER export signals linking with differential af®nity to a
common ER export receptor, or is there bulk ¯ow of product
with sorting occurring via retention signals by which
proteins are deviated from the default route at different
levels of the pathway? Support for the second model, at least
for soluble proteins, came from the cloning of an ERD2
homologue from Arabidopsis thaliana (Lee et al., 1993).
This protein acts as a transmembrane receptor that binds to a
speci®c sorting tetrapeptide (H/KDEL) at the carboxyl
terminus of ER-resident proteins in yeast and mammals
(Lewis and Pelham, 1990; Lewis et al., 1990). The
arabidopsis ERD2 is capable of functionally complementing
a yeast null mutant (Lee et al., 1993), and its GFP fusion
locates to the ER and to Golgi stacks (Boevink et al., 1998).
Recent data from quantitative biochemical in vivo assays
measuring ER export of well-characterized cargo molecules
in Nicotiana tabacum protoplasts provided good evidence
for the bulk ¯ow theory (Phillipson et al., 2001).
In vivo observations of membrane protein transport from
ER to Golgi have recently been facilitated by the use of
¯uorescent protein chimeras. Selective photobleaching
experiments using two ¯uorescent marker proteins locating
to the Golgi (ST-YFP and ERD2-GFP) have permitted
confocal imaging of ER-to-Golgi protein transport
(Brandizzi et al., 2002b). Fluorescence recovery in individual Golgi stacks after photobleaching of ST-YFP or ERD2GFP reached 80±90 % of the pre-bleach level after only
5 min in cells treated with latrunculin B alone to disrupt
actin ®laments or in conjunction with colchicine to affect
microtubule integrity. This indicated a rapid exchange with
pools of the fusion protein from other parts of the cell,
independent from an intact actin or microtubule cytoskeleton. The rapid cycling of both fusion proteins was dependent
on energy, as could be shown by loss of ¯uorescence
recovery in cells after ATP depletion.
COPII vesicles vs. direct connections
In animal cells, protein transport between the ER and the
Golgi apparatus occurs through intermediate compartments
known as vesicular±tubular clusters (VTCs) (or the ER±
Golgi intermediate compartment, ERGIC). These compartments represent the ®rst site of segregation of anterograde
(forward from the ER to the Golgi) and retrograde
(backwards from the Golgi to ER) protein transport
(reviewed by Klumperman, 2000). Transport between the
ER and VTCs is supposedly mediated by specialized
protein-coated vesicles, the so-called COPII vesicles.
Cargo packaging and vesicle formation require sequential
binding to the ER membrane of the GTPase Sar1p and two
heterodimeric coat protein complexes, Sec23/24p and
Sec13/31p (reviewed by Barlowe, 2002). Transport between
the VTCs and the Golgi apparatus seems not to be mediated
by distinct vesicles but by the fusion of peripheral VTCs to
form the cis-Golgi cisternae (Klumperman, 2000). The
involvement of another set of coated vesicles (COPI
vesicles formed under the in¯uence of the small GTPase
Arf1p), normally thought to act as retrograde protein
transporters, between the Golgi cisternae and from VTCs
back to the ER (see below), in anterograde protein transport
170
Neumann et al. Ð Protein Transport in Plant Cells
F I G . 3. Models of ER-to-Golgi protein transport. A, The `vacuum cleaner model' (Boevink et al., 1998) suggests that Golgi stacks move over the ER
constantly picking up cargo. According to this model, the whole ER surface is capable of forming export sites, resulting in their random distribution.
In contrast, the `stop-and-go' model (B) hypothesizes that Golgi stacks stop at ®xed ER export sites to take up cargo from the ER, before moving onto
the next stop. In the more dynamic `mobile export sites' model (C), Golgi stacks and ER export sites move together as `secretory units' (Brandizzi
et al., 2002b) allowing cargo to be transported from the ER towards the Golgi at any time during movement.
is still hotly debated (Klumperman, 2000; Spang, 2002).
Finally, delivery of cargo to the Golgi complex involves the
small GTPase Rab1, which controls tethering and fusion
events at the Golgi level (Allan et al., 2000).
In plant cells, ER-to-Golgi protein transport might follow
a simpler route. To date, no equivalent of the VTCs of animal
cells has been identi®ed. On the contrary, recent ®ndings
based on GFP expression and transmission electron microscopy (TEM) indicate that direct connections in the form of
tubular extensions may exist between the ER and the cis-face
of Golgi stacks in tobacco leaf cells (Brandizzi et al., 2002b),
suggesting that vesicles might not mediate protein transport
between the two organelles. At ®rst glance, this ®nding
seems to contradict the fact that components of the COPII
machinery have been identi®ed in plants by EST database
searches (Andreeva et al., 1998a), are associated with the ER
(Bar-Peled and Raikhel, 1997; Movafeghi et al., 1999), and
are necessary for transport of proteins to the Golgi (Andreeva
et al., 2000; Phillipson et al., 2001). This has been interpreted
as evidence that ER±Golgi protein transport shows structural
and functional similarities between animal and plant cells.
