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The Plant Journal (2006) 46, 95–110 doi: 10.1111/j.1365-313X.2006.02675.x In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery Giovanni Stefano1,†, Luciana Renna1,†, Laurent Chatre2,†,‡, Sally L. Hanton1, Patrick Moreau2, Chris Hawes3 and Federica Brandizzi1,* 1 Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada, 2 Laboratoire de Biogenèse Membranaire, UMR5200, CNRS, Université de Bordeaux 2, Bordeaux, France, and 3 Research 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 between the endoplasmic reticulum (ER) and the Golgi apparatus (Brandizzi et al., 2002; Denecke et al., 1992; Hanton 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 (Andreeva et al., 2000; Bar-Peled and Raikhel, 1997; Phillipson ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd 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 retrograde 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 95 96 Giovanni Stefano et al. 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 (Movafeghi 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 nonGolgi 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 reabsorbance of Golgi membrane proteins into the ER (Brandizzi 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 differentiation 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 (Robineau 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 Arf1mediated 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 epidermal cells, a green fluorescent fusion (GFP) of an Arabidopsis 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 coexpression experiments with a yellow fluorescent protein (YFP) fusion to the Golgi marker ERD2 (ERD2-YFP; Brandizzi 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 eventually detached from it (Figure 1b and Supplementary data 1). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 97 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. 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 Arf1GFP structures (Figure 2b). This suggests that COPI coatomer 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 exchange of protein cargo between the ER and Golgi apparatus, 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 exchanging 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 calculated 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 continuously 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 effects 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 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 98 Giovanni Stefano et al. 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. markers (Brandizzi et al., 2002; daSilva et al., 2004). Therefore, 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 sequences 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 comparable results, we used the same optical density of Agrobacterium 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. Figure 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). Coexpression of Arf1GDP with ERD2-YFP caused re-absorbance 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 distributed 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 membranes appeared enlarged in those cells where Golgi membranes 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 sialyltransferase transmembrane domain (ST-YFP; Brandizzi et al., 2002) or an Arabidopsis b1,2-xylosyltransferase cytosolic 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 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 99 (a) 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 (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). 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 To determine whether the Arf1 mutants perturbed the distribution 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 (Supplementary data 4). GFP fusions of the inactive and active mutants of Arf1 (Arf1GDP-GFP and Arf1GTP-GFP, respectively) were expressed 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 distributed to punctate structures in most cells (Figure 5b), corresponding well with observations by Xu and Scheres (2005). To determine the identity of the structures highlighted by Arf1GTP-GFP, we co-expressed the fusion with ERD2YFP. 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 coincided 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 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 100 Giovanni Stefano et al. (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. 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 untagged Arf1GDP with either Arf1-YFP or eCOP-YFP. This caused a cytosolic distribution of both markers (Figure 7b,d), in comparison with the controls (Figure 7a,c; see also Supplementary data 5). These results suggest that Arf1GDP 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 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 101 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. 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 apparatus. Photobleaching of ERD2-YFP fluorescence in the presence of the mutant was followed by recovery (halftime 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. ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 102 Giovanni Stefano et al. 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). 