In tobacco leaf epidermal cells, the integrity of protein export from

In tobacco leaf epidermal cells, the integrity of protein exportfrom the endoplasmic reticulum and of ER export sitesdepends on active COPI machinery

Giovanni Stefano1,†, Luciana Renna1,†, Laurent Chatre2,†,‡, Sally L. Hanton1, Patrick Moreau2, Chris Hawes3 and

Federica Brandizzi1,*

1Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada,2Laboratoire de Biogenese Membranaire, UMR5200, CNRS, Universite de Bordeaux 2, Bordeaux, France, and3Research School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, UK

Received 5 October 2005; revised 2 December 2005; accepted 14 December 2005.*For correspondence (fax þ1 306 966 4461; e-mail [email protected]).†These authors have contributed equally.‡Present address: Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada.

Summary

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on

coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and

dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage

between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences

COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the

dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching

analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which

COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically

associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export

sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional

structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also

has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport

is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for

ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the

ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the

balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for

forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of

the ER export domains formed by the Sar1-COPII system.

Keywords: COPI, anterograde transport, endoplasmic reticulum export sites.

Introduction

In plant cells, proteins destined for the early secretory

pathway attain a steady-state localization by cycling be-

tween the endoplasmic reticulum (ER) and the Golgi

apparatus (Brandizzi et al., 2002; Denecke et al., 1992; Han-

ton et al., 2005). Our understanding of the mechanisms

regulating protein cycling at the ER/Golgi interface in plants

is limited. Emerging evidence indicates that anterograde ER/

Golgi protein transport is mediated by COPII vectors (An-

dreeva et al., 2000; Bar-Peled and Raikhel, 1997; Phillipson

et al., 2001; Ritzenthaler et al., 2002; daSilva et al., 2004;

Takeuchi et al., 2000; Yang et al., 2005), although their

morphology is still undetermined. It is assumed that retro-

grade Golgi/ER protein transport is mediated by COPI

machinery. The existence of COPI components such as

coatomer and the GTPase Arf1, which is responsible for

coatomer recruitment (Teal et al., 1994), has been proved in

plants (Contreras et al., 2000; Couchy et al., 2003; Movafeghi

et al., 1999; Pimpl et al., 2000; Takeuchi et al., 2002), and

ª 2006 The Authors 95Journal compilation ª 2006 Blackwell Publishing Ltd

The Plant Journal (2006) 46, 95–110 doi: 10.1111/j.1365-313X.2006.02675.x

COPI-coated vesicles have been observed predominantly at

cis and medial Golgi cisternae (Pimpl et al., 2000). However,

a full characterization of the dynamics of COPI at the Golgi

apparatus in living plant cells has yet to emerge.

It has been shown that Arf1 localizes at the Golgi

apparatus (Takeuchi et al., 2002) with the coatomer (Mova-

feghi et al., 1999; Pimpl et al., 2000; Ritzenthaler et al., 2002).

However, a distribution of Arf1 on non-Golgi structures has

also been verified (Xu and Scheres, 2005), and it has been

shown that Arf1 has a role in the sequence-specific vacuolar

sorting route to the lytic vacuole in tobacco (Pimpl et al.,

2003). This raises the question of whether Arf1 requires

coatomer at other organelles besides the Golgi apparatus, or

whether Arf1 interacts with different proteins in its non-

Golgi locations.

Mutations that hamper the GTPase activity of Arf1 have

been used to manipulate anterograde ER to Golgi protein

transport (Lee et al., 2002; Pimpl et al., 2003; Ritzenthaler

et al., 2002; Saint-Jore et al., 2002; daSilva et al., 2004;

Takeuchi et al., 2002). Dominant negative mutants of Arf1

that are impaired in GTP/GDP exchange affect the secretion

of soluble markers (Pimpl et al., 2003), and, along with

brefeldin A (BFA)-induced inhibition of Arf1, cause a re-

absorbance of Golgi membrane proteins into the ER (Bran-

dizzi et al., 2002; Lee et al., 2002; Ritzenthaler et al., 2002;

Saint-Jore et al., 2002; daSilva et al., 2004; Takeuchi et al.,

2000). The information available on the mechanisms by

which COPI may regulate COPII-mediated protein export

from the ER in plant cells is in general speculative, based on

studies in vitro and in non-plant systems (Hanton et al.,

2005). It remains to be shown in vivo how perturbation of

COPI-mediated traffic affects protein export from the ER in

plant cells.

Protein export from the ER occurs at the ER export sites

(ERES), the subcellular distribution of which has been

recently investigated in plants (daSilva et al., 2004; Yang

et al., 2005). It has been demonstrated that ERES differen-

tiation can be induced upon co-expression of the COPII

initiator Sar1 and Golgi membrane markers in tobacco leaf

epidermal cells. In this system, ERES tracked with the

Golgi bodies in close proximity (daSilva et al., 2004). It has

also been shown that a fluorescent protein fusion of a

COPII component, Sec13, labelled stationary punctate

structures on the surface of the ER in the absence of

Golgi markers in tobacco BY-2 cells (Yang et al., 2005).

These structures considerably outnumbered the Golgi

stacks, although some were seen to associate with the

rims of Golgi stacks (Yang et al., 2005). These data suggest

that the Golgi apparatus is not continually linked to a

single ERES. On the basis of these observations, it cannot

be excluded that in tobacco leaf epidermal cells COPII coat

markers may have a different distribution in comparison

to cargo-induced Sar1 punctae. Therefore, an examination

of COPII coat distribution is needed to determine

unambiguously the distribution of ERES in tobacco leaf

epidermal cells.

The drug BFA, which blocks the activation of Arf1 (Robi-

neau et al., 2000), prevents cargo-induced recruitment of a

fluorescent fusion of Sar1 at the ERES in tobacco leaf

epidermal cells (daSilva et al., 2004). In contrast, BFA does

not have an effect on the distribution of ERES labelled by

Sec13-GFP in tobacco BY-2 cells (Yang et al., 2005). Thus, it

is important to define whether the integrity of ERES depends

on active COPI machinery in the absence of cargo-induced

ERES formation in order to complete the characterization of

the requirements for differentiation of ERES in tobacco leaf

epidermal cells.

Here, we have analyzed the dynamic distribution of COPI

and COPII machineries in living tobacco leaf epidermal cells.

In particular, we focused on the manipulation of Arf1-

mediated protein transport to address the relevance of an

intact COPI machinery in control of the integrity of ER export

sites and of efficient ER protein export in vivo. Our results

indicate that Arf1 and coatomer are dynamically associated

with Golgi bodies but that Arf1 also binds to Golgi-derived

structures that lack coatomer, and that COPII components,

such as Sec23 and Sec24, localize at punctate structures that

track with Golgi stacks. We also found that blockage of Arf1

activity results in either inhibition or retardation of the

assembly of COPI machinery, in disruption of the integrity of

the ER export sites, and in the collapse of protein export

from the ER. Thus, our data suggest that the Arf1–coatomer

system is required for forward protein trafficking out of the

ER due to its role in recycling essential components to the ER

that are needed to differentiate ER export domains formed

by the COPII system.