However, it is conceivable that COPII components simply
determine the site of formation of ER-to-cis-Golgi-connections, and that vesicle vectors are not a prerequisite of
transport (Hawes et al., 1999). Considering that protein
transport in mammalian cells between the VTCs and the cisGolgi seems to be by fusion of peripheral VTCs to form the
cis-cisternae, and that VTCs have not been identi®ed in plant
cells to date, direct fusion events between the ER and the
Golgi are not implausible.
Similar to its mammalian counterpart, the A. thaliana
small GTPase AtRab1b, seems to be involved in protein
transport at early steps of the secretory pathway. A
dominant-inhibitory mutant of AtRab1b inhibited the
secretion of a ¯uorescent marker protein as well as the
Golgi localization of a Golgi-targeted ¯uorescent marker,
leading to ¯uorescence accumulating in the ER in both cases
(Batoko et al., 2000).
Retention and distribution of proteins in the Golgi
The means by which Golgi-resident proteins are retained
in the stack is still largely a matter of debate. Whilst many of
the ER-resident processing proteins are soluble, Golgi-
Neumann et al. Ð Protein Transport in Plant Cells
171
F I G . 4. Confocal laser scanning micrographs showing the location of different regulatory and structural proteins of the secretory pathway in relation to
Golgi stacks. A, Optical section through the cortical cytoplasm of a tobacco BY2 cell stably transformed with the Golgi marker GmMan1-GFP
(Glycine max a-mannosidase 1-GFP, green channel) after ®xation and immunolocalization with anti-AtArf1p antibodies (red channel). The merged
image clearly reveals that anti-AtArf1p labelling is associated with the Golgi, forming a ring-shaped pattern con®ned to the periphery of each stack
(Ritzenthaler et al., 2002). B, Optical section through the centre of a transgenic GmMan1-GFP BY2 cell after ®xation (GFP signal in green channel)
and immunolocalization with anti-Atg-COP antibodies (red channel). As can be seen in the merged image, the COPI coatomer subunit co-localizes
with Golgi-associated GFP-¯uorescence. As for Arf1p, anti-Atg-COP ¯uorescence is restricted to the margins of the Golgi stacks (Ritzenthaler et al.,
2002). C, Projection of 15 optical sections (1 mm per section) through a pea root tip cell after ®xation and double-immunolabelling with anti-VSR
antibodies (17F9, green channel) and JIM 84, a trans-Golgi marker (red channel). As shown in the merged image, more than 90 % of the prevacuolar
organelles labelled by 17F9 are separate from Golgi stacks (Li et al., 2002). Occasionally, ¯uorescence labelling by the two antibodies co-localizes
(merged image, open arrow). D, Optical section through the cortical cytoplasm of a leaf epidermal cell of a transgenic ST-GFP tobacco plant (GFP
signal in green channel) transiently transformed with YFP-AtRab2a (YFP signal in red channel). As can be seen in the merged image, both ¯uorescent
fusion proteins co-localize in Golgi stacks. In addition to the Golgi, YFP-AtRab2a labels small spherical structures (sometimes measuring up to 3 mm
in diameter) in which no GFP signal can be detected (merged image, arrows). Bars = 5 mm (A±C, insert D) and 20 mm (D). Micrographs for A and B
kindly provided by Christophe Ritzenthaler and that for C by Liwen Jiang.
Nicotiana tabacum BY2
cells stably transformed
with a GFP-fusion of
AtRER1B (full length)
Pisum sativum root tips
Arabidopsis thaliana roots
Arabidopsis thaliana and
Zea mays roots
Nicotiana tabacum BY2
cells
Nicotiana tabacum pollen
tubes transiently and stably
transformed with a
GFP-fusion of NtRab2
(full length)
Nicotiana tabacum leaves
transiently transformed with
a GFP-fusion of AtRab2a
(full length)
Nicotiana tabacum BY2
cells stably transformed
with a GFP-fusion of Pra2
and Pra3 (full length)
Nicotiana tabacum pollen
tubes transformed with a
GFP-fusion of AtRac7
(full length)
BP-80 (also called VSRPS-1)
(Pisum sativum)
AtELP (Arabidopsis thaliana)
AtArf1p (Arabidopsis thaliana)
Rho-like protein (N/A)
NtRab2 (Nicotiana tabacum)
AtRab2a (Arabidopsis thaliana)
Pra2, Pra3 (Pisum sativum)
AtRac7 (Arabidopsis thaliana)
Nicotiana clevelandii
leaves transiently
transformed with a
GFP-fusion of ERD2
(full length)
Experimental system
AtRER1B (Arabidopsis thaliana)
Regulatory proteins
ERD2 (Arabidopsis thaliana)
Protein (species of origin)
Golgi
Golgi
Rims of Golgi
cisternae and
COPI vesicles
Golgi, `transGolgi network'.
