Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites.
ABSTRACT Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.
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ABSTRACT: Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants.Frontiers in Plant Science 08/2014; 5:411. · 3.64 Impact Factor
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ABSTRACT: The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.Journal of Biological Chemistry 06/2014; · 4.60 Impact Factor
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ABSTRACT: The Golgi apparatus plays essential roles in intracellular trafficking, protein and lipid modification, and polysaccharide synthesis in eukaryotic cells. It is well known for its unique stacked structure, which is conserved among most eukaryotes. However, the mechanisms of biogenesis and maintenance of the structure, which are deeply related to ER-Golgi and intra-Golgi transport systems, have long been mysterious. Now having extremely powerful microscopic technologies developed for live-cell imaging, the plant Golgi apparatus provides an ideal system to resolve the question. The plant Golgi apparatus has unique features that are not conserved in other kingdoms, which will also give new insights into the Golgi functions in plant life. In this review, we will summarize the features of the plant Golgi apparatus and transport mechanisms around it, with a focus on recent advances in Golgi biogenesis by live imaging of plants cells.International review of cell and molecular biology 01/2014; 310:221-87. · 4.52 Impact Factor
Dynamics of COPII Vesicles and the Golgi Apparatus in
Cultured Nicotiana tabacum BY-2 Cells Provides Evidence
for Transient Association of Golgi Stacks with
Endoplasmic Reticulum Exit Sites
Yao-dong Yang,a,1Rabab Elamawi,b,1Julia Bubeck,aRainer Pepperkok,cChristophe Ritzenthaler,b
and David G. Robinsona,2
aDepartment of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg, Germany
bInstitut de Biologie Mole ´culaire des Plantes, 67084 Strasbourg, France
cCell Biology Cell Biophysics Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the
organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi
stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment
are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the
formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been
difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana
tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the
location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus.
On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-
coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green
fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein–labeled ER or Golgi stacks. COPII binding
to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both
after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi
stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of
membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When
a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other
hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus
has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to
investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily
associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully
occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended
to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were
also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus
is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently
with several ERES as they move.
The endoplasmic reticulum (ER) is a highly versatile membrane
compartment that extends throughout the cytoplasm of eukary-
otic cells. Probably the most important of its numerous functions
is that it acts as the port of entry for newly synthesized proteins
organelles constituting the secretory and endocytic pathways.
This characteristic feature was first established more than
30 years ago in classic studies on the intracellular transport of
secretory proteins in mammalian cells (Palade, 1975) and has
been continually elaborated on ever since (amongst numerous
recent reviews, for animal cells, see Klumperman, 2000; Lee
et al., 2004; for plants, see Ju ¨rgens, 2004; Ward and Brandizzi,
It is universally accepted that ER-to-Golgi protein transport
in mammalian cells is mediated by the sequential action of
COPII- and COPI-coat protein complexes (Duden, 2003; Lee
et al., 2004). This is because a pleiomorphic structure known
alternatively astheER–Golgiintermediate compartment (ERGIC)
and vesicular tubular clusters (VTCs) transits along microtubules
1These authors contributed equally to this work.
2To whom correspondence should be addressed. E-mail david.
firstname.lastname@example.org; fax 49-6221-546406.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: David G. Robinson
WOnline version contains Web-only data.
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 17, 1513–1531, May 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
from the ER to the perinuclear-located Golgi apparatus with the
help of a dynein/dynactin motor (Murshid and Presley, 2004).
Characteristically, VTCs have COPI coats (Horstmann et al.,
2002). However, there is general agreement that only the COPII
machinery is responsible for the actual transport of cargo out of
the ER (Barlowe, 1998, 2003).
In mammalian cells, export competent soluble and trans-
SNAREs Sed5, Bos1, Sec22, and Bet1, and several integral
membrane proteins, including members of the p24 family and
the Erv41/46 complex (Otte et al., 2001; Miller et al., 2002;
Mossessova et al., 2003). The coat proteins are the GTPase
31 at the cytosolic surface of the membrane (Antonny and
Schekman, 2001). COPII-coat protein recruitment starts by the
binding of Sar1p to the guanine nucleotide exchange factor
(GEF) Sec12p, an integral ER membrane protein (Barlowe and
Schekman, 1993), and is followed by the sequential attachment
of Sec23/24 and then Sec13/31 dimers (Aridor and Balch, 2000).
The Sec23/24 dimer has been implicated in the selection of
cargo molecules into COPII vesicles (Bi et al., 2002; Miller et al.,
2002) by interacting with diacidic (DXE) and diaromatic (FF)
motifs in the cytoplasmic tails of transmembrane cargo mole-
cules (Aridor et al., 2001; Otte and Barlowe, 2002).
In mammalian gland cells engaged in regulated secretion,
ERES collect at specialized domains of the ER known as
transitional ER (tER). Such domains are characterized by a high
density of vesicle/tubule budding profiles in thin sections (for
example, see Sesso et al., 1994; Bannykh et al., 1996; Ladinsky
et al., 1999). The presence of COPII-coat proteins at these sites
has been confirmed by immunogold labeling (Orci et al., 1991;
Tang et al., 2000, 2001; Horstmann et al., 2002). tER is also often
recognized in microorganisms. A clear example is that of the
model alga Chlamydomonas reinhardtii, where tER and adjacent
Golgi stacks are held in an ER amplexus attached to the nuclear
envelope (Zhang andRobinson, 1986).Another well-known case
is that of the fission yeast Pichia pastoris, which in contrast with
Saccharomyces cerevisiae possesses a stacked Golgi appara-
tus (Mogelsvang et al., 2003). Pichia has several discrete tER
1999). The reason for such aggregations of COPII budding sites
was thought to lie in the oligomerization status of Sec12p, which
for the interaction of adjacent molecules (Bevis et al., 2002).
However, other scaffolding proteins, possibly Sec16p (Supek
et al., 2002), now seem to be required for this event because
COPII budding sites in P. pastoris still form when the localization
of Sec12p to the tER is disrupted (Soderholm et al., 2004). In
cultured cells and those mammalian cells exhibiting constitutive
secretion, ERES are randomly located on the surface of the ER
(Hammond and Glick, 2000; Stephens et al., 2000).