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 fluorescence 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-mediated protein transport with a reduced activity in comparison with wild-type Arf1. on the interaction of a recombinant GST-Sec23 with plantexpressed 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 (Figure 10a, arrowhead) and in the cytosol (Figure 10a). Coexpression 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 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 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). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 103 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, ERD2GFP (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 YFPSec24 (f) and as merged images (g). Scale bar in (a), (b) ¼ 5 lm; (e) ¼ 2 lm. peri-Golgi area (6.7% of YFP-Sec24-labelled structures; Figure 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 expression 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 (Supplementary data 7d). Similar results were achieved with a fluorescent fusion of another COPII component, Sec23 (YFPSec23; Supplementary data 7d and 8, and data not shown), although the percentage of Sec23 structures that did not colocalize was lower than that of Sec24 (3.7%). These data reveal that in tobacco leaf epidermal cells COPII markers can label ERES without over-expression of Golgi marker proteins, 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). However, YFP-Sec24 was distributed into the cytosol in coexpression 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 maintained 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 (Supplementary data 8). These data indicate that collapse of COPImediated 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 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 104 Giovanni Stefano et al. 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. Arf1, suggesting that coatomer dissociation does not coincide 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 mammalian cells (Presley et al., 2002). Arf1-GFP was found to localize at the Golgi apparatus with coatomer, and also at additional structures that lack coatomer and originate from the Golgi apparatus. These structures may represent the trans Golgi network (TGN) or TGN-derived structures that detach from the Golgi apparatus, 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 structures, and it is also possible that the Golgi-derived structures may eventually mature to become, or merge with, endosomal 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 ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 105 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 involvement of Sec24 in cargo selection for incorporation into COPII vectors (Miller et al., 2002). We cannot exclude the possibility 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, accumulate 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 correlative 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 recruitment (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-Jeunemaitre, 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 demonstrated 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). Therefore, it is reasonable to suggest that Arf1 mutants may block the retrograde pathway. The interference with the anterograde transport of membrane proteins to the Golgi apparatus may then be an indirect effect mediated by Arf1 mutants on protein export from the ER. ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 106 Giovanni Stefano et al. 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 generally 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 movement 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 membranes, 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 differentiation 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 SatiatJeunemaitre, 2005). Experimental procedures Molecular cloning Standard molecular techniques were used for subcloning. The fluorescent proteins used in this study were based on fusions with either mGFP5 (Haseloff et al., 1997), ECFP or EYFP (Clontech Inc., Palo Alto, CA, USA). The spectral properties of mGFP5 allow efficient spectral separation from YFP (Brandizzi et al., 2002). As a Golgi 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-YFP construct, the DNA sequence encompassing the cytoplasmic tail and transmembrane region of an Arabidopsis b1,2-xylosyltransferase (DNA kindly provided by H. Steinkellner, Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Austria) that have been shown to target the Golgi apparatus in Nicotiana benthamiana as a GFP fusion (Dirnberger et al., 2002) was amplified by PCR and subcloned into pVKH18En6 upstream of a YFP sequence using XbaI and SalI sites of the binary vector. For coatomer labelling, we generated a YFP fusion with the Arabidopsis homologue of eCOP, a component of the COPI coatomer (Kreis et al., 1995). The coding sequence for eCOP (Genbank accession number AF370325) was obtained from RIKEN and amplified by PCR with primers containing the XbaI and SalI sites for subcloning upstream of YFP in the binary vector pVKH18-En6. To generate fluorescent fusions of Arf1 proteins (Pimpl et al., 2003), we used the DNA of the GTPases kindly provided by J. Denecke (University of Leeds, Leeds, UK) and spliced it upstream of a fluorescent protein sequence in the binary vector