PVC
GFP imaging
GFP imaging
Golgi, plasma
membrane
(not at the
pollen tube tip)
Pra2: Golgi
and `endosomes';
Pra3: `TGN'
and/or PVC
GFP imaging, TEM
Golgi, cytosol
immunogold labelling
and small
with anti-GFP antibodies spherical bodies
GFP imaging, TEM
Golgi stack
immunogold labelling
periphery
with anti-GFP antibodies
Immuno¯uorescence
using anti-human
Rac1 antibodies
TEM immunogold
labelling with antiAtArf1p antibodies
TEM immunogold
labelling with antiAtELP antibodies
Immuno¯uorescence and Golgi, PVC
TEM immunogold
labelling using antiVSRPS-1 antibodies
GFP imaging
GFP imaging, TEM
ER, all Golgi
immunogold labelling
cisternae
with anti-GFP antibodies
Microscopical technique Localization
Boevink et al. (1998)
Rac-like GTPase
Rab GTPases (Rab11
homologues)
Small GTPase involved
in post-Golgi traf®cking
Small GTPase involved
in ER-to-Golgi traf®c
Small GTPase involved
in regulating the
cytoskeleton organization
Small GTPase
involved in COPI
vesicle formation
Sorting receptor for
the lytic vacuole
Sorting receptor for
the lytic vacuole
Cheung et al. (2003)
Inaba et al. (2002)*
U. Neumann, I, Moore, C. Hawes and H. Batoko
(unpubl. res.)
Cheung et al. (2002)
Couchy et al. (1998)*
Pimpl et al. (2000)
Sanderfoot et al. (1998)*
Paris et al. (1997)*
Recycling receptor
Takeuchi et al. (2000)
for membrane-bound ER proteins
H/KDEL receptor for
retrieval of escaped
soluble ER-resident proteins
Putative function
Original reference
(providing location
data)
Table 1. Examples of proteins microscopically located to the Golgi and the `trans-Golgi network' in cells of higher plants
172
Neumann et al. Ð Protein Transport in Plant Cells
Lilium davidii, pollen
and pollen tubes
a-actinin-like protein (N/A)
Arabidopsis thaliana roots:
wild type or from plants
transformed with HA-tagged
AtTLG2a and T7-tagged
AtTLG2b
Arabidopsis thaliana roots
from plants stably
transformed with T7tagged AtVTI1a
PM and adjacent
vesicles, vesicles
of the `TGN',
PCR
`Trans-Golgi
network', dense
vesicles, PVC
Rims of Golgi
cisternae and
COPI vesicles
AtSYP51:
`TGN', PVC;
AtSYP61: `TGN'
TEM immunogold
`Trans-Golgi
labelling using antinetwork'
AtVPS45, antiAtTLG2a, anti-HA
and/or anti-T7 antibodies
TEM immunogold
labelling using
anti-T7 antibodies
TEM immunogold
labelling using
antibodies raised
against the different
COP components
Electron microscopy
Golgi, partially
(identi®cation of clathrin coated reticulum,
interlocking triskelions) multivesicular
bodies, plasma
membrane,
clathrin-coated
vesicles
Immuno¯uorescence,
Membranes of
TEM immunogold
Golgi-associated
labelling using
vesicles
commercially available
anti-a-actinin antibodies
TEM immunogold
labelling with antiAtSH3P1 antibodies
Immuno¯uorescence
Golgi
using anti-ADL6
antibodies (root tips);
GFP imaging (protoplasts)
Microscopical technique Localization
AtSYP51, AtSYP61 (Arabidopsis Arabidopsis thaliana roots:
TEM immunogold
thaliana)
wild type or from plants
labelling
transformed with HA-AtSYP41
and T7-AtSYP42
AtVPS45, AtTLG2a, -b (now
AtSYP41, AtSYP42)
(Arabidopsis thaliana)
SNAREs
AtVTI1a (now AtVTI11)
(Arabidopsis thaliana)
Atg-COP (Arabidopsis thaliana) Arabidopsis thaliana and
Zmd-COP, Zme-COP (Zea mays) Zea mays roots
N/A
Arabidopsis thaliana
pollen grains
AtSH3P1 (Arabidopsis thaliana)
Structural proteins
Clathrin (N/A)
Arabidopsis thaliana root
tips; A. thaliana protoplasts
transformed with a
GFP-fusion of ADL6
(full length)
Experimental system
ADL6 (Arabidopsis thaliana)
Protein (species of origin)
TA B L E 1. Continued
Syntaxins (SNAREs)
SNAREs
SNARE involved in
protein transport from
the Golgi to the PVC
Coatomer subunit of
COPI vesicles
Coat protein of clathrin
coated vesicles
Budding and sorting of
Golgi-associated vesicles
Protein involved in the
®ssion and uncoating of
clathrin-coated vesicles