Like the yeasts, higher plant cells have a polydisperse Golgi
apparatus and do not possess VTCs (Pavelka and Robinson,
2003). In addition, the Golgi apparatus moves along actin
filaments that run parallel and close to the ER (Boevink et al.,
1998; Ward and Brandizzi, 2004). Despite these clear morpho-
logical differences in the early secretory pathway, COPI/COPII
vesiculating machineries appear to be quite conserved amongst
the various eukaryotic cell types. Thus, COPI homologs can be
found in the Arabidopsis database (Andreeva et al., 1998), and
some have been identified in plant extracts (Movafeghi et al.,
1999; Contreras et al., 2000, 2004). The presence of COPI
proteins at the surface of vesicles budding from the periphery of
Golgi cisternae has also been demonstrated by immunolabeling
at both light (Ritzenthaler et al., 2002a) and electron microscopy
levels (Pimpl et al., 2000). Many plant COPII homologs have also
Raikhel, 1997; Sar1 and Sec23, Movafeghi et al., 1999). More-
over, a functional Sar1 has been shown to be necessary for
successful ER-to-Golgi transport in plant cells (Takeuchi et al.,
2000; Phillipson et al., 2001).
tER in higher plant cells is poorly characterized, and vesicu-
lation profiles at the ER in thin sections have only rarely been
recorded in the literature (e.g., Craig and Staehelin, 1988;
Staehelin, 1997; Ritzenthaler et al., 2002a), suggesting that
ERES in this cell type are short lived and randomly distributed.
To visualize ERES in tobacco (Nicotiana tabacum) BY-2 cells, we
have employed two different approaches: (1) direct visualization
of endogenous COPII proteins (Sar1, Sec13, and Sec23) by
immunofluorescence microscopy in cell lines stably expressing
ER- and Golgi-localized green fluorescent protein (GFP) markers
and (2) visualization of ER-bound Sec13 by expression of
a LeSec13:GFP construct in cells transiently expressing ER-
and Golgi-localized red fluorescent protein (RFP) markers. In
both cases, COPII is seen as punctate fluorescence over the
surface of the ER. These point sources considerably outnumber
Golgi stacks, although some are seen to associate with the rims
of Golgi stacks. COPII labeling does not change or disappear
with BFA, despite considerable morphological changes in the
Golgi apparatus. Prevention of ER export through expression of
a Sar1 mutant locked in the GDP state leads to disturbances in
the ability to visualize COPII at the ER.
Generation of Plant COPII Antisera
We previously generated antibodies against an AtSec23 frag-
ment (Movafeghi et al., 1999). To increase the certainty of valid
identification of plant ERES by antibody labeling, we expressed
fusion proteins and subsequently prepared and purified anti-
bodies against two further COPII components: the GTPase
AtSar1 and the coat protein AtSec13. Similarly, we prepared
an antibody against AtSec12, the GEF required for Sar1 recruit-
ment. We tested in protein gel blots all of these antibodies on
membrane and cytosol fractions obtained from suspension
cultured Arabidopsis thaliana and tobacco BY-2 cells (Figure
1A). With the exception of AtSec12, each antiserum recognized
a single polypeptide in both subcellular fractions. This poly-
peptide corresponded to the expected molecular mass for the
protein in question: 87 kD for AtSec23, 33 kD for AtSec13, and
22 kD for AtSar1. For AtSec12, a polypeptide of ;42 kD was
detected only in the membrane fraction, as expected for a type I
integral membrane protein (Bar-Peled and Raikhel, 1997).
1514 The Plant Cell
Because ourmicroscopyinvestigations weretobeperformed on
BY-2 cells expressing various fluorescent ER and Golgi (X)FP
constructs, we also prepared protein gel blots from fractions
isolated from BY-2 cells. The same polypeptides were identified
in both fractions; however, based on relative concentrations of
applied proteins, the signals were estimated to be 40 to 50%
weaker in the case of the BY-2 antigens. Interestingly, the
AtSec12 antiserum consistently failed to cross-react with mem-
brane preparations from BY-2 cells. Because a complete se-
quence for this protein from tobacco is not available, we cannot
provide a reason for this.
We also tested the COPII antisera on fractions collected from
a linear sucrose density gradient of Arabidopsis membranes
to verify their association with the ER. As seen in Figure 1B,
AtSec12, AtSec13, and AtSar1 have distribution profiles very
similar to the ER marker calnexin and are clearly different to that
of the Golgi marker reversibly glycosylated polypeptide. These
results are consistent with our previous demonstration that the
behavior of AtSec23-bearing membranes from cauliflower in-
florescence in response to Mg2þions is typical for ER in sucrose
COPII Immunostaining in Tobacco BY-2 Cell Lines
Expressing Fluorescent ER and Golgi Markers
Immunofluorescence labeling of wild-type and transgenic BY-2
cells with COPII antisera gave rise in all cases to a punctate
pattern (Figure 2). A similar picture was produced with all three
antisera: anti-AtSec23 (Figures 2A and 2K), anti-AtSec13 (Figure
2B), and anti-AtSar1 (Figures 2E, 2H, and 2M). The signal density
was lower in median sections (Figures 2A to 2C) than in optical
sections through the cell cortex parallel to the cell surface
(Figures 2D to 2M), presumably because of the higher incidence
of ER in surface view. Colocalization of labeling when using any
In BY-2 cells expressing the Golgi marker GmMan1:GFP,
immunostaining with anti-AtSar1 gave rise to a homogeneous
density of this labeling was found to be 3.9, 4.4, and 3.5 point
sources/mm2in Figures 2F, 2I, and 2L, respectively, with diam-
eters of 540 6 174 nm, 568 6 230 nm, and 512 6 166 nm (n >
100), respectively. By comparison, Golgi stacks with an average
apparent diameterof 8806164nm(n¼ 77)had adensity ofonly
Figure 1. Cross-Reactivities of Antisera Raised against Recombinant Arabidopsis COPII Proteins.
(A) Cytosolic proteins and total membranes were isolated from the suspension cultures of Arabidopsis and tobacco BY-2 cells as described in Methods
and probed with the antisera indicated. Equal amounts of protein were applied to the lanes in each gel blot (20 mg per lane for the Arabidopsis gel; 30 mg
per lane for the BY-2 gel). M, membrane; C, cytosol.