pVKH18-En6 with the XbaI and SalI sites. For labelling of ER export sites, the Arabidopsis homologues of Sec24 (locus At3g07100) and Sec23 (locus At3g23660) were obtained as ABRC clones and fused to the N-terminus of a YFP using the BamHI and SacI sites of the binary ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 COPI influences ER protein export 107 vector pVKH18-En6. For Agrobacterium tumefaciens transient expression of untagged Arabidopsis Arf1 and its GDP and GTP restricted mutants (Pimpl et al., 2003), we used a modified binary vector pVKH18-En6 bearing two 35S-pNOS reading cassettes in direct orientation (see also Supplementary material 2). In one cassette we subcloned the DNA coding sequence of the proteins within the unique XbaI and SacI sites, and in the other cassette we subcloned a CFP-SKL fusion to ensure visualization of peroxisomes. The primer sequences used for the subcloning indicated above 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) were used. Plant material and transient expression systems Four-week-old Nicotiana tabacum (cv. Petit Havana) greenhouse plants grown at 25C were used for Agrobacterium tumefaciens (strain GV3101)-mediated transient expression (Batoko et al., 2000). The bacterial optical density (OD600) used for plant transformation was 0.02–0.05 for tagged and untagged versions of Arf1 and its mutants, 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 and 2% sucrose in a controlled room at 25C with a 16 h light/8 h dark regime at a light irradiance of 200 mE m)2 sec)1. Tobacco leaf protoplast preparation and subsequent DNA transfection via electroporation were performed as described by Phillipson et al. (2001), and the plasmid concentrations used are given in Figure 4(b). For these experiments we used untagged Arf1 proteins subcloned in expression vectors described by Pimpl et al. (2003) and tagged Arf1 proteins (Supplementary data 4) that were subcloned into the binary vector pVKH18-En6. After incubation for 24 h in the dark, the protoplast suspension was spun for 5 min at 100 g in a swingout centrifuge (4K15; Sigma, Oakville, Canada), which results in the floating of the cells. Using an extra-fine Pasteur pipette, 1 ml of clear supernatant from below the floating cell layer was removed. The remainder of the suspension (1 ml) was brought to 10 ml with 250 mM NaCl and mixed gently by inverting the tube twice. After a second spin of 5 min at 100 g, the supernatant was removed with a peristaltic pump, and the cell pellet was placed on ice. The cells were extracted in a final volume of 250 ll. Equal volumes of cell extract and culture medium were analysed by protein gel blotting or by enzymatic analysis. a-Amylase assay Protoplasts were extracted in a-amylase extraction buffer (Crofts et al., 1999) via sonication for 5 sec. The extracts were centrifuged for 10 min at 25 000 g at 4C and the supernatant was recovered. The culture medium was also spun for 10 min at 25 000 g at 4C to remove residual cell debris. The a-amylase assays and calculation of the secretion index were performed as described previously (Phillipson et al., 2001). The secretion index represents the ratio between the extracellular and intracellular activity (Phillipson et al., 2001). Sampling, imaging and spot fluorescence recovery after photobleaching (FRAP) analysis Transformed leaves were analysed 44–48 h after infection of the lower epidermis. Imaging was performed using an upright Zeiss Laser Scanning Confocal Microscope LSM510 META (Zeiss, Jena, Germany), and a 63· water immersion objective. For imaging expression of either GFP constructs or YFP constructs or both, we used imaging setting as described by Brandizzi et al. (2002) with a 3 lm pinhole diameter. Time-lapse scanning was acquired with imaging system software of the microscope. Comparison of different levels of expression between cells expressing tagged Arf1 mutants was carried out by visualizing cells with the same imaging settings of the confocal microscope (i.e. laser intensity, pinhole diameter and settings of the imaging detectors) as described by daSilva et al. (2004). For each image, we used the palette function of the microscope software, which measures the fluorescence intensity value for each image pixel. Differences in the number of saturated pixels between cells were an indication of higher or lower concentrations of a GFP fusion. Subsequently, only for presentation purposes in this paper, the images may have been acquired with different settings. Spot FRAP experiments for fluorochrome photobleaching and half-time computation were performed as described by Brandizzi et al. (2002). Significance was determined using a Student two-tailed t-test for two samples assuming equal variance. For FRAP experiments, a steady-state protein distribution at the Golgi bodies was assumed. Such an assumption is reasonable as FRAP experiments have not produced appreciable differences when performed at either 2 or 3 days after transformation when the levels of expression are highest. In vitro expression Production of GST-Sec23 subcloned in pGEX vector was induced in Esherichia coli BL21(DE3) lysogens. Positive clones were selected for low-scale protein production. A single colony was inoculated initially into 5 ml of Luria Bertani containing ampicillin (100 ll ml)1), further expanded into a 100 ml shaker culture in 250 ml