Dynamin-like protein
involved in vesicle
formation for vacuolar
traf®c at the `TGN'
Putative function
Sanderfoot et al. (2001)*
Bassham et al. (2000)*
Zheng et al. (1999)*
Pimpl et al. (2000)
for original references
see Coleman et al. (1988)*
Li and Yen (2001)*
Lam et al. (2001)*
Jin et al. (2001)*
Original reference
(providing location
data)
Neumann et al. Ð Protein Transport in Plant Cells
173
Golgi
Nicotiana benthamiana leaves GFP imaging
transiently transformed with
GFP-fusions of different b
1,2-xylosyltransferase domains
(CTS, CT, T, C)
Phaseolus vulgaris hypocotyl
b 1,2-xylosyltransferase
(Arabidopsis thaliana)
Xylan synthase (Phaseolus
vulgaris)
TEM immunogold
labelling with
anti-bean xylan
synthase antibodies
Onion epidermal cells transientlyYFP imaging
transformed with a YFP-fusion
of GONST1 (full length)
GONST1 (Arabidopsis thaliana)
Glycosyltransferase
Nucleotide sugar
(GDP-mannose) transporter
N-linked oligosaccharide
processing enzyme
Glycosyltransferase
Glycosyltransferase
Glycosyltransferase
Syntaxin (SNARE)
Putative function
Golgi and post-Golgi Synthesis of secondary
vesicles of developing wall xylan
xylem cells
Golgi
Golgi
GFP imaging, TEM
Cis-face of
immunogold labelling
the Golgi
with anti-GFP antibodies
Nicotiana tabacum BY2 cells
stably transformed with a
GFP-fusion of mannosidase I
(deletion of C-terminal
11 amino acids)
Golgi
Trans-face
of the Golgi
a-1,2 mannosidase I soybean
(Glycine max)
Immuno¯uorescence
and TEM immunogold
labelling using 9E10
and A14 anti-Myc
antibodies
GFP imaging, TEM
Trans-face
immunogold labelling
of the Golgi
with anti-GFP antibodies
GFP imaging
Microscopical technique Localization
GFP imaging
Arabisopsis thaliana
(roots and callus) stably
transformed with Myc-tagged
sialyl transferase (full length)
Nicotiana clevelandii leaves
transiently transformed
with a GFP-fusion of the
52 N-terminal amino acids
of sialyl transferase
Nicotiana tabacum BY2
cells transformed with a
GFP-fusion of AtSed5
(full length)
Experimental system
N-acetylglucoso-aminyltransferase INicotiana benthamiana leaves
(GnTI) (Nicotiana tabacum)
transiently transformed with
a GFP-fusion of the GnTI
cytoplasmic transmembrane
stem (CTS) domain
a-2,6-sialyl transferase (rat)
Processing proteins
a-2,6-sialyl transferase (rat)
AtSed5 (now called AtSYP31)
(Arabidopsis thaliana)
Protein (species of origin)
TA B L E 1. Continued
Gregory et al. (2002)*
Dirnberger et al. (2002)
Baldwin et al. (2001)*
NebenfuÈhr et al. (1999)
Essl et al. (1999)
Wee et al. (1998)*
Boevink et al. (1998)
Takeuchi et al. (2002)
Original reference
(providing location
data)
174
Neumann et al. Ð Protein Transport in Plant Cells
Phaseolus vulgaris hypocotyls
and root tips
Nicotiana tabacum leaves
transiently transformed with
a GFP-fusion of galactosyl
transferase (60 C-terminal
amino acids)
Root protoplasts of
Arabidopsis thaliana plants
stably transformed with a
GFP-fusion of MUR4
(full length)
b 1,3-glucan (callose) synthase
(Phaseolus vulgaris)
b-1,4-galactosyl transferase
(human)
MUR4 (Arabidopsis thaliana)
GFP imaging
GFP imaging
TEM immunogold
labelling with
anti-b 1,3-glucan
synthase antibodies
Putative function
Golgi
ER and Golgi
UDP-D-Xyl 4-epimerase
Glycosyltransferase
Golgi of root tip
Callose synthesis
meristematic cells
during cell plate
formation, surface
of secondary wall
thickenings and PM in
pits in developing
xylem cells
Microscopical technique Localization
Burget et al. (2003)*
Saint-Jore et al. (2002)
Gregory et al. (2002)*
Original reference
(providing location
data)
* Literature cited in this table only, not in the main text:
Baldwin TC, Handford MG, Yuseff MI, Orellana A, Dupree P. 2001. Identi®cation and characterization of GONST1, a Golgi-localized GDP-mannose transporter in Arabidopsis. The Plant Cell 13:
2283±2295.
Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV. 2000. AtVPS45 complex formation at the trans-Golgi network. Molecular Biology of the Cell 11: 2251±2265.
Burget EG, Verma R, Mùlhùj M, Reiter W-D. 2003. The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the
MUR4 gene of Arabidopsis. The Plant Cell 15: 523±531.
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Experimental system
Protein (species of origin)
TA B L E 1. Continued
Neumann et al. Ð Protein Transport in Plant Cells
175
176
Neumann et al. Ð Protein Transport in Plant Cells
resident enzymes are transmembrane proteins (Table 1).
Information regarding the retention of membrane proteins at
various points of the secretory pathway has recently been
revealed by a study investigating the default pathway of type
I membrane-bound proteins (Brandizzi et al., 2002c). In this
study, GFP was fused to proteins with transmembrane
domains of variable length and the fusion proteins were
shown to be distributed along the organelles of the secretory
pathway in a transmembrane length-dependent manner. As
for animal cells (Munro, 1995), it is expected that the
membrane thickness increases in plant cells from the ER to
the Golgi and ®nally to the plasma membrane/tonoplast
owing to a sterol gradient in the lipid composition (Moreau
et al., 1998). Accumulation in the Golgi occurred when GFP
was fused to transmembrane domains of 19 or 20 amino
acids (Brandizzi et al., 2002c). Whether this retention
mechanism is universally valid for true Golgi residents
remains to be elucidated, although the signal anchor
sequence of a rat sialyl transferase targets GFP to the plant
Golgi (Boevink et al., 1998; Saint-Jore et al., 2002), and
equivalent sequences of plant glycosyltransferases give the
same result (Essl et al., 1999; Dirnberger et al., 2002; Pagny
et al., 2003). Phe residues, suggested to play a role in Golgi
retention in mammalian cells, only seem to play a minor role
in plants, as indicated by comparison of the number and
position of Phe residues of the transmembrane domain of
proteins within the BP80 family (Brandizzi et al., 2002c).
As mentioned earlier, enzymatic activities change gradually from the cis-face to the trans-face of the Golgi stack,
re¯ecting a sequential processing down the stack. Therefore,
a logical question is what controls the distribution of resident
enzymes across the different cisternae. This has only been
addressed experimentally a couple of times in plant cells
(Fitchette et al., 1999), and models were ®rst formulated for
mammalian Golgi enzymes. Again, the bilayer/membrane
thickness model provides one possible explanation.
Alternatively, as in mammalian cells, retrograde transport
of processing machinery in vesicle vectors may both reduce
loss and control positioning of enzymes whilst cisternae
continually mature from the cis- to the trans-face (Opat et al.,
2001). More evidence is needed before ruling out one or
other model for plant Golgi enzymes.
Intra-Golgi transport and retrograde transport (COPI
machinery)
The fact that each Golgi stack is divided into a number of
cisternae leads to the question of how cargo is transported
down the stack. In analogy to mammalian cells, two models
have been proposed, the `vesicle shuttle' and the `cisternal
maturation' models (reviewed by Hawes and SatiatJeunemaitre, 1996; NebenfuÈhr and Staehelin, 2001).
According to the vesicle shuttle model, Golgi cisternae are
stable entities with a speci®c set of processing proteins, and
cargo is sequentially transported down the stack in vesicular
shuttles. The second model suggests that cisternae progressively move down the stack and mature from the cis-face to
the trans-face of the Golgi. New cis-cisternae are formed by
fusion of ER-to-Golgi transport intermediates and retrograde transport vesicles, which shuttle back the processing
enzymes from the trans-cisternum. Experimental evidence
exists for both models and it cannot be ruled out that either
mechanism or a mixture of both is active. In addition to the
algal scale argument (Becker et al., 1995), recent data on the
ultrastructural morphology of Golgi stacks in BY2 cells and
the redistribution of a ¯uorescent cis-Golgi marker protein
after brefeldin A treatment (a fungal toxin widely used to
study protein transport along the secretory pathway;
NebenfuÈhr et al., 2002) seem to favour the cisternal
maturation model (Ritzenthaler et al., 2002). Finally, it
cannot be excluded that cisternae of the stack are all joined
by interconnecting tubules, necessitating a different model
to explain the regulation of cis-to-trans-transport.