(B) Arabidopsis total cell membranes separated on a linear isopycnic sucrose density gradient (as described in Methods). Individual fractions
were probed with COPII antisera and with standard antisera for ER (calnexin) and Golgi (reversibly glycosylated polypeptide [RGP]) marker
COPII and Golgi 1515
Figure 2. Confocal Immunofluorescence Images of BY-2 Cells Labeled with AtCOPII Antisera.
(A) Anti-AtSec23 staining.
(B) Anti-AtSec13 staining.
(C) Merge image of (A) and (B); median optical section, wild-type cell.
(D) Golgi stacks visualized in a GmMan1:GFP transformed cell; cortical section.
(E) Anti-AtSar1 staining of cell in (D).
(F) Merge image of (D) and (E).
(G) Cortical ER visualized in a GFP:HDEL transformed cell.
(H) Anti-AtSar1 staining of cell in (G).
(I) Merge image of (G) and (H).
(J) Cortical ER in GFP:HDEL transformed cell.
(K) Anti-AtSec13 staining of cell in (J).
(L) Merge image of (J) and (K).
(M) High magnification of cortical ER (green, GFP:HDEL) with COPII visualized with anti-AtSar1 (red; colocalization indicated in yellow).
Bars ¼ 5 mm for all panels except (M) (1 mm).
1516The Plant Cell
preferentially associated with cisternae of the cortical ER net-
work (Figures 2G to 2L). At high magnifications, individual
punctate COPII fluorescent signals were often found sitting
directly on strands of ER (see arrows in Figure 2M). Using the
same parameters for the estimations obtained from Figure 2F,
the average diameter of these signals was 456 6 18 nm (n ¼ 43).
Interestingly, and in agreement with immunostaining for COPII
in mammalian cells (Rust et al., 2001), the colocalization (yellow)
of green (GFP:HDEL) and red (anti-AtSar1) signals is restricted
to narrow semicircular profiles on the ER. Punctate fluorescent
signals of a similar size were also seen lying adjacent to the ER
tubules. These could represent either ER-bound COPII out of
the plane of section or individual released COPII vesicles, as
suggested by Rust et al. (2001).
Establishment of a Tobacco BY-2 Cell Line
To visualize and study the dynamics of ERES in living cells, we
transformed BY-2 cells with Lycopersicon esculentum (Le)-
Sec13:GFP. To minimize potential toxic side effects related to
LeSec13-GFP overproduction, an inducible promoter system
was preferred to a 35S stable expression system for BY-2
transformation. However, to rule out the possibility that the
expression of this construct might influence the manifestation of
ERES, for example, by enhancing secretory activity through an
increased number of ERES, we examined the effects of the
Sec13:GFP construct on the secretory index (ratio of extracel-
lular to intracellular activities of a secretory enzyme; Phillipson
et al., 2001). To do this, we electroporated tobacco mesophyll
protoplasts with constant amounts of plasmid encoding for
mid DNA. As shown in Figure 3, Sec13:GFP had no effect on the
secretion index, even at high concentrations of plasmid DNA. As
in the GTP form. In agreement with the results of daSilva et al.
(2004), this mutant proved to be a potent inhibitor of secretion.
Thus, it would appear that the secretory pathway in tobacco is
not influenced by the overexpression of (X)FP-tagged COPII-
coat proteins (here, Sec13, and Sar1 in daSilva et al., 2004).
Beginning ;24 h after exposure to dexamethasone, the BY-2
cells were expressing the Sec13:GFP fusion construct in suffi-
cient quantities to allow for detection by confocal microscopy. In
optical sections taken through the cortex (Figure 4A), a dense
punctate image was obtained, similar to the antibody staining
generated with COPII antisera (Figure 2). When cells expressing
the GFP fusion construct were fixed and immunostained with
(Figures 4B to 4D), confirming the Sec13 identity of the protein
carrying the GFP signal. During cytokinesis, the LeSec13:GFP
signal in mitotic BY-2 cells is clearly seen to aggregate in and
around the phragmoplast (Figure 4E), a region in which Golgi
stacks are excluded (Figures 4H and 4I). Interestingly, when the
LeSec13:GFP signal is compared with that of coexpressed
BiP:DsRed (immunoglobulin binding protein cognate, a lumenal
of the two (Figures 4F and 4G). Although there is a clear coloc-
alization of the two fluorescent signals in the phragmoplast,
BiP:DsRed is absent from the reformed nuclear envelope. In
contrast with LeSec13:GFP, BiP:DsRed is localized to the di-
vision plane, suggesting that the ER that gets trapped in the
LeSec13:GFP reveal, in addition to punctate fluorescence
throughout the cytoplasm, an intense staining of the nuclear
envelope and a diffuse staining of the nuclear matrix except for
the nucleoli (Figure 4J). This, for us initially unexpected result, is
fully in keeping with the known behavior of Sec13 in other
eukaryotic cells. In yeast, it has been shown that, in addition to
being a COPII-coat protein, Sec13p is also incorporated into the
nuclear pore complex Nup84p (Siniossoglou et al., 2000). More-
over, in mammalian cells, Sec13 has recently been shown to
shuttle between the cytosol and nuclear matrix (Enninga et al.,
2003). As shown in Figure 5, we have performed on the
LeSec13:GFP BY-2 cell line the same kind of fluorescence
recovery after photobleaching (FRAP) experiments that were
nuclear rim was still not observed after 60 min when a region of
the nuclear envelope that also included part of the ER, cytosol,
and nuclear matrix was photobleached (Figures 5D and 5F). By
contrast, the pool of cytosolic and ER-associated LeSec13:GFP
recovered very rapidly (Figure 5C). When most of the area of
the nucleus was photobleached, a near complete recovery of
Figure 3. Expression of Sec13-GFP Is without Effect on Secretion.
Tobacco protoplasts were electroporated with a constant amount of
plasmid encoding for a-amylase together with increasing amounts of
plasmid encoding for LeSec13-GFP or for the GTP-blocked mutant
Sar1[H74L]. Standard deviations, as indicated by error bars, were
calculated from six separate experiments. Protein gel blots of total
cytosolic proteins, corresponding to the individual panels of the secre-
tion index histogram, are also given to document the gradual increase in
expressed effector protein in the protoplast homogenates. Note that in
marked contrast with LeSec13-GFP, the expression of the Sar1 mutant
leads to a drastic reduction in secretory activity.