flasks. The cells were incubated with shaking at 30C until an OD600 of approximately 1.0 was reached. Protein production was induced by the addition of 1 mM IPTG and further incubation of the culture for 5 h at 30C. Cells were then pelleted and lysed according to the instructions provided by the manufacturer of the glutathione resin columns (BD Biosciences, Mississauga, Canada) for binding of GSTtagged proteins. Protein binding, removal of endogenous proteins and elution of GST-tagged proteins were performed according to the manufacturer’s instructions. Glutathione–agarose affinity chromatography of leaf extracts A sample (1 g) of leaves transformed with YFP-Sec24 was subjected 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 protease inhibitor cocktail for plant cell extracts (Sigma) in liquid N2. The resulting suspension was then centrifuged at 4C, 14 000 g for 15 min. An aliquot (1 ml) of the supernatant was added to 150 ll of a glutathione–agarose beads suspension (see below) [72% in NS buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2)] previously mixed with bacterial lysates containing GST-Sec23 and washed from unbound proteins. The mix was kept for 3 h at 4C with gentle rotation. The beads were centrifuged at 4C, 500 g for 1 min and then washed three times with NS buffer. Bound proteins were eluted from the beads with an appropriate volume of 5x SDS–PAGE sample buffer [0.225 M Tris-HCl, pH 6.8; 50% glycerol; 5% SDS; 0.05% bromophenol blue; 0.25 M DTT (QIAGENQIAexpressionist kit; Qiagen, Mississauga, Canada) in a ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 95–110 108 Giovanni Stefano et al. 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 extracts after sonication of protoplasts in a-amylase extraction buffer, and on leaf extracts in NE buffer. Protein samples were resuspended in a-amylase extraction buffer, and were loaded in equal volumes after twofold dilution with 2x SDS loading buffer prior to boiling (Crofts et al., 1999). Proteins in SDS–polyacrylamide gels were transferred onto a nitrocellulose membrane and then blocked with PBS, 0.5% Tween-20, and 5% milk powder for 1 h. The filter was then incubated in blocking buffer with primary antibody at a dilution of 1:2000 for the anti-GFP serum (AbCam) and 1:5000 for the antiIgG antibody. All the antisera were from rabbits, and further steps were performed as described by Crofts et al. (1999). Acknowledgements We are grateful to Dr E.L. Snapp (Einstein College of Medicine, New York, NY, USA) for valuable discussion. We acknowledge for financial support the University of Saskatchewan and the Department of Biology, U of S, CFI and Canada Research Chair (CRC) grants to F.B for the development of this work. S.H. is supported by a CRC Provincial Operating Fund and a Department of Biology Post-Doctoral Award. CRC Provincial Operating Fund and Graduate College Studies Award are acknowledged for the support of G.S. For her MSc studentship, L.R. is indebted to a University of Saskatchewan New Faculty Award. L.C. is recipient of a Government of Canada Award CIEC-ICCS, International Council for Canadian Studies, spent in F.B. laboratory. We are grateful to Dr J. Denecke (Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, UK) for the generous gift of Arf1 DNA, and Dr H. Steinkellner (Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Austria) for the generous gift of the Arabidopsis b1,2-xylosyltransferase DNA. Supplementary Material The following supplementary material is available for this article online: Figure S1. Arf1-GFP localizes at the Golgi apparatus and at additional structures that originate from this organelle. Figure S2. Generation of a bi-cistronic vector that allows visualization of cells expressing an untagged protein via monitoring the presence of CFP-SKL, a fluorescent protein fusion targeted to the peroxisomes. Figure S3. Effect of Arf1 proteins on the subcellular distribution of ST-YFP or Xylo-YFP. Figure S4. Tagged Arf1 proteins have a similar effect on the secretion of a soluble cargo in comparison to the untagged counterparts, and are present in cells as intact fusions. Figure S5. The peroxisomal marker CFP-SKL does not have an effect on the subcellular distribution of Arf1-YFP. Figure S6. FRAP experiments on eCOPI-YFP and Arf1GTP-GFP show YFP fluorescence recovery in the presence of Arf1GTP-GFP. Figure S7. Time-lapse microscopy on Sec24- and Sec23-labelled ERES shows that these structures are highly motile. Figure S8. The subcellular distribution of fluorescent Sec23 is influenced by the integrity of COPI. This material is available as part of the online article from http:// www.blackwell-synergy.com References Andreeva, A.V., Zheng, H., Saint-Jore, C.M., Kutuzov, M.A., Evans, D.E. and Hawes, C.R. (2000) Organization of transport from endoplasmic reticulum to Golgi in higher plants. Biochem. Soc. Trans. 28, 505–512. Antonny, B. and Schekman, R. (2001) ER export: public transportation by the COPII coach. Curr. Opin. Cell Biol. 13, 438– 443. Bar-Peled, M. and Raikhel, N.V. (1997) Characterization of AtSEC12 and AtSAR1. Proteins likely involved in endoplasmic reticulum and Golgi transport. Plant Physiol. 114, 315–324. Barlowe, C. (2002a) COPII-dependent transport from the endoplasmic reticulum. Curr. Opin. Cell Biol. 14, 417–422. Barlowe, C. 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