In addition to anterograde protein transport from the ER
to the Golgi and down the Golgi stack, retrograde protein
transport occurs between the Golgi and the ER (e.g.
transport of escaped ER residents) as well as inside the
Golgi stack, from the trans- towards the cis-face. In
mammalian cells, retrograde transport is likely to be
mediated by COPI vesicles and regulated by the Arf1p
GTPase (Spang, 2002). Components of the COPI machinery
have been located to the Golgi by means of immuno¯uorescence (Ritzenthaler et al., 2002) (Fig. 4A and B). More
precisely, a study combining biochemistry with cryosection immunogold labelling techniques, revealed that in
plant cells, components of the COPI coat as well as Arf1p
mainly locate to the cis-half of Golgi stacks and that COPI
coat proteins are present on small vesicles budding off from
cis-cisternae (Pimpl et al., 2000). In addition, in vitro COPI
vesicle induction from ER/Golgi membranes of transgenic
tobacco plants overproducing the soluble secretory marker
a-amylase fused to HDEL (the C-terminal sorting peptide
of ER residents), showed that COPI vesicles contained the
modi®ed secretory marker as well as the ER-resident
calreticulin. This was the ®rst indication in plants that
COPI vesicles might be involved in retrograde transport
from the Golgi (Pimpl et al., 2000). The involvement of
AtArf1 in this particular transport event was also deduced
from the effect of two dominant negative mutant forms of
AtArf1 on the distribution of three ¯uorescent Golgi
markers (Takeuchi et al., 2002). To date, the demonstration
of the role of COPI vesicles in intra-Golgi retrograde
transport remains a challenge, mainly owing to the lack of
marker molecules for this speci®c transport event.
Recycling through the Golgi: protein import from
destinations other than the ER
During secretion, vesicle-derived membrane is continuously added to the plasma membrane. To balance this
increase in membrane surface area, clathrin-coated endocytic vesicles pinch off from the plasma membrane towards
the inside of the cell (reviewed by Holstein, 2002). The
different endocytic compartments in plant cells are still not
fully characterized. In analogy to mammalian cells, the term
`endosome' is often found in the plant literature indicating a
compartment containing endocytosed material (JuÈrgens and
Geldner, 2002). Ultrastructurally, the `plant endosome'
most likely corresponds to the partially coated reticulum
(PCR), a compartment that may originate from the `trans-
Neumann et al. Ð Protein Transport in Plant Cells
Golgi network' (Hillmer et al., 1988). From this compartment, protein might be recycled towards the Golgi or
transported towards the lytic vacuole via multi-vesicular
bodies (MVBs) for degradation. This was shown by elegant
time-course studies following the uptake of cationized
ferritin (CF) in soybean protoplasts (Fowke et al., 1991),
re¯ecting vesicle-mediated plasma membrane recycling
(and not receptor-mediated endocytosis). This marker is
quickly endocytosed through coated pits into coated
vesicles. Within the cytoplasm, CF sequentially labelled
the following organelles: tubular elements of the PCR;
periphery of Golgi cisternae; MVBs; and ®nally the central
vacuole. Labelling of the Golgi by CF almost certainly
re¯ects membrane recycling from the plasma membrane,
presumably through the PCR (Fowke et al., 1991). Whether
direct plasma membrane recycling to the Golgi can occur in
plant cells still has to be elucidated.
In addition to maintaining the plasma membrane surface,
protein import into the Golgi through the `plant endosome'
could also be important for regulating, by endocytosis at the
plasma membrane, the number of ion channels, proton
pumps (Crooks et al., 1999) and carrier proteins, such as
PIN1 (auxin ef¯ux carrier; see Geldner et al., 2001).
Remodelling the distribution of such proteins at the plasma
membrane via vesicle traf®cking must certainly be regarded
as an important way in which a plant can react to changes in
environmental conditions (Levine, 2002).
OUT OF T HE GOL GI
The major destinations for proteins exiting the Golgi
apparatus are the plasma membrane and the vacuolar
system. During cytokinesis, another protein traf®cking
route leads towards the developing cell plate (reviewed by
Bednarek and Falbel, 2002). Here we focus on how sorting
of proteins to the plasma membrane or the vacuolar system
occurs and where this sorting takes place.