COPII and Golgi1517
intranuclear LeSec13:GFP was detected after 10 min with a half-
time of ;2 min (Figures 4G to 4J). This was significantly slower
than the recovery of free GFP (half-time of 30 s). These results
suggest a very rapid exchange of LeSec13:GFP molecules
between the cytoplasm and the ER, a slower exchange between
the nucleus and the cytoplasm, and a high stability of the nuclear
pore complex-bound LeSec13:GFP. More importantly, they are
almost identical to those obtained by Enninga et al. (2003),
underlining yet again the credibility of the fluorescence signal we
obtained with the LeSec13:GFP construct.
Figure 4. Visualization of COPII in BY-2 Cells Expressing AtSec13:GFP.
(A) Punctate GFP signal in cell cortex. Inset is a high magnification.
(B) to (D) Antibody staining of AtSec13 in LeSec13:GFP cell line; GFP signal (B), immunofluorescence labeling (C), merged image (D).
(E) LeSec13:GFP fluorescence pattern in a mitotic cell (late anaphase). Inset is high magnification of area indicated in brackets.
(F) and (G) As for (E) but for telophase. (F), LeSec13:GFP fluorescence; (G), as for (F) but revealing coexpressed BiP:DsRed.
(H) and (I) Distribution of Golgi stacks during mitosis. (H), LeSec13 fluorescence; (I), Golgi stacks revealed by expression of GmManI-RFP.
(J) Median optical section revealing a distinct staining of the nuclear envelope and nuclear matrix, in addition to punctate signals in the cytoplasm.
Bars ¼ 5 mm.
1518 The Plant Cell
To gain further insights into the dynamics of the LeSec13:GFP
fluorescence, LeSec13:GFP expressing cells were visualized by
time-lapse microscopy (Figure 6). Because of the rapidity at
based confocal microscopy proved superior to conventional
confocal laser scanning microscopy for this analysis. With this
imaging technique we were able to continuously observe
changes in cortical LeSec13:GFP for several minutes without
any apparent photobleaching (Figure 6; see Supplemental Video
1 online). Taken separately, individual frames appeared very
similar to the images obtained with fixed cells (cf. Figures 5 and
4A): numerous punctate fluorescent structures that averaged
430 6 220nm (n> 900) in diameter with a density of 5.0 punctate
structures?mm?2were observed. A comparison between suc-
cessive frames revealed that only very few point light sources
could be followed for any significant length of time, making an
estimation of their movement impossible. Thus, on successive
frames (arrowheads and circles, Figure 6A), the point light
sources appeared immobile. We therefore conclude that COPII
binding has a very short half-life (at the most 10 s). This results in
a blinking rather than moving appearance of LeSec13:GFP
Effects of Secretory Inhibitors on COPII Immunostaining
and LeSec13:GFP Fluorescence
The physiological relevance of the COPII visualizations de-
scribed above can be tested by analyzing the effects of sub-
stances known to perturb the secretory pathway. Because the
dependency of ER export on Sar1-GTP had already been
Figure 5. Analysis of LeSec13:GFP Dynamic by FRAP.
(A) to (D) FRAP analysis of LeSec13:GFP present within the nuclear envelope, ER, and cytosol. LeSec13:GFP fluorescence was monitored before (A),
immediately after photobleaching (B), as well as 1 min (C) and 60 min (D) after recovery.
(E) and (F) Detailed views of the boxed regions shown in (A) and (D), respectively.
(G) to (J) FRAP analysis of nuclear LeSec13:GFP.
(G) Plots show fluorescence recovery of nuclear LeSec13:GFP versus free GFP in the bleached area. Images were acquired every 30 s. Measurements
of fluorescence intensity were subtracted from background fluorescence and normalized from loss of fluorescence during bleaching and imaging. Error
bars are standard deviations (n ¼ 3).
(H) to (J) The images show single optical sections of LeSec13:GFP present within the nucleus before (H) and immediately after photobleaching (I) as
well as 10 min after recovery (J). Bleached areas are circled in white.
Bars ¼ 5 mm.
COPII and Golgi 1519
established (Takeuchi et al., 2000; Phillipson et al., 2001), we
decided to examine the consequences of expression of
Sar1[T39N], a mutated form of Sar1 fixed in the GDP form. To
confirm that Sar1[T39N] blocks secretion in BY-2 cells, we
coexpressed a secretory form of RFP (SP:RFP). In control cells,
SP:RFP is secreted into the membrane but also accumulates in
the vacuole (Figures 7A to 7C). When Sar1[T39N] is expressed,
SP:RFP is retained in the ER (Figures 7D to 7F). Under these
conditions, a smeared rather than a clear punctate image for
attribute this imperfect COPII image to impaired recruitment of
Sec13/23 as a result of insufficient Sar1-GTP.
The macrocyclic lactone brefeldin A (BFA) is often used to
block secretion (reviewed in Nebenfu ¨hr et al., 2002). In addition
to perturbing the Golgi apparatus, it has been suggested that
BFA might also directly affect ER export (Brandizzi et al., 2002).
This claim has recently found support in the observation that
the distribution of punctate Sar1-YFP structures on the ER of
tobacco epidermis is changed upon addition of BFA (daSilva
et al., 2004). We therefore decided to investigate what effects
BFA has on the COPII structures wehave visualized in BY-2 cells
by applying the drug to the LeSec13:GFP cell line expressing the
cis-Golgi marker GmManI-RFP. Treatment with 10mg?mL?1lead
to a complete redistribution of the RFP-tagged protein into the
structures (Figures 8A to 8F). Similarly, BFA had no effect on
COPII visualization when performed by immunostaining with
AtSec13 antibodies (Figures 8G to 8I).
Figure 6. Analysis of LeSec13:GFP Dynamic by Time-Lapse Microscopy.
(A) to (D) Cortical LeSec13:GFP was visualized using an UltraVIEW RS confocal microscope. Single optical sections images were acquired every 1.2 s
for 27.6 s (see Supplemental Video 1 online). Images taken at 0, 1.2, 13.2, and 27.6 s are presented ([A] to [D], respectively). Arrowheads and circles
indicate LeSec13:GFP punctate structures that remained immobile for at least 1.2 and 13.2 s, respectively. Bars ¼ 5 mm.
(E) Number of fluorescent foci in each individual frames of the video were calculated using the analyze particles command in ImageJ and were plotted
on a graph.