Results

Arf1 localizes at the Golgi apparatus and Golgi-derived

structures

To determine the distribution of Arf1 in tobacco leaf epi-

dermal cells, a green fluorescent fusion (GFP) of an Ara-

bidopsis Arf1 (Phillipson et al., 2001; Arf1-GFP) was used

for confocal microscopy analysis. Arf1-GFP was distributed

on punctate structures of heterogeneous size (£1 lm in

diameter; Figure 1a,b). The larger Arf1-GFP structures

corresponded to Golgi bodies (Figure 1a), as shown by co-

expression experiments with a yellow fluorescent protein

(YFP) fusion to the Golgi marker ERD2 (ERD2-YFP; Bran-

dizzi et al., 2002). To establish the origin of the smaller

Arf1-labelled structures, we monitored the distribution of

Arf1 with respect to the Golgi apparatus over time in cells

co-expressing Arf1-GFP and ERD2-YFP. We observed that

small Arf1-structures formed on a Golgi stack and even-

tually detached from it (Figure 1b and Supplementary

data 1).

96 Giovanni Stefano et al.

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110

Our localization data support additional roles of Arf1

besides regulation of COPI assembly (Pimpl et al., 2003). To

provide further evidence for this, we investigated whether

coatomer was distributed on the non-Golgi structures

labelled by Arf1. Co-expression experiments using either

ERD2-GFP or Arf1-GFP with a YFP fusion to an Arabidopsis

eCOP (eCOP-YFP), a component of the COPI coatomer (Kreis

et al., 1995), demonstrated that coatomer co-localized with

the Golgi marker (Figure 2a) but not with the additional Arf1-

GFP structures (Figure 2b). This suggests that COPI coatom-

er components only appear to play a role in intra-Golgi and

Golgi to ER transport, although Arf1 may have additional

roles besides COPI assembly at the Golgi apparatus.

Arf1, coatomer and protein cargo exhibit different dynamics

at the Golgi apparatus

To provide evidence of a dynamic cycle of COPI on and off

the Golgi membranes in living plant cells and to correlate the

residence time of COPI on Golgi membranes with the ex-

change of protein cargo between the ER and Golgi appar-

atus, we used fluorescence recovery after photobleaching

(FRAP) analysis on fluorescent fusions of eCOP, Arf1 or ERD2

expressed in tobacco leaves. FRAP allows measurement of

the rate of movement of a fluorescent marker towards and

from defined areas of a cell. A significant fluorescence

recovery indicates that the bleached molecules are ex-

changing with fluorescent pools of the protein localized in

other parts of the cell, assuming that chimaeric protein

levels in the bleached area had reached a steady state at the

start of each experiment (Lippincott-Schwartz and Patterson,

2003). By photobleaching eCOP-YFP fluorescence, we cal-

culated that coatomer associates and dissociates from Golgi

membranes with a half time of 20.7 � 3.0 sec (Figure 3a,b).

Photobleaching of Arf1-GFP fluorescence on the Golgi

bodies (Figure 3a) was followed by a rapid fluorescence

recovery (half time ¼ 12.1 � 2.1 sec; Figure 3a,b), which

was significantly faster than that of eCOP-YFP (P < 0.05).

FRAP experiments on Golgi bodies labelled with ERD2-YFP

(Figure 3a), which is known to distribute all over the Golgi

stack (Boevink et al., 1998), indicated a half-time recovery of

fluorescence of 123.0 � 14.8 sec (Figure 3b), much slower

than the half-time values measured for coatomer and Arf1.

These data show that not only does a Golgi membrane

protein move to and from the Golgi apparatus, as described

earlier and for other Golgi proteins, such as ST-GFP and

GONST1-GFP (Brandizzi et al., 2002; daSilva et al., 2004), but

also reveal that cytosolic COPI components exchange con-

tinuously at the surface of the Golgi apparatus, providing

evidence for constant remodelling of the membranous and

soluble components of this organelle.

GTPase-defective mutants of Arf1 disrupt Golgi protein

distribution to different extents

To analyze in vivo whether interference with the normal

cycling of Arf1 on and off Golgi membranes would influence

Golgi membrane protein distribution, we examined the ef-

fects of active and inactive forms of Arf1 on the distribution

of a fluorescent fusion of ERD2, which is exported from the

ER by COPII carriers and cycles continuously between ER

and Golgi at the same rate as other Golgi membrane

Figure 1. Arf1 localizes at the Golgi apparatus and at additional structures that originate from the Golgi apparatus.

(a) A confocal image of a tobacco leaf epidermal cell co-expressing Arf1-GFP and the Golgi marker ERD2-YFP shows that Arf1-GFP is localized at Golgi bodies and at

smaller additional structures (inset in merged image, arrowhead). Scale bar ¼ 5 lm.

(b) Sequence of images showing a cell co-transformed with ERD2-YFP (pseudo-coloured magenta) and Arf1-GFP (pseudo-coloured green). Co-localization of the two

markers appears white. Time (sec) of acquisition of frames is indicated at the top left corner. The arrowheads indicate Arf1-GFP structures that are detached from the

Golgi apparatus. Scale bar ¼ 4 lm.

COPI influences ER protein export 97


markers (Brandizzi et al., 2002; daSilva et al., 2004). There-

fore, we co-expressed this marker with either wild-type Arf1

or Arf1 sequences bearing point mutations that create

dominant negative mutants of the GTPase by enhancing its

affinity for either GDP or GTP [T31N or Q71L, respectively

(Teal et al., 1994)]. To identify cells expressing untagged

Arf1 or its mutant forms, we subcloned their coding se-

quences in a bi-cistronic vector encoding a cyan fluorescent

protein (CFP) spliced to the tripeptide serine-lysine-leucine

(CFP-SKL) for targeting to peroxisomes (Sparkes et al., 2003;

see also Supplementary data 2). We then co-transformed

tobacco leaf epidermal cells with these bi-cistronic vectors

and with a plasmid encoding ERD2-YFP. To produce com-

parable results, we used the same optical density of Agro-

bacterium containing the different constructs for tobacco

leaf transient transformation, and we analyzed samples by

confocal microscopy at the same time after transformation.

In the presence of wild-type Arf1, we observed a typical

punctate distribution of the ERD2-YFP (Figure 4a; cf. Fig-

ure 1), in agreement with reports that over-expression of

Arf1 does not affect the distribution of Golgi markers (Lee

et al., 2002; Takeuchi et al., 2002; Xu and Scheres, 2005), and

has no effect on protein secretion (Pimpl et al., 2003). Co-

expression of Arf1GDP with ERD2-YFP caused re-absorb-

ance of the Golgi marker into the ER in the majority (95%) of

cells co-expressing the mutant and ERD2-YFP (Figure 4a).

However, the fluorescence of the marker was mostly distri-

buted in the Golgi in the majority of cells co-expressing

Arf1GTP and ERD2-YFP (85% of the cells co-expressing the

mutant and the Golgi marker; Figure 4a). The ER mem-

branes appeared enlarged in those cells where Golgi mem-

branes were re-absorbed into the ER in comparison to

control cells. This is probably due to accumulation of a Golgi

pool of fluorescent ERD2 whose export is inhibited by the

mutants (see also Lee et al., 2002).