As to the place of sorting, the term `trans-Golgi network'
(TGN) from the mammalian `endomembrane nomenclature' is becoming more common in the plant literature.
However, in plant cells, no discrete protein-sorting compartment with a characteristic set of proteins, downstream
from the trans-face of the Golgi has yet been described. In
addition, in plants, protein sorting and secretory vesicle
production can take place as early as at the cis-cisternae of
the Golgi stack (see below). Therefore, it might be better to
suggest that production of clathrin-coated vesicles destined
for the lytic vacuolar system is limited to the trans-face of
the Golgi. Whether this face of the Golgi, which certainly
can comprise a network of tubules and vesicles, is
homologous to the mammalian TGN is open for debate.
Secretion: transport towards the plasma membrane and the
cell wall
The default destination of soluble proteins and complex
carbohydrates has been suggested to be the plasma membrane (Denecke et al., 1990). Secretory proteins usually
carry an amino terminal signal peptide for insertion into the
ER, which is clipped off upon translocation across the ER
177
membrane (Vitale and Denecke, 1999). Soluble secretory
proteins are not known to carry any positive targeting
information, which would divert them from the default
pathway to the plasma membrane, either to the vacuolar
system or back to the ER (Hadlington and Denecke, 2000).
In contrast, the situation with membrane proteins is less
clear. Until recently, the tonoplast had been regarded as the
default destination (Barrieu and Chrispeels, 1999).
However, a VSR-based (vacuolar sorting receptor) GFPfusion with a lengthened transmembrane domain accumulated on the plasma membrane, indicating that positive
sorting information might be necessary to direct membrane
proteins from the Golgi towards the tonoplast (Brandizzi
et al., 2002c). Nevertheless, transport of proteins to speci®c
areas of the plasma membrane, nicely illustrated by proteins
such as the auxin ef¯ux carrier PIN1, which locates to the
distal part of the plasma membrane in root cells of
arabidopsis seedlings (Geldner et al., 2001; JuÈrgens and
Geldner, 2002), is dif®cult to imagine without some form of
either positive targeting or retention mechanism.
Transport towards the vacuolar system: different types of
vacuoles, vesicles, sorting signals, receptors and sorting
sites
As mentioned earlier, sorting signals are necessary for
transport to the vacuolar system. In plant cells, two main
types of vacuoles [distinguishable by different sets of TIPs
(tonoplast intrinsic proteins) and lumenal contents] may coexist, and sorting towards either of them depends on
different peptide targeting signals and is mediated by
different sets of transport vesicles (Paris et al., 1996; Jiang
and Rogers, 1998; Hinz et al., 1999).
Transport to the lytic vacuole, characterized by g-TIPs,
occurs via an intermediate compartment known as the
prevacuolar compartment (PVC) and relies on aminoterminal, sequence-speci®c propeptides (NPIR or equivalent), which are recognized by VSRs. The ®rst VSR to be
identi®ed was BP80 from Pisum sativum (now called
VSRPS-1; reviewed by Paris and Neuhaus, 2002). VCRs are
thought to cycle between the Golgi apparatus and the PVC
(Mitsuhashi et al., 2000) where they are preferentially
located, as was shown by confocal immuno¯uorescence for
several VSRPS-1 homologues (Li et al., 2002) (Fig. 4C). It is
thought that VSRs mediate the packaging of cargo destined
to the lytic vacuole in trans-Golgi-located clathrin-coated
vesicles (Hinz et al., 1999).
In contrast, proteins delivered to the protein storage
vacuole (a-TIP vacuoles) reach their destination in vesicles
apparently devoid of any speci®c protein coating and, in
legume seeds, have been termed dense vesicles (Hohl et al.,
1996). Sorting relies on a carboxyl-terminal propeptide and
an as yet unidenti®ed sorting receptor, the existence of
which is deduced from the observation that transport
towards the storage vacuole can be saturated (Frigerio
et al., 1998). Immunogold labelling indicates that sorting of
some storage proteins can occur as early as the cis-cisternae
of the Golgi in pea cotyledons (Hillmer et al., 2001). Other
storage vacuole proteins as well as integral membrane
proteins are sorted even earlier, at the ER level, where they
178
Neumann et al. Ð Protein Transport in Plant Cells
are packed into so-called precursor-accumulating (PAC)
vesicles and transported to the storage vacuole via a route
bypassing the Golgi (Mitsuhashi et al., 2001). A vacuolar
sorting receptor for this pathway has been identi®ed in
pumpkin seeds (Shimada et al., 2002).