1520The Plant Cell
Sec13:GFP Fluorescence in Relation to the ER and Golgi
in Living BY-2 Cells
Simultaneous fluorescence imaging of the ER and Golgi appa-
ratus in cells expressing LeSec13:GFP is possible when these
organelles are made visible with YFP or RFP constructs. We
therefore prepared BiP:DsRed and GmMan1:RFP as ER and
Golgi markers, respectively. The validity of BiP:DsRed as an ER
marker was confirmed by bombarding transgenic BY-2 cells
expressing GFP:HDEL. The typical cortical ER network was
revealed by both fluorescent markers and showed complete
colocalization (data not shown). An identical image was also
obtained when BY-2 cells transiently expressed Sec12-YFP,
the GEF for Sar1 (Figures 9A to 9C). When the inducible
LeSec13:GFP cell line was bombarded with BiP:DsRed, the
punctate green COPII coats were highlighted against the tubular
red ER network (Figures 9D to 9F). However, as was the case
with the immunostaining for COPII-coat proteins (see Figures 2G
to 2M), many of the LeSec13:GFP punctate signals lay adjacent
to the ER tubules.
To confirm the validity of GmMan1-RFP as a Golgi marker, we
bombarded transgenic BY-2 cells expressing GmMan1:GFP
with GmMan1-RFP. Again, a perfect colocalization with both
cell line was then bombarded with GmMan1-RFP. Optical
sections in the cortical region of a cell are presented in Figures
9G and 9H . As with the antibody staining data presented above,
the LeSec13:GFP fluorescent points greatly outnumbered the
Golgi stacks (Figure 9I). However, some of the Golgi stacks were
densely surrounded at their periphery by LeSec13:GFP fluores-
cence, giving rise to a kind of halo (see the group of Golgi stacks
in the middle of Figure 9I, and Figure 9K), whereas others were
relatively free (Figure 9J).
Figure 7. Effects of Sar1[T39N] Secretory Inhibitors on LeSec13:GFP.
(A) to (F) LeSec13:GFP-expressing cells were transformed by biolistics so as to express SP:RFP alone ([A] to [C]) or together with Sar1[T39N] ([D] to
[F]). LeSec13:GFP distribution ([A] and [D]); SP:RFP distribution ([B] and [E]). Note that SP:RFP is vacuolar in the absence of Sar1[T39N] (B) and is
retained in the ER upon Sar1[T39N] expression (E). (C) and (F) are merged images corresponding to (A) and (B) and (D) and (E), respectively.
(G) to (J) Effect of Sar1[T39N] in cells coexpressing LeSec13:GFP and GmManI-RFP. LeSec13:GFP signal (G); GmManI:RFP signal (H). Note that
GmManI:RFP is redistributed into the ER. (I) and (J) are merged images showing a median (I) and a cortical single optical section (J).
Bars ¼ 5 mm.
COPII and Golgi 1521
COPII Associates with Mobile Golgi Stacks at Their Rims
To analyze the relationship between COPII and Golgi stacks at
greater resolution, we have observed live BY-2 cells expressing
LeSec13:GFP and GmMan1-RFP by dual wavelength micros-
copy in a Nipkov confocal laser scanning microscope. The
results are presented in the form of a movie of 183-s duration
(see Supplemental Videos 2 and 3 online), from which three
frames (0, 61, and 183 s) are presented in Figures 10A to
10C. Three features are immediately apparent from these se-
quences. First, in agreement with data presented above,
LeSec13:GFP fluorescent points greatly exceeded Golgi stacks
(RFP-fluorescence) in number. Second, in confirmation of the
observations of Nebenfu ¨hr et al. (1999), not all Golgi stacks were
simultaneously in movement. Thirdly, Golgi stacks were more
often encountered with peripheral COPII fluorescence than not.
Nevertheless, there are clear examples where a single Golgi
stack moved into the plane of vision lacking associated Le-
Sec13:GFP fluorescence but then was seen to be completely
surrounded by them a few seconds later (cf. frames 34.4, 37.1,
and 39.7 s in the third row of Figure 10B). We have also seen
examples where a single Golgi stack, immobile for a period of
many seconds, was present with and without COPII fluores-
LeSec13:GFP fluorescence surrounding an individual Golgi
stack was plotted against the speed of movement of the stack
(Figure 11), it became apparent that the slower a Golgi stack
moves, the greater was the degree of COPII association. In
surface view (i.e., looking at a stack from above or below) Golgi-
associated COPII fluorescence appeared in the form of a partial
or complete corona (see the third row of frames in Figure 10B,
and Figure 10C). Rarely did we find images where the GFP and
RFP signals were superimposed. Golgi stacks were also fre-
quently seen in side view, where the RFP signal took the form of
a cigar. Very often, however, the signal was only partially visible
as red, and more often was yellow—the merge color. This
observation indicates that COPII–Golgi interactions take place
results also showthatGolgi stacks do not havea fixedorientation
with respect to the ER: as already reported by Nebenfu ¨hr et al.
(1999), they were observed to tumble as they move.
Figure 8. Effect of BFA on LeSec13:GFP.
(A) to (F) LeSec13:GFP cells were transformed by biolistics so as to coexpress GmManI:RFP and were monitored before ([A] to [C]) and after a 20-min
treatment with 10 mg?L?1BFA ([D] to [F]). BFA treatment led to the complete redistribution of GmManI:RFP from the Golgi (A) into the ER (D).
Covisualization of LeSec13:GFP (green) and GmManI:RFP (red) at low magnification and high magnification is shown in (B) and (E) and (C) and (F),
(G) to (I) Immunodetection of Sec13 in GmMan1:GFP cells treated for 60 min with BFA. GmMan1:GFP signal (G); anti-AtSec13 immunolabeling (H);
corresponding merged image (I).
Bars ¼ 5 mm.
1522 The Plant Cell
Figure 9. Labeling of ER and Golgi in BY-2 Cells Expressing LeSec13:GFP.
(A) and (B) Cortical ER in a cell coexpressing AtSec12-YFP (A) and AtBiP:DsRed (B).
(C) Merge image for the cell depicted in (A) and (B).
(D) An LeSec13:GFP expressing cell bombarded with AtBiP:DsRed; channel selected for green fluorescence.
(E) The same cell as in (D), but channel selected for red fluorescence.
(F) Merge image for cell depicted in (D) and (E).
(G) COPII labeling in a cell expressing LeSec13:GFP, which was bombarded with GmManI-RFP; channel selected for green fluorescence.