To determine whether Arf1 mutants could have a similar

effect on other Golgi markers, we co-expressed wild-type

Arf1 and its mutants with YFP fusions of either a rat sialyl-

transferase transmembrane domain (ST-YFP; Brandizzi

et al., 2002) or an Arabidopsis b1,2-xylosyltransferase cyto-

solic tail and transmembrane domain (Dirnberger et al.,

2002; Xylo-YFP). Under these conditions, we verified that the

effect of Arf1 mutants on the distribution of these proteins is

similar to that of ERD2-YFP (Supplementary data 3).

These data show that disruption of the COPI machinery at

the Golgi apparatus mediated by Arf1 mutants is followed by

collapse of the Golgi membranes into the ER. As the Golgi

markers used in these experiments cycle continuously in and

out of the Golgi apparatus, most likely from and to the ER

(Brandizzi et al., 2002; daSilva et al., 2004), the redistribution

Figure 2. Coatomer localizes at the Golgi apparatus but not at additional Arf1 structures.

(a) A confocal image of a cell co-expressing ERD2-GFP and eCOP-YFP shows that coatomer localizes at the Golgi apparatus (inset).

(b) A confocal image of a tobacco leaf epidermal cell co-expressing Arf1-GFP and eCOP-YFP shows co-localization of the two markers at the Golgi apparatus, and the

presence of smaller additional Arf1-GFP structures which lack eCOP-YFP labelling (inset in merged image, arrowhead). Scale bars ¼ 5 lm.



of the Golgi markers into the ER suggests an interruption

of protein exchange between the ER and the Golgi

apparatus.

To confirm further that Arf1GDP has a stronger negative

effect on protein export from the ER than does Arf1GTP, we

co-transfected tobacco leaf protoplasts with the secretory

marker a-amylase (Phillipson et al., 2001) and a dilution

series of untagged Arf1, and Arf1GDP or Arf1GTP mutants.

Arf1 did not affect a-amylase secretion (Figure 4b), but the

Arf1GDP and Arf1GTP mutants exhibited a negative effect

on the secretion of a-amylase (see also Pimpl et al., 2003).

Arf1GDP exhibited a stronger effect than Arf1GTP on

secretion of the soluble marker. These data mirror our

microscopy results showing that Arf1GDP affected the ER

export of a Golgi marker more strongly than Arf1GTP under

the same experimental conditions, and suggest that the two

mutants may block ER export by different means.

Differences in the cycling of the Arf1 mutants on and off

Golgi membranes is the basis of their effect on Golgi

membrane protein distribution

To determine whether the Arf1 mutants perturbed the dis-

tribution of membrane proteins at the Golgi apparatus with

different mechanisms, we first aimed to determine the

subcellular localizations of their fluorescent protein fusions

by confocal microscopy (Figure 5). The activity of these

proteins was also tested by a biochemical assay (Supple-

mentary data 4).

GFP fusions of the inactive and active mutants of Arf1

(Arf1GDP-GFP and Arf1GTP-GFP, respectively) were ex-

pressed in tobacco leaf epidermal cells using the same

Agrobacterium optical density and confocal microscope

imaging settings to compare the levels of expression

between cells (see also Experimental procedures). We

hypothesized that Arf1GDP should be localized in the

cytosol, as although it should be able to bind to membranes

via its interaction with a guanine nucleotide exchange factor

(GEF), its inability to exchange GDP for GTP would be

followed by fast release from the membranes into the

cytosol, causing its association with Golgi membranes to be

undetectable. Consistent with our hypothesis, permanently

inactive Arf1 (Arf1GDP-GFP) was localized in the cytosol

(Figure 5a) at any level of expression (see also Xu and

Scheres, 2005). The GTP-trapped mutant was instead distri-

buted to punctate structures in most cells (Figure 5b),

corresponding well with observations by Xu and Scheres

(2005). To determine the identity of the structures highligh-

ted by Arf1GTP-GFP, we co-expressed the fusion with ERD2-

YFP. We found that Arf1GTP-GFP mostly co-localized with

the Golgi marker but also highlighted additional structures

(Figure 6a, inset in merged image, arrowhead), which coin-

cided with those marked by wild-type Arf1-YFP (data not

shown). We also detected a cytosolic distribution of

Arf1GTP-GFP in those cells that were over-expressing the

mutant (Figures 5c and 6b). In these cells, ERD2-YFP was

distributed in the ER (Figure 6b).

These data suggest that the Arf1 mutants may affect COPI

distribution via different mechanisms. In fact, the cytosolic

(a)

(b)

Figure 3. COPI machinery and Golgi enzymes cycle to and from the Golgi

apparatus continuously.

(a) Qualitative FRAP experiment on a cortical section of tobacco leaf

epidermal cells expressing eCOP-YFP, Arf1-GFP or ERD2-YFP treated with

the actin depolymerizing agent, latrunculin B (Brandizzi et al., 2002) to stop

Golgi movement. In the ERD2-YFP panel, the fluorescence of all Golgi stacks

was photobleached but only two Golgi stacks are indicated by arrowheads.

Time from the photobleaching event (bleach) is expressed in seconds at the

bottom left of the two subsequent frames. Scale bars ¼ 2 lm.

(b) Histograms of the time required for the fluorescence in the photobleached

region to recover to 50% of the recovery asymptote (half-time) measured in

FRAP experiments in cells expressing either eCOP-YFP (n ¼ 15), Arf1-GFP

(n ¼ 12) or ERD2-YFP (n ¼ 11). n, number of bleached Golgi stacks. The

difference between the groups was significant at P < 0.05 (student’s test).



distribution of Arf1GDP indicates that this mutant is not able

to initiate the assembly of COPI machinery at the Golgi

membranes. Golgi-associated Arf1GTP may be capable of

initiating the assembly of COPI machinery on the Golgi. To

explore these hypotheses further, we co-expressed un-

tagged Arf1GDP with either Arf1-YFP or eCOP-YFP. This

caused a cytosolic distribution of both markers (Fig-

ure 7b,d), in comparison with the controls (Figure 7a,c; see

also Supplementary data 5). These results suggest that Arf1-

GDP most likely titrates out any Arf1-GEFs needed for the

activation of wild-type Arf1 at the Golgi apparatus, and

consequently impedes the association of coatomer to the

Golgi membranes. In the presence of Arf1GTP bound

mutant, coatomer was capable of cycling on and off Golgi

membranes (Supplementary data 6). This result prompted

us to test the extent to which protein exchange between ER

and Golgi could be affected by this mutant. Therefore, we

performed FRAP analysis on Golgi membranes labelled by

ERD2-YFP. As this marker cycles between the ER and Golgi

apparatus (Brandizzi et al., 2002), a difference in the recovery

(a)

(b)

Figure 4. Arf1 is determinant for the integrity of Golgi membranes.

(a) Merged confocal images of tobacco leaf epidermal cells co-expressing ERD2-YFP (pseudo-coloured magenta) with untagged Arf1, Arf1GDP and Arf1GTP

mutants. The presence of the untagged GTPase in cells is revealed by the visualization of peroxisomes (pseudo-coloured green) with imaging settings for CFP. The

percentage values correspond to the number of cells with punctate ERD2-YFP distribution divided by the total number of observed cells and multiplied by 100. The

total number was the sum of the cells with punctate fluorescence distribution of ERD2-YFP plus the number of cells with ERD2-YFP re-distributed exclusively in the

ER. Sample size ¼ 100 cells for each experiment with one Arf1 protein. Scale bars ¼ 5 lm.

(b) Secretion assay using tobacco leaf protoplasts. Histogram showing the secretion index of a-amylase [ratio of extracellular and intracellular activities, Phillipson

et al. (2001)] in protoplasts expressing untagged Arf1, Arf1GDP [Arf1(T31N)], and Arf1GTP [Arf1(Q71L)]. Concentrations of DNA (lg) for each sample are indicated

along the x-axis. Error bars ¼ standard error of the mean for three independent repetitions.

Figure 5. The subcellular localization of Arf1 depends on its GTPase status.

Confocal images of tobacco leaf epidermal cells expressing GFP fusions of Arf1GDP (a) or Arf1GTP (b,c). Arf1GDP-GFP is cytosolic (a), while Arf1GTP-GFP localizes

at punctate structures (b), which are lost in over-expressing cells (c). Note that for presentation purposes the settings for confocal imaging (laser output and detector

gain) to acquire image (c) were lowered with respect to image (b) in order not to over-saturate the image (see also Experimental procedures). Scale bars ¼ 5 lm.



of fluorescence of ERD2 in the Golgi apparatus in comparison

to a control would indicate that the Arf1GTP mutant may

affect protein cycling between the ER and the Golgi appar-

atus. Photobleaching of ERD2-YFP fluorescence in the pres-

ence of the mutant was followed by recovery (half-

time ¼ 204.3 � 40.6 sec; Figure 8b,c), indicating that ER to

Golgi protein transport was occurring. However, comparison

with the recovery rate of the fluorescence of the same

Golgi marker in control cells (half-time ¼ 123.0 � 14.8 sec;

Figures 3 and 8a,c) indicated that the mutant significantly

reduced the recovery of fluorescence of ERD2-YFP into the

Golgi apparatus. Therefore, constitutive activation of Arf1 on

Golgi membranes most likely retards protein cargo exchange

at the ER/Golgi interface.

Figure 6. Constitutively active Arf1 localizes at Golgi membranes as long as the integrity of Golgi bodies is maintained.

(a) Confocal image of a cell co-expressing ERD2-YFP (pseudo-coloured magenta) and Arf1GTP-GFP (pseudo-coloured green), showing that the active form of Arf1

localizes at the Golgi apparatus and additional structures (inset in merged image, arrowhead).

(b) Confocal image of a cell co-expressing ERD2-YFP and high levels of Arf1GTP-GFP. Note the distribution of ERD2-YFP fluorescence in the ER and that the

fluorescence of Arf1GTP-GFP is cytosolic. Scale bars ¼ 5 lm.

Figure 7. When Arf1 is inactive, the COPI machinery is cytosolic.

Confocal images of tobacco leaf epidermal cells expressing either Arf1-YFP alone as a control (a), or (b) Arf1-YFP (pseudo-coloured magenta) and untagged Arf1GDP

encoded in a bi-cistronic vector bearing the CFP-SKL sequence for labelling peroxisomes (pseudo-coloured green) for identification of cells co-expressing the

mutant protein.

(c) Control cell expressing eCOP-YFP alone.

(d) Confocal image of a cell co-expressing eCOP-YFP and untagged Arf1GDP in a bi-cistronic vector as in (b). Co-expression of CFP-SKL with Arf1-YFP did not affect

the distribution of the Arf1-YFP in comparison to the control (Supplementary data 5). Scale bar ¼ 5 lm.



The slower recovery of ERD2-YFP fluorescence at the

Golgi apparatus suggested that GTPase activity of the

Arf1GTP mutant was not entirely abolished. To test this,

we bleached the fluorescence of Arf1GTP-GFP on Golgi

stacks. Upon photobleaching Arf1GTP-GFP, the fluores-

cence recovered with a half-time of 35.7 � 3.4 sec (n ¼ 11),

which is threefold slower than the Arf1-GFP half-time

(12.1 � 2.1 sec, n ¼ 12, see Figure 3a,b) in accordance with

results in mammalian cells and in vitro (Teal et al., 1994;

Vasudevan et al., 1998).

Taken together, our results indicate that inactive Arf1

prevents COPI formation at the Golgi apparatus while the

active Arf1 mutant allows COPI formation and COPI-medi-

ated protein transport with a reduced activity in comparison

with wild-type Arf1.

The differentiation of ER export sites depends on active

retrograde protein export

We next aimed to test whether impaired COPI activity affects

the integrity of the ER export machinery. For this, we used a

YFP fusion of an Arabidopsis Sec24, a COPII coat component

(YFP-Sec24), to label the ERES. To ensure that fluorescent

Sec24 retained its functionality, we tested its ability to

interact with another COPII coat component Sec23 and form

a heterodimeric complex (Antonny and Schekman, 2001) in

a glutathione–agarose affinity chromatography assay based

on the interaction of a recombinant GST-Sec23 with plant-

expressed YFP-Sec24 (Figure 9). YFP-Sec24 was found to

interact with GST-Sec23 (Figure 9, lane 5) but not with GST

alone (Figure 9, lane 3). Therefore, YFP-Sec24 is a functional

marker capable of interacting with Sec23 as shown in

mammalian cells (Stephens et al., 2000). At a subcellular

level, YFP-Sec24 was localized at punctate structures (Fig-

ure 10a, arrowhead) and in the cytosol (Figure 10a). Co-

expression experiments using YFP-Sec24 with ERD2-GFP

indicated that these structures localized at the peri-Golgi

area, with the exception of additional rare bright YFP-Sec24

structures of variable size that did not localize at the

Figure 8. In the presence of Arf1GTP, ER protein export to the Golgi is impaired.

(a,b) Half-time recovery curves of ERD2-YFP fluorescence in cells expressing ERD2-YFP alone (a) or in the presence of Arf1GTP (b) and treated with latrunculin B to

depolymerize actin. In (b), the presence of untagged Arf1GTP was ensured by the visualization of peroxisomes labelled with CFP-SKL.

(c) Histogram of the half-times of recovery of YFP fluorescence after photobleaching in cells expressing ERD2-YFP alone (ERD2-YFP, n ¼ 11) or in the presence of

Arf1GTP (ERD2-YFP þ Arf1GTP; n ¼ 11). The difference between the groups was significant at P < 0.05 (Student’s t-test).

Figure 9. YFP-Sec24 is capable of interaction with the COPII component

Sec23

Extracts of tobacco leaves expressing Sec24-YFP were incubated with

recombinant GST-Sec23 bound onto glutathione–agarose beads. Proteins

retained by the GST-Sec23 agarose beads were eluted and then boiled in SDS

sample buffer for immunoblot analysis with anti-GFP serum. Sec24-YFP (lane

5) was retained by GST-Sec23. Negative controls: extracts of untransformed

tobacco leaves [()); lane 1]; GST beads alone did not retain YFP (lane 2) nor

Sec24-YFP (lane 3); Sec23-GST did not interact with YFP (lane 4).



peri-Golgi area (6.7% of YFP-Sec24-labelled structures; Fig-

ure 10b–d). We also verified that the YFP-Sec24-labelled

ERES tracked together with Golgi stacks (Figure 10e–g; see

also Supplementary data 7a–c). Furthermore, the expres-

sion of a Golgi marker did not appear to be necessary for the

movement of YFP-Sec24 punctate structures as these were

motile in cells expressing the Sec24 construct alone (Sup-

plementary data 7d). Similar results were achieved with a

fluorescent fusion of another COPII component, Sec23 (YFP-

Sec23; Supplementary data 7d and 8, and data not shown),

although the percentage of Sec23 structures that did not co-

localize was lower than that of Sec24 (3.7%). These data re-

veal that in tobacco leaf epidermal cells COPII markers can

label ERES without over-expression of Golgi marker pro-

teins, and support previous findings that ERES are in close

association with the Golgi stacks (daSilva et al., 2004).

To explore the influence of COPI machinery on the

differentiation of ERES, we co-expressed YFP-Sec24 with

fluorescent fusions of wild-type and mutant Arf1 proteins. In

the presence of Arf1-GFP, YFP-Sec24 was distributed at the

peri-Golgi area but not at the additional smaller structures

labelled by Arf1-GFP (Figure 11a, inset arrowhead). How-

ever, YFP-Sec24 was distributed into the cytosol in co-

expression with Arf1GDP-GFP (Figure 11b). In the presence

of the active Arf1 mutant, the punctate appearance of the

ERES at the Golgi area labelled by YFP-Sec24 was main-

tained in most cells (Figure 11c). In cells that were highly

over-expressing Arf1GTP-GFP, YFP-Sec24 fluorescence was

distributed in the cytosol (Figure 11d). Similar results were

obtained with the other COPII marker, YFP-Sec23 (Supple-

mentary data 8). These data indicate that collapse of COPI-

mediated protein transport perturbs differentiation of ER

export sites and distribution of membrane proteins at the

Golgi apparatus. Taken together, our findings also suggest

that ERES maintain their integrity as long as COPI-mediated

protein transport takes place.

Discussion

The association of COPI at membranes is highly dynamic

and implies additional functions for Arf1 beside ER/Golgi

protein transport

In this work, localization analyses of Arf1-GFP mutants have

indicated that active Arf1 associates with Golgi membranes

while inactive Arf1 resides in the cytosol, consistent with

cycles of binding and release of the GTPase to and from

Golgi membranes demonstrated by FRAP experiments.

FRAP experiments also indicated a transient association of

coatomer with Golgi bodies with a slower turnover than

Figure 10. ER export sites and Golgi bodies behave as single mobile secretory units.

(a)–(d) Confocal images of tobacco leaf epidermal cells expressing either the ER export site marker YFP-Sec24 alone (a), or YFP-Sec24 (b) with a Golgi marker, ERD2-

GFP (c). (d) Merged image of (b) and (c). Note that YFP-Sec24 punctate structures localize mostly at the peri-Golgi area. Rare additional punctate structures are

indicated (arrowheads).

(e)–(g) High-magnification time-lapse of a cell co-expressing YFP-Sec24 and ERD2-GFP presented in vertical display as single channel for ERD2-GFP (e), for YFP-

Sec24 (f) and as merged images (g).

Scale bar in (a), (b) ¼ 5 lm; (e) ¼ 2 lm.



Arf1, suggesting that coatomer dissociation does not coin-

cide with Arf1 GTP hydrolysis. These results imply that

several cycles of binding and release of Arf1 may be required

to form a COPI domain at the ER/Golgi interface and to

determine COPI uncoating, analogous to findings in mam-

malian cells (Presley et al., 2002).

Arf1-GFP was found to localize at the Golgi apparatus with

coatomer, and also at additional structures that lack coa-

tomer and originate from the Golgi apparatus. These

structures may represent the trans Golgi network (TGN) or

TGN-derived structures that detach from the Golgi appar-

atus, analogous to findings in mammalian cells (Waguri

et al., 2003). A separate nature of the plant TGN from the

Golgi apparatus has also been suggested in a study of the

localization of TGN SNAREs in Arabidopsis cells (Uemura

et al., 2004). These structures showed heterogeneous size

and it cannot be excluded that a population of larger ones

may be composed of groups of smaller structures. It has

been suggested that the non-Golgi Arf1 structures may be

endocytic compartments in Arabidopsis and onion cells

based on labelling with a Rab5 homologue, ARA7-GFP (Xu

and Scheres, 2005), although this protein has also been

shown to identify pre-vacuolar compartments in tobacco

leaf cells (Kotzer et al., 2004). As we followed the formation

of the structures only at the Golgi apparatus, we cannot

exclude the co-existence of Arf1-associated endocytic struc-

tures, and it is also possible that the Golgi-derived structures

may eventually mature to become, or merge with, endo-

somal or pre-vacuolar structures. The distribution of Arf1

verified in this study and by Xu and Scheres (2005) may be

linked to the subcellular location of various GEFs for Arf1

activation that are present in the Arabidopsis genome

Figure 11. The integrity of the ERES depends on a functional COPI-mediated protein transport.

Confocal images of cells co-expressing YFP-Sec24 and GFP fusions of Arf1 (a), Arf1 GDP mutant (b), or Arf1GTP at lower (c) and higher expression (d). The

arrowhead in the inset in the merged image in (a) indicates an Arf1-GFP structure that lacks YFP-Sec24 labelling.

(c,d) Confocal images of cells expressing Arf1GTP-GFP. In cells where the punctate appearance of Arf1GTP-GFP was maintained, YFP-Sec24 localized at the Golgi

area but not at the additional Arf1GTP-GFP structures (inset, arrowhead), analogously to wild-type Arf1-GFP (a). Note that the punctate distribution of YFP-Sec24 is

lost in cells expressing high levels of Arf1GTP-GFP (d, arrowhead). Scale bars ¼ 5 lm.



database, the best characterized being the EMB30/GNOM

gene product (Geldner et al., 2003; Grebe et al., 2000;

Memon, 2004; Shevell et al., 2000). The lack of distribution

of coatomer on the non-Golgi organelles supports the

findings that Arf1 has cellular functions besides intra-Golgi

transport and Golgi/ER protein transport in plant cells (Pimpl

et al., 2003). These functions may also include the formation

of clathrin-coated vesicles as shown in non-plant systems

(Puertollano et al., 2001; Robinson and Kreis, 1992; Stamnes

and Rothman, 1993).

The distribution of fluorescent fusions of components of the

COPII coat confirms that ER export sites move with the Golgi

apparatus in tobacco leaf epidermal cells

To provide further evidence of the subcellular distribution of

the ERES in tobacco leaf epidermal cells, we used active

fluorescent fusions of the COPII coat components Sec24 and

Sec23, which are known ERES markers in non-plant systems

(Stephens, 2003; Stephens et al., 2000). YFP fusions of Sec24

and Sec23 localize at punctate structures that tracked with

Golgi stacks. We have also verified the existence of rare

additional bright YFP-Sec24 and -Sec23 structures, which do

not localize to nor track with Golgi bodies. A similar result

was obtained with a fluorescent fusion of Sar1 and of an

active mutant of Sar1 (daSilva et al., 2004). These structures

may represent embryonic ERES where new Golgi bodies

may differentiate.

Our findings confirm that the distribution of ERES shown

by Golgi-cargo-induced accumulation of Sar1 (daSilva et al.,

2004) is not artefactual, and provides further support for the

model which suggests that Golgi stacks and associated

ERES function as mobile secretory units in this system. The

dimer Sec23/24 may accumulate at ERES in the absence of

over-expression of Golgi proteins for the suggested involve-

ment of Sec24 in cargo selection for incorporation into COPII

vectors (Miller et al., 2002). We cannot exclude the possibil-

ity that co-expression of a Golgi marker may induce higher

accumulation of these COPII components at the ERES in

comparison to cells expressing these COPII markers alone. It

is possible that basal levels of cargo export from the ER are

sufficient to make Sec24, and consequently Sec23, accumu-

late visibly at the ERES. It may also be that case that Sec24

and Sec23 are more visible than Sar1 at the ER export sites

as they may cycle on and off the ERES more slowly than

Sar1, analogous to the dynamics of eCOP and Arf1 at the

Golgi membranes shown in this paper.

Recently, it has been proposed that ERES associate

intermittently with Golgi bodies in tobacco BY-2 cells (Yang

et al., 2005), although it was not possible to equate the

Sec13-GFP structures with ERES in the absence of correla-

tive imaging data on the export of membrane or lumenal ER

cargo (Yang et al., 2005). It cannot be excluded that the

different dynamics of ERES observed in BY-2 cells and

tobacco leaf epidermal cells may be linked to the different

experimental systems (see also Yang et al., 2005, for similar

discussion).

The close spatial association of Golgi with ERES in

tobacco leaf epidermal cells is comparable to that observed

in Pichia pastoris (Mogelsvang et al., 2003; Rossanese et al.,

1999), Trypanosoma brucei (He et al., 2004), and Drosophila

melanogaster (Herpers and Rabouille, 2004; Kondylis and

Rabouille, 2003), although the exact organization of plant

ERES has yet to be determined at an ultrastructural level.

Biochemical studies have shown that Sar1 binds to the ER

and not to the Golgi membranes by virtue of its N-terminal

hydrophobic domain, and that the subsequent assembly of

the multi-subunit COPII complex occurs after Sar1 recruit-

ment (Bar-Peled and Raikhel, 1997; Barlowe, 2002a,b; Bi

et al., 2002; Matsuoka et al., 2001). It has been suggested

that, in tobacco leaf epidermal cells, the Golgi and ER may

be physically linked although the extent of this association is

unknown (Brandizzi et al., 2002; Hawes and Satiat-Jeune-

maitre, 2005). We cannot exclude the possibility that COPI

and COPII may create domains at the ER/Golgi interface that

allow separation of the direction of transport. Future studies

utilizing electron microscopy will elucidate which model

applies to the ER/Golgi interface in tobacco leaf epidermal

cells.

The GTPase activity of Arf1 influences ER protein export to

the Golgi apparatus

Here we have shown that impaired GTPase activity of Arf1

disrupts the distribution of membrane markers in the Golgi

apparatus. FRAP experiments on these markers have dem-

onstrated that these proteins cycle in and out of the Golgi

apparatus (this paper, Brandizzi et al., 2002; daSilva et al.,

2004; Brandizzi, unpublished results), and most likely

between this organelle and the ER (see daSilva et al., 2004,

for a discussion). Therefore, a disruption of the localization

of membrane proteins in the Golgi operated by Arf1 mutants

is most likely linked to a block in protein export from the ER.

It cannot be excluded that Arf1 and COPI components are

directly required for successful construction or functioning

of the ERES. It has been shown that in mammalian cells COPI

may be required at a pre-Golgi step in transport from the ER

(Stephens and Pepperkok, 2002). Although our data do not

allow us to distinguish whether the Arf1 mutants act directly

by blocking anterograde protein movement or retrograde

protein transport, Arf1 and coatomer have been localized on

the Golgi apparatus (Movafeghi et al., 1999; Pimpl et al.,

2000; Ritzenthaler et al., 2002; Takeuchi et al., 2002). There-

fore, it is reasonable to suggest that Arf1 mutants may block

the retrograde pathway. The interference with the antero-

grade transport of membrane proteins to the Golgi appar-

atus may then be an indirect effect mediated by Arf1 mutants

on protein export from the ER.



We have shown that impaired retrograde transport from

the Golgi apparatus mediated by Arf1 mutants affects the

differentiation of the ER export sites and Golgi protein

distribution, although inactive and active Arf1 mutants

appear to have different mechanisms. Inactive Arf1 gener-

ally stabilizes the cytosolic pool of the GTPase, inhibits

association of the coatomer with the Golgi bodies and

disrupts the distribution of Golgi markers and the integrity of

ERES (see also Pimpl et al., 2000; Ritzenthaler et al., 2002;

daSilva et al., 2004; Xu and Scheres, 2005). In contrast, in

conditions of low expression, the Arf1GTP mutant binds to

Golgi membranes, Golgi bodies remain intact, the move-

ment of a Golgi marker in and out of the Golgi apparatus is

reduced and the integrity of ER export sites is unaffected.

However, at a higher expression of this mutant, the mutant is

cytosolic, and Golgi membranes and ER export sites are

disrupted. FRAP experiments on Arf1GTP-GFP show that

this protein is capable of exchanging between cytosol and

membranes but at a slower rate than the wild-type Arf1.

FRAP experiments have also indicated that COPI assembly

and dissociation at the Golgi can still occur in the presence

of active Arf1 mutant but that Arf1 mutant-associated

membranes accumulate before fusion with acceptor mem-

branes, probably due to a slow shedding of the COPI coat.

Such retardation of fusion of COPI vectors with acceptor

membranes results in a loss of Golgi integrity in conditions

of over-expression of the Arf1GTP mutant, possibly caused

by a loss of other essential components that normally

recycle from the Golgi body back to the ER, but are now

titrated out in fusion-incompetent COPI vectors. Therefore,

we suggest that the slower GTPase activity of the mutant

allows recycling of COPII components at the ERES from the

Golgi apparatus, while complete blockage of COPI coatomer

assembly at the Golgi membranes mediated by the Arf1GDP

mutant inhibits this recycling completely. This effect may

explain how, under the same conditions of expression,

Arf1GDP has a stronger effect on protein export from the ER

than Arf1GTP does.

Our data on the dependence of ERES differentiation on a

functional COPI machinery highlight an important difference

between the organization of the ERES in tobacco leaf

epidermal cells and in mammalian cells. In vertebrate cells

treated with BFA, the COPII coat on the ERES dynamically

exchanges on and off membranes in cells. Vertebrate ERES

maintenance is dependent on ER export activities, as COPII

labelling of ERES was lost in the presence of an inactive

mutant of Sar1 (Ward et al., 2001). Furthermore, it has been

shown that, in the presence of BFA, ERES recruit some

membrane proteins (e.g. p58, GRASP65, and GM130) but not

others (e.g. Golgi enzymes and secretory cargo such as

vesicular stomatitis virus G protein) (Ward et al., 2001). This

has been interpreted to mean that the differentiation of ERES

requires the activity of Sar1–COPII prior to activity of the

Arf1–COPI system to enable recruitment of the diverse array

of secretory proteins (Ward et al., 2001). Our data instead

suggest that events consequent to an active retrograde

transport operating at ERES are crucial for their differenti-

ation and, as a consequence, for protein cargo movement to

the Golgi apparatus. Therefore, we propose a model that, in

tobacco leaf epidermal cells, COPII and COPI transport

routes that control the trafficking of proteins between the

ER and the Golgi strictly depend on each other, most likely

because of the recycling of necessary transport machinery

for ER protein export. Inhibition of one transport route leads

to the collapse of its matching retrograde route and vice

versa, and a sequential activity of the Sar1–COPII and Arf1–

coatomer systems jointly serves to form and maintain ERES

and Golgi structures, whose components continuously

circulate through the ER. Without the joint activities of both

Sar1–COPII and Arf1–coatomer, forward trafficking into the

Golgi cannot occur. In this hypothesis, Arf1–coatomer is

required for forward trafficking out of the ER due to its role in

differentiating ER export domains formed by the Sar1–COPII

system. This model suggests that the Golgi apparatus is an

outgrowth of the ER whose identity depends on the active

process of secretion and whose positioning is influenced by

the localization of the ER export sites (Hawes and Satiat-

Jeunemaitre, 2005).

Experimental procedures

Molecular cloning

Standard molecular techniques were used for subcloning. Thefluorescent proteins used in this study were based on fusions witheither mGFP5 (Haseloff et al., 1997), ECFP or EYFP (Clontech Inc.,Palo Alto, CA, USA). The spectral properties of mGFP5 allow effi-cient spectral separation from YFP (Brandizzi et al., 2002). As aGolgi marker, we used the H/KDEL receptor (ERD2, Lee et al., 1993)fused to GFP (Boevink et al., 1998) or YFP (Brandizzi et al., 2002),ST-YFP (Brandizzi et al., 2002) and Xylo-YFP. For the Xylo-YFPconstruct, the DNA sequence encompassing the cytoplasmic tailand transmembrane region of an Arabidopsis b1,2-xylosyltransf-erase (DNA kindly provided by H. Steinkellner, Zentrum furAngewandte Genetik, Universitat fur Bodenkultur Wien, Austria)that have been shown to target the Golgi apparatus in Nicotianabenthamiana as a GFP fusion (Dirnberger et al., 2002) was ampli-fied by PCR and subcloned into pVKH18En6 upstream of a YFPsequence using XbaI and SalI sites of the binary vector. For coa-tomer labelling, we generated a YFP fusion with the Arabidopsishomologue of eCOP, a component of the COPI coatomer (Kreiset al., 1995). The coding sequence for eCOP (Genbank accessionnumber AF370325) was obtained from RIKEN and amplified byPCR with primers containing the XbaI and SalI sites for subcloningupstream of YFP in the binary vector pVKH18-En6. To generatefluorescent fusions of Arf1 proteins (Pimpl et al., 2003), we usedthe DNA of the GTPases kindly provided by J. Denecke (Universityof Leeds, Leeds, UK) and spliced it upstream of a fluorescentprotein sequence in the binary vector pVKH18-En6 with the XbaIand SalI sites. For labelling of ER export sites, the Arabidopsishomologues of Sec24 (locus At3g07100) and Sec23 (locusAt3g23660) were obtained as ABRC clones and fused to theN-terminus of a YFP using the BamHI and SacI sites of the binary



vector pVKH18-En6. For Agrobacterium tumefaciens transientexpression of untagged Arabidopsis Arf1 and its GDP and GTPrestricted mutants (Pimpl et al., 2003), we used a modified binaryvector pVKH18-En6 bearing two 35S-pNOS reading cassettes indirect orientation (see also Supplementary material 2). In onecassette we subcloned the DNA coding sequence of the proteinswithin the unique XbaI and SacI sites, and in the other cassette wesubcloned a CFP-SKL fusion to ensure visualization of peroxi-somes. The primer sequences used for the subcloning indicatedabove are available upon request. For a-amylase experiments,plasmids bearing the sequences encoding the wild-type Arf1,Arf1GDP and GTP mutants described in Pimpl et al. (2003) wereused.

Plant material and transient expression systems

Four-week-old Nicotiana tabacum (cv. Petit Havana) greenhouseplants grown at 25�C were used for Agrobacterium tumefaciens(strain GV3101)-mediated transient expression (Batoko et al., 2000).The bacterial optical density (OD600) used for plant transformationwas 0.02–0.05 for tagged and untagged versions of Arf1 and itsmutants, and 0.2 for ERD2-, ST- and Xylo-constructs.

For transient expression in protoplasts, Nicotiana tabacum plants(cv. Petit Havana) were grown in Murashige and Skoog medium and2% sucrose in a controlled room at 25�C with a 16 h light/8 h darkregime at a light irradiance of 200 mE m)2 sec)1. Tobacco leafprotoplast preparation and subsequent DNA transfection via elec-troporation were performed as described by Phillipson et al. (2001),and the plasmid concentrations used are given in Figure 4(b). Forthese experiments we used untagged Arf1 proteins subcloned inexpression vectors described by Pimpl et al. (2003) and tagged Arf1proteins (Supplementary data 4) that were subcloned into thebinary vector pVKH18-En6. After incubation for 24 h in the dark,the protoplast suspension was spun for 5 min at 100 g in a swing-out centrifuge (4K15; Sigma, Oakville, Canada), which results in thefloating of the cells. Using an extra-fine Pasteur pipette, 1 ml of clearsupernatant from below the floating cell layer was removed. Theremainder of the suspension (1 ml) was brought to 10 ml with250 mM NaCl and mixed gently by inverting the tube twice. After asecond spin of 5 min at 100 g, the supernatant was removed with aperistaltic pump, and the cell pellet was placed on ice. The cellswere extracted in a final volume of 250 ll. Equal volumes of cellextract and culture medium were analysed by protein gel blotting orby enzymatic analysis.

a-Amylase assay

Protoplasts were extracted in a-amylase extraction buffer (Croftset al., 1999) via sonication for 5 sec. The extracts were centri-fuged for 10 min at 25 000 g at 4�C and the supernatant wasrecovered. The culture medium was also spun for 10 min at25 000 g at 4�C to remove residual cell debris. The a-amylaseassays and calculation of the secretion index were performed asdescribed previously (Phillipson et al., 2001). The secretion indexrepresents the ratio between the extracellular and intracellularactivity (Phillipson et al., 2001).

Sampling, imaging and spot fluorescence recovery after

photobleaching (FRAP) analysis

Transformed leaves were analysed 44–48 h after infection of thelower epidermis. Imaging was performed using an upright Zeiss

Laser Scanning Confocal Microscope LSM510 META (Zeiss, Jena,Germany), and a 63· water immersion objective. For imagingexpression of either GFP constructs or YFP constructs or both, weused imaging setting as described by Brandizzi et al. (2002) with a3 lm pinhole diameter. Time-lapse scanning was acquired withimaging system software of the microscope. Comparison of differ-ent levels of expression between cells expressing tagged Arf1 mu-tants was carried out by visualizing cells with the same imagingsettings of the confocal microscope (i.e. laser intensity, pinholediameter and settings of the imaging detectors) as described bydaSilva et al. (2004). For each image, we used the palette function ofthe microscope software, which measures the fluorescence inten-sity value for each image pixel. Differences in the number of sat-urated pixels between cells were an indication of higher or lowerconcentrations of a GFP fusion. Subsequently, only for presentationpurposes in this paper, the images may have been acquired withdifferent settings. Spot FRAP experiments for fluorochrome pho-tobleaching and half-time computation were performed as des-cribed by Brandizzi et al. (2002). Significance was determined usinga Student two-tailed t-test for two samples assuming equal vari-ance. For FRAP experiments, a steady-state protein distribution atthe Golgi bodies was assumed. Such an assumption is reasonableas FRAP experiments have not produced appreciable differenceswhen performed at either 2 or 3 days after transformation when thelevels of expression are highest.

In vitro expression

Production of GST-Sec23 subcloned in pGEX vector was induced inEsherichia coli BL21(DE3) lysogens. Positive clones were selectedfor low-scale protein production. A single colony was inoculatedinitially into 5 ml of Luria Bertani containing ampicillin (100 ll ml)1),further expanded into a 100 ml shaker culture in 250 ml flasks. Thecells were incubated with shaking at 30�C until an OD600 ofapproximately 1.0 was reached. Protein production was induced bythe addition of 1 mM IPTG and further incubation of the culture for5 h at 30�C. Cells were then pelleted and lysed according to theinstructions provided by the manufacturer of the glutathione resincolumns (BD Biosciences, Mississauga, Canada) for binding of GST-tagged proteins. Protein binding, removal of endogenous proteinsand elution of GST-tagged proteins were performed according tothe manufacturer’s instructions.

Glutathione–agarose affinity chromatography of leaf

extracts

A sample (1 g) of leaves transformed with YFP-Sec24 was sub-jected to protein extraction in 1.25 ml of NE buffer (20 mM HEPES,pH 7.5, 100 mM NaCl, 10 mM EDTA, 5 mM MgCl2) with proteaseinhibitor cocktail for plant cell extracts (Sigma) in liquid N2. Theresulting suspension was then centrifuged at 4�C, 14 000 g for15 min. An aliquot (1 ml) of the supernatant was added to 150 llof a glutathione–agarose beads suspension (see below) [72% inNS buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2)]previously mixed with bacterial lysates containing GST-Sec23 andwashed from unbound proteins. The mix was kept for 3 h at 4�Cwith gentle rotation. The beads were centrifuged at 4�C, 500 g for1 min and then washed three times with NS buffer. Bound pro-teins were eluted from the beads with an appropriate volume of5x SDS–PAGE sample buffer [0.225 M Tris-HCl, pH 6.8; 50%glycerol; 5% SDS; 0.05% bromophenol blue; 0.25 M DTT (QIAGEN-QIAexpressionist kit; Qiagen, Mississauga, Canada) in a



proportion sample:buffer ¼ 1:0.4, respectively] and run on a 10%SDS–PAGE gel.

Western blot analysis

Western blot analysis of proteins was conducted on protoplast ex-tracts after sonication of protoplasts in a-amylase extraction buffer,and on leaf extracts in NE buffer. Protein samples were resuspendedin a-amylase extraction buffer, and were loaded in equal volumesafter twofold dilution with 2x SDS loading buffer prior to boiling(Crofts et al., 1999). Proteins in SDS–polyacrylamide gels weretransferred onto a nitrocellulose membrane and then blocked withPBS, 0.5% Tween-20, and 5% milk powder for 1 h. The filter wasthen incubated in blocking buffer with primary antibody at a dilutionof 1:2000 for the anti-GFP serum (AbCam) and 1:5000 for the anti-IgG antibody. All the antisera were from rabbits, and further stepswere performed as described by Crofts et al. (1999).

Acknowledgements

We are grateful to Dr E.L. Snapp (Einstein College of Medicine, NewYork, NY, USA) for valuable discussion. We acknowledge forfinancial support the University of Saskatchewan and the Depart-ment of Biology, U of S, CFI and Canada Research Chair (CRC) grantsto F.B for the development of this work. S.H. is supported by a CRCProvincial Operating Fund and a Department of Biology Post-Doc-toral Award. CRC Provincial Operating Fund and Graduate CollegeStudies Award are acknowledged for the support of G.S. For herMSc studentship, L.R. is indebted to a University of SaskatchewanNew Faculty Award. L.C. is recipient of a Government of CanadaAward CIEC-ICCS, International Council for Canadian Studies, spentin F.B. laboratory. We are grateful to Dr J. Denecke (Centre for PlantSciences, School of Biology, University of Leeds, Leeds, UK) for thegenerous gift of Arf1 DNA, and Dr H. Steinkellner (Zentrum furAngewandte Genetik, Universitat fur Bodenkultur Wien, Austria) forthe generous gift of the Arabidopsis b1,2-xylosyltransferase DNA.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Arf1-GFP localizes at the Golgi apparatus and atadditional structures that originate from this organelle.Figure S2. Generation of a bi-cistronic vector that allows visualiza-tion of cells expressing an untagged protein via monitoring thepresence of CFP-SKL, a fluorescent protein fusion targeted to theperoxisomes.Figure S3. Effect of Arf1 proteins on the subcellular distribution ofST-YFP or Xylo-YFP.Figure S4. Tagged Arf1 proteins have a similar effect on thesecretion of a soluble cargo in comparison to the untaggedcounterparts, and are present in cells as intact fusions.Figure S5. The peroxisomal marker CFP-SKL does not have an effecton the subcellular distribution of Arf1-YFP.Figure S6. FRAP experiments on eCOPI-YFP and Arf1GTP-GFP showYFP fluorescence recovery in the presence of Arf1GTP-GFP.Figure S7. Time-lapse microscopy on Sec24- and Sec23-labelledERES shows that these structures are highly motile.Figure S8. The subcellular distribution of fluorescent Sec23 isinfluenced by the integrity of COPI.This material is available as part of the online article from http://www.blackwell-synergy.com

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