REGULATORY PROTEINS AT THE HEART
OF THE SECRETORY PATHWAY
In recent years, it has become apparent that the different
transport events along the secretory/biosynthetic and
endocytic pathway are orchestrated by a plethora of
proteins. The functional importance of the plant Golgi
apparatus in the secretory pathway is re¯ected by the fact
that numerous regulatory proteins are structurally and
functionally linked to the Golgi and the `trans-Golgi
network' (Hawes et al., 1999; Sanderfoot and Raikhel,
1999; NebenfuÈhr, 2002; Rutherford and Moore, 2002; see
Table 1). For example, small GTPases act as molecular
switches involved in the formation, transport and fusion of
transport vesicles, while SNAREs are integral membrane
proteins involved in determining the speci®city of fusion
events along the endomembrane system, residing on
transport vesicles (R-SNAREs) and target membranes (QSNAREs). Considerable effort has been put into comparing
various genomes to identify plant homologues of proteins
that have been shown to regulate various traf®cking events
along the secretory pathway in yeast and animals (Andreeva
et al., 1998a; Sanderfoot et al., 2000; JuÈrgens and Geldner,
2002). Statements such as `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' (JuÈrgens and Geldner,
2002) may be correct as to the functional aspect. However,
even though homologous Rab proteins may show the same
subcellular location, it cannot be necessarily concluded that
they exert the same function. For instance, despite the
Golgi-location of two mammalian splice-variants of Rab6,
which differ only in three amino acid residues, Rab6A and
Rab6A¢ seem to function in different membrane-traf®cking
events (Echard et al., 2000). Likewise, two plant Rab2
isoforms have been found to locate to Golgi stacks (Fig. 4D)
but seem to regulate quite different transport steps. In
tobacco pollen tubes, NtRab2 seems to sustain ER-to-Golgi
traf®c (Cheung et al., 2002), while studies conducted in our
group seem to indicate that an arabidopsis Rab2 isoform
regulates vesicle traf®c between the Golgi and a post-Golgi
compartment (U. Neumann, I. Moore, C. Hawes and H.
Batoko, unpubl. res.).
It is outside the scope of this current Brie®ng to consider
the putative roles of the various small GTPases and
SNAREs that have been identi®ed in plants. A summary
of the major regulatory proteins locating to the plant Golgi
identi®ed to date is given in Table 1.
CONCLUSIONS
Immense progress has been made in recent years to
elucidate the various transport events along the secretory
and endocytic pathways in eukaryotic cells. This is certainly
true for the Golgi apparatus, which has seen a renaissance in
research popularity since the 100th anniversary of its
discovery by Camillo Golgi in 1898. New data regarding
the molecular machinery that drives and regulates traf®cking events at the Golgi level are published almost weekly. In
this context, comparative genomic analyses have helped to
identify plant homologues of yeast and animal proteins
regulating Golgi-related transport steps. One of the main
challenges of the post-genomic era is to provide functional
and structural evidence for the speci®c roles of plant
proteins putatively playing a role in the secretory/endocytic
pathway. As to structural data at the subcellular level,
technological progress in light microscopy such as confocal
microscopy and deconvolution technology for improving
images, combined with developments in immunolabelling
and ¯uorescent protein technology have revolutionized the
study of cell biology (Brandizzi et al., 2002a). However,
especially with regard to the Golgi apparatus, confocal
microscopy combined with GFP technology in order to
locate proteins is not without its pitfalls. It becomes more
and more common to assume that a punctate distribution of
GFP ¯uorescence in the cytoplasm is suf®cient to establish
that a speci®c protein is located to the Golgi without
additional con®rmatory evidence such as co-localization
with a known Golgi marker at the light microscopical level
(e.g. a ¯uorescent protein marker or by immunocytochemical labelling with `anti-Golgi' antibodies) or immunogold
labelling at the TEM level. Cryotechniques for specimen
preparation, such as ultra-rapid freezing at ambient or high
pressure and freeze-substitution, allow for excellent ultrastructural preservation, but even without these more
sophisticated microscopical techniques, immunogold labelling at the TEM level is an excellent way to localize proteins
at the subcellular level.
Despite the progress made in recent years regarding the
functioning of the secretory/endocytic pathway in general
and the plant Golgi apparatus in particular, many aspects
still remain to be discovered. The necessary advances will
be made only if we are able to link molecular with structural
and functional data.
ACKNOWLEDGEMENTS
We thank David Evans for critical reading of the manuscript, Barry Martin for skilful assistance with high-pressure
freezing and Liwen Jiang and Christophe Ritzenthaler for
kindly providing micrographs. We acknowledge the
Biotechnology and Biological Sciences Research Council,
UK, for supporting the work undertaken in our laboratory.
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