(H) The same cell as in (G), but channel selected for red fluorescence.
(I) Merge image for cell depicted in (G) and (H).
(J) and (K) High magnifications of two regions in (I) showing low and high density association of LeSec13:GFP with Golgi stacks.
Bars ¼ 5 mm for all panels except (J) and (K) (1 mm).
COPII and Golgi1523
Figure 10. Analysis of LeSec13:GFP in Relation to the Golgi Apparatus by Time-Lapse Microscopy.
LeSec13:GFP-expressing cells were transformed by biolistics to coexpress GmManI:RFP and were monitored using an UltraVIEW RS confocal
(A) and (B) Single optical sections images were acquired approximately every 2.6 s for 182.7 s (see Supplemental Video 2 online). LeSec13:GFP (green
signal); GmManI:RFP (red signal).
(A) Images taken at 0, 60.9, and 182.7 s.
(B) Detailed views of the boxed area visible in Supplemental Video 2 online and in (A). Numbers correspond to the time (in seconds). Arrowheads point
to a single Golgi stack during four consecutive frames. Note that at 34.4 s very little LeSec13:GFP is associated with the Golgi stack, whereas at 42.4 s
the same Golgi stack is completely surrounded by a halo of LeSec13:GFP.
(C) High-magnification pictures of Golgi stacks in face and side view (left and right panels, respectively), showing variable levels of association with
(D) Maximum intensity projection images of a cell coexpressing LeSec13:GFP and GmMan1:GFP viewed at a 18.4-s interval. Arrows point to the same
Golgi stack that shows variable levels of association with LeSec13:GFP during time.
Bars ¼ 5 mm in (A) and (D) and 1 mm in (B) and (C).
1524The Plant Cell
COPII Visualization and Higher Plant ERES
The COPIIcoat on ERESinmammalian cells isknownto develop
sequentially: first by recruitment of Sar1-GTP to Sec12, then the
sequestration of Sec23/24, followed by the binding of Sec13/31
(Barlowe, 2003; Bonifacino and Glick, 2004; Lee et al., 2004).
Membrane-bound Sec13/31 should therefore represent fully
assembled ERES and is the reason why the bulk of our ob-
servations have been made with Sec13 antibodies or with a
transiently expressed LeSec13:GFP construct. Nevertheless,
antibody staining with anti-Sar1 and anti-Sec23 gave rise to
very similar patterns of COPII visualization. The validity of
LeSec13:GFP as a marker for COPII is not only supported by
its colocalization with Sec13 antibodies and by its localization to
the nuclear envelope and nuclear matrix, which is the same for
Sec13 in mammalian cells (Enninga et al., 2003), but also by its
FRAP behavior. In addition, the expression of Sec13:GFP has no
effect on the secretion index of protoplasts, whereas the ex-
pression of Sar1 mutants prevent export out of the ER and in the
case of the Sar1-GDP mutant lead to an altered pattern of
Although the interaction between Sec12 and Sar1-GTP is
pivotal to the formation of ERES, Sec12 is excluded from COPII
vesicles induced in vitro (Barlowe et al., 1994; Barlowe, 2002). In
experiments when Sec12-GFP is expressed transiently, it would
appear that the COPII GEF is distributed uniformly throughout
has been described for S. cerevisiae (Rossanese et al., 1999), for
mammalian cells (Weissman et al., 2001), and most recently for
tobacco epidermal (daSilva et al., 2004) and BY-2 cells (this
article). However, such fluorescent patterns are not in accor-
dance with the discrete punctate visualization of COPII coat
components as seen here and reported on numerous occasions
elsewhere (Shugrue et al., 1999; Hammond and Glick, 2000;
Stephens et al., 2000; Rust et al., 2001). An explanation for this
discrepancy is not immediately apparent.
In contrast with Sec12, which is an integral membrane protein,
Sar1 and the other COPII-coat proteins cycle on and off the ER
membrane. As a consequence, these proteins can be detected
proteins, and theoretically on released COPII vesicles. Because
of their lower local concentrations and higher diffusion rates,
fluorescently labeled cytosolic COPII proteins give a weak,
uniform, and diffuse signal, as seen in the nuclear matrix for
Sec13 (Figures 4J and 5H to 5J). A similar diffuse cytosolic
fluorescence can be observed for COPI components, especially
after their release from Golgi membranes upon BFA treatment
(Ritzenthaler et al., 2002a). This type of fluorescence is different
to the punctate fluorescence seen for all COPII components.
represent released COPII vesicles, as suggested by Rust et al.
A consistent observation in our investigation, and one which
contradicts the data presented by daSilva et al. (2004), is that
COPII binding sites that we have visualized at the surface of the
ER greatly outnumber that of the Golgi stacks. Although Golgi
stacks temporarily associate with COPII principally at their rims,
it is not clear whether the visualization of COPII binding is
sufficient to allow these sites to be defined as ERES. This can
or lumenal) collect and exit from the ER at these sites. For the
moment, we can therefore only regard the punctate COPII sites
as being putative ERES. If each were an ERES, this would mean
that COPII vesicles are formed and released with extreme
rapidity because our live cell imaging data suggest that COPII
cycles on and off the ER within seconds. However, the continual
formation and release of COPII vesicles does not appear to be
very plausible because it is difficult to understand how mobile
Golgi stacks could efficiently collect this released cargo. An
alternative scenario is that the punctate COPII sites are potential
ERES, but the completion of vesicle budding and release is only
triggered upon arrival and docking of a Golgi stack. As a conse-
quence, this would mean that a large portion of the COPII-coat
proteins are involved in futile cycles of binding and dissociation.
At the least, this would indicate that Sec13, and the other COPII-
coat proteins for that matter, are not a limiting factor in the ER-
to-Golgi transport in plants. Indeed, their overexpression, as
demonstrated here and by daSilva et al. (2004), is without effect
Randomly distributed ERES in mammalian cells are relatively
immobile (displacement time of 5 to 15 mm?h?1; Stephens, 2003)
in comparison with the rate of cargo transport between the
cortical ER and the perinuclear Golgi apparatus (0.5 to 1 mm?s?1;
Stephens et al., 2000). As previously mentioned, ERGIC/VTCs
are responsible for this long-range transport, and these are
generally considered to arise from the homotypic fusion of COPII
vesicles (Stephens and Pepperkok, 2001;Duden, 2003).It would
appear that each ERGIC/VTC is formed from a single ERES
(Stephens et al., 2000). Upon completion of mitosis in mamma-
lian cells, ERES form de novo (frequency: 2 h?1?100 mm?2) and
Figure 11. Analysis of Golgi COPII Association in Relation to Golgi
The net velocity of five individual Golgi stacks (from Supplemental Video
2 online) was measured at different times and plotted against the
associated LeSec13:GFP fluorescence intensity in a fixed circular
5-mm2area around each Golgi stack.
