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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Sec22 and Memb11 Are v-SNAREs of the Anterograde Endoplasmic Reticulum-Golgi Pathway in Tobacco Leaf Epidermal Cells¹

Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200, Centre National de la Recherche Scientifique-Université Bordeaux 2, 33076 Bordeaux, France (L.C., A.H., P.M.); Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada (F.B.); Ecole Supèrieure de Technologie des Biomolécules de Bordeaux, Université Bordeaux 2, 33076 Bordeaux, France (A.H.); and Research School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (C.H.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.

In eukaryotic cells, the molecular machinery involved in the secretory pathway appears to be highly conserved. Genomic sequences encoding for several families of proteins, such as coat proteins, small GTPases, ATPases, and SNAREs that have been found to mediate secretory processes in animal and yeast cells (Chen and Scheller, 2001; Spang, 2002; Barlowe, 2003; Bonifacino and Lippincott-Schwartz, 2003; Sannerud et al., 2003), are also present in the Arabidopsis (Arabidopsis thaliana) genome (Pimpl et al., 2000; Takeuchi et al., 2000, 2002; Pelham, 2001; Phillipson et al., 2001; Tai and Banfield, 2001; Jürgens and Geldner, 2002; Lee et al., 2002; Nebenführ, 2002; Rutherford and Moore, 2002; Surpin and Raikhel, 2004). SNAREs are engaged in several processes in plants that can be related to different extents to their fusogenic properties (for review, see Pratelli et al., 2004; Surpin and Raikhel, 2004).

In particular, SNAREs interact through their coiled-coil domains that lead to the formation of protein complexes between a vesicle and a target membrane prior to membrane fusion. On the basis of the functional location, SNAREs were originally divided into v-SNAREs and t-SNAREs. To avoid ambiguity in the case of homotypic membrane fusion, SNAREs have been reclassified either as R-SNAREs or Q-SNAREs depending on the presence of a conserved Arg or Gln in a central position of the helix bundle (the zero layer) of the coiled-coil domain (Fasshauer et al., 1998). SNAREs are subdivided in five subfamilies: Qa-SNAREs (syntaxin), Qb-SNAREs (SNAP Ns), Qc-SNAREs (SNAP Cs), R-SNAREs (VAMPs), and SNAP25 (SNAP Ns and Cs; Bock et al., 2001). Members of each subfamily are found in all eukaryotes, albeit the number of genes for each subfamily varies. More than 35 SNARE genes have been identified in Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae, whereas the Arabidopsis genome contains 54 genes (Sanderfoot et al., 2000; Uemura et al., 2004). Some of the Arabidopsis SNAREs show a high