COPII and Golgi1525
minutes (Stephens, 2003). During this time, COPII proteins
continually cycle on and off the membrane but with different
and Sec13/31; R. Forster, D. Stephens, and R. Pepperkok,
fuse with one another and divide. In common with mammalian
cells, ERES in BY-2 cells are quite stationary, but they appear to
be more dynamic structures than their mammalian counterparts:
individual ERES were rarely visible for periods longer than a few
seconds. Because of this property, it was not possible to as-
certain whether plant ERES aggregate and/or divide. It could
also be the reason why the visualization of COPII budding in
plants by electron microscopy has been so elusive.
It is well known that during mitosis the Golgi apparatus in
mammalian cellsbreaksdownintovesicles (ShorterandWarren,
2002). It has been claimed that these vesicles, together with
Golgi matrix proteins that are required as a scaffold for the
reconstitution of the Golgi apparatus at the onset of the sub-
sequent interphase, lie in close proximity to ERES whose
function is arrested during mitosis (Prescott et al., 2001;
Seemann et al., 2002). Immunostaining with COPII antisera has
(Prescott et al., 2001). However, the recently published data of
Stephens (2003) indicates that this visualization is artifactual in
nature: live cell imaging with YFP-Sec23 clearly showed a dis-
placement of COPII into the cytosol during mitosis. In plants, the
Golgi apparatus does not fragment during mitosis, and in BY-2
cells, many Golgi stacks appear to be immobilized in the
immediate vicinity of the mitotic spindle (Nebenfu ¨hr et al.,
2000). However, during telophase, the plant Golgi apparatus is
particularly active in secreting to the forming cell plate. As shown
by Segui-Simarro et al. (2004), Golgi stacks enter the phragmo-
plast between the daughter nuclei during late telophase, where
increasing amounts of ER are also to be found. Such a stage is
comparable to that depicted in Figures 3F and 3G. Consistent
with this are our results, obtained by live cell imaging with
LeSec13:GFP, which demonstrate that ERES are visible within
the phragmoplast and are presumably functionally intact.
BFA and ERES
BFA has been a most useful tool in investigations into the
secretory and endocytic pathways (reviewed in Nebenfu ¨hr
et al., 2002). Research on mammalian and fungal cells has
established that this drug interacts with a complex formed
between the GTPase ADP ribosylation factor (ARF) and its GEF
(Peyroche et al., 1999). The discovery that the Arabidopsis
protein GNOM, which is important for the correct targeting of
the auxin efflux carrier PIN1 to the plasma membrane, is a BFA-
likely that the molecular target for BFA is the same for all
ARF-GEFs have so far not been reported at the ER in any cell
type, so claims that BFA can act at the level of ERES (Brandizzi
et al., 2002; daSilva et al., 2004; Hawes and Brandizzi, 2004)
require experimental substantiation. Our results showing that
are in agreement with anti-Sec31 staining data obtained on NRK
cells (Puri and Linstedt, 2003). However, as such these results
say nothing about the export competence of the ERES so
visualized. Ward et al. (2001) previously showed that COPII
components still cycle at ERES after addition of BFA. More
recent FRET measurements performed on Vero cells indicate
that BFA interferes with the kinetics of the interaction between
Sec23 and Sec31, whereas the interaction between Sar1
and Sec23 remained unaltered (R. Forster, D. Stephens, and R.
Pepperkok, unpublished data). However, treatment with BFA for
short periods, during which time COPI assembly was inhibited,
did not alter the steady state distribution of any COPII compo-
nent. Thus, it seems likely that any effect of BFA on ER export is
an indirect one resulting from a perturbation in the fine tuning of
2000; Ward et al., 2001), upon whose maintenance successful
ER–Golgi transport depends.
ERES and the Golgi Apparatus: Models and Data
Currently, there are three models that have been put forward to
explain ER-to-Golgi transport in plants (Neumann et al., 2003). In
the earliest of these, Golgi stacks were considered to sweep up
export vesicles as they moved over the surface of the ER (the
vacuum cleaner model; Boevink et al., 1998). By contrast, the
stop-and-go model of Nebenfu ¨hr et al. (1999) foresees cargo
collection restricted only to those Golgi stacks that have tem-
porarily come to a halt over an ERES. According to the third,
mobile secretory unit concept (Neumann et al., 2003), each
individual Golgi stack has its own ERES and both travel together
across the surface of the ER. This means that ERES and Golgi
stacks must be identical in number and intracellular distribution,
that both are motile, and, finally, that ERES should be long-lived
Support for this latter model has recently been presented
by daSilva et al. (2004), who have investigated the distribution
of AtSar1-YFP, AtERD2-GFP, and the Golgi marker ST-GFP by
transient expression in tobacco epidermis. According to these
authors, these two constructs were constantly located together
into ‘‘distinct but overlapping structures,’’ and that this tandem
structure was mobile in an actin-dependent manner. A degree
of permanency for this structure was suggested by selective
photobleaching of the YFP signal that inevitably recovered in
the immediate vicinity of the GFP signal. In complete contradic-
ERES can attach to a single Golgi stack at any one time and that
ERES are not constantly associated with the Golgi apparatus,
assuming of course that each punctate COPII labeling repre-
sents one ERES (see above). Moreover, Golgi-ERES associa-
tions are not permanent but are continually changing in number
and position at the rims of the stack as the Golgi moves. Another
important distinction between our data and that of daSilva et al.
images. In the latter article, these two structures appear to be of
similar size, but in our work, ERES appear significantly smaller.
Thus, our observations are more in keeping with a kiss-and-run
model for ER-to-Golgi transport.
1526The Plant Cell
A feature common to the vacuum cleaner and stop-and-go
models is that ERES outnumber Golgi stacks and are relatively
stationary. This is supported by the data presented here.
However, in the sense that ERES are seen at the rims of both
stationary and moving Golgi stacks in BY-2 cells, our data do not
exactly conform with the latter model. Indeed, we have demon-
period both with and without associated peripheral ERES. Thus,
Golgi motility per se does not seem to be a precondition for
successful ER-to-Golgi transport, and this is in agreement with
FRAP measurements dealing with the recovery of photo-
bleached Golgi marker proteins on immobilized (Brandizzi et al.,
2002) and moving (Brandizzi and Hawes, 2004) Golgi stacks.
Mobility is a crucial feature of the mobile secretory unit model,
even though it remains unclear as to how a Golgi stack and its
associated ERES remain together during movement. However,
this is important because if true it would mean that a Golgi stack
would have to drag its ERES through a lipid membrane. In our
opinion, this can only be achieved through direct physical
continuities (tubular ER-Golgi connections) or through the exis-
tence of some kind of scaffolding elements linking the two
together. The former possibility has previously been suggested
(Brandizzi and Hawes, 2004; Hawes and Brandizzi, 2004), but
always in neglect of retrograde COPI vesicle transport, which
2002a). Evidence for the latter is lacking. With regard to Golgi
motility we would like, in addition, to point out some inconsis-
tencies in the data published using the tobacco leaf epidermis
and BY-2 cell systems. According to Boevink et al. (1998), Golgi
stacks in epidermal cells move with speeds of up to 0.76 mm?s?1
along stationary cortical ER and in excess of 2.2 mm?s?1within
transvacuolar cytoplasmic strands. In the article by daSilva
et al. (2004), the speed of Golgi stacks lies between 0.1 and
0.3 mm?s?1(calculated from the values given in Figure 9 of
that article). These latter values contrast with those determined
for Golgi stacks in BY-2 cells (;3 mm?s?1) by Nebenfu ¨hr et al.
(1999) and confirmed here. In fact, such low velocities are in the
range of the almost stationary wiggling motion described by
Nebenfu ¨hr et al. (1999) for BY-2 cells. Because our data suggest
that the degree of Golgi-COPII association increases the slower
the Golgi moves, which is understandable, we would therefore
plead for more caution in the interpretation of the temporal
aspect of ERES-Golgi associations.
The differences in the depiction of the ERES and Golgi stacks
as given in our article and that of daSilva et al. (2004) may well lie
in the relative secretory status of the cell types employed in the
two studies. Tobacco BY-2 cellsrepresent a rapidly growing and
dividing cell system with a high rate of secretion. Tobacco leaf
epidermal cells, on the other hand, hardly grow, do not divide,
and will obviously be secreting at a much lower level. Thus, one
might consider the leaf epidermis to represent a kind of minimal
system, with membrane trafficking to the cell surface and within
the endomembrane system being reduced to a housekeeping
role. In this situation, ER export will not be comparable to that in
BY-2 cells: the number of ERES may well be reduced to a level
where their number approximates that of the Golgi stacks. This
being the case, it is probably more efficient to have a Golgi stack
hovering in the vicinity of a more or less stationary ERES than to
be rapidly wandering across the surface of an ER with few exit
sites. By contrast, a situation where ERES greatly outnumber
Golgi stacks would have advantages for a rapidly secreting
to be sorted at the ER and secreted per unit of time. It also would
be more robust toward distortions because missing a few events
would not matter, whereas the secretory unit model would re-
quire much more stringent regulatory mechanisms.
Suspension cultures of Bright Yellow 2 (BY-2) tobacco (Nicotiana
tabacum) were maintained at 278C in MS medium (Sigma-Aldrich,
Taufkirchen, Germany). Arabidopsis thaliana var Columbia cell suspen-
sions were cultured in Gamborg’s B5 Medium (Sigma-Aldrich G5893).
Cells were maintained in the log phase by subculturing weekly into fresh
medium at a dilution of 1:50. In addition to wild-type cells, two stably
transformed BY-2 cell lines were also employed: one expressing the
Golgi marker a-mannosidase 1-GFP from Glycine max (GmMan1:GFP;
Nebenfu ¨hr et al., 1999); the other expressing the ER-localized fusion
construct GFP:HDEL (Nebenfu ¨hr et al., 2000). For secretory index
determinations (see below), tobacco plants (N. tabacum cv Petite
Havana) were grown on agar under sterile conditions as given in Crofts
et al. (1999) and daSilva et al. (2004).
Generation of Recombinant Proteins and Preparation of Antisera
of affinity-purified antibodies (from rats, AtSec13; from rabbits, AtSec23)
have previously been given (Movafeghi et al., 1999; Contreras et al.,
2004). AtSec12b (accession number BAB09140, Arabidopsis Biological
Resource Center, The Ohio State University, Columbus, OH) was
amplified from the CD4-7 cDNA library. The cytosolic domain was cloned
into the EcoRI and SmaI sites in pGEX4T3 (Amersham Biosciences
Europe, Freiburg, Germany). A GST fusion protein was produced and
purified from Escherichia coli as previously described (Movafeghi
et al., 1999). Polyclonal antibodies in rats were generated commercially
(Biosciences, Go ¨ttingen, Germany) and affinity purified using a GST-
AtSec12b-Sepharose 4B column.
Stable Transformation of BY-2 Cells with LeSec13:GFP
A tomato (Lycopersicon esculentum) EST clone (accession number
AI776423) closely homologous to AtSec13 was ordered from Clemson
amplified by PCR and cloned into pCK(X/S) LTEV-EGFP (Ritzenthaler
et al., 2002b) to obtain pCK-LeSec13-EGFP. The LeSec13-EGFP was
then subcloned into the pTA7002 vector (Aoyama and Chua, 1997) for
creating an inducible LeSec13-EGFP stable transformant. PTA7002-
LeSec13-EGFP was introduced into the LBA4404 strain of Agrobacte-
rium tumefaciens by the freeze-thaw method (An et al., 1988).
Transformation of BY-2 cells was done by coculturing a 3-d-old BY-2
culture with a 36-h-old LBA4404 culture. The coculture was incubated in
the dark without shaking for 2 d at 258C. The cells were pelleted and
washed three times in fresh medium containing 250 mg/mL of carbeni-
cilin. After a final wash, the suspension was poured on the solid medium
containing carbenicilin and hygromycin. Positive calli (obtained after
several weeks) were screened by fluorescence microscopy to select the
best cell lines. Expression of LeSec13:GFP in the BY-2 cells was initiated
COPII and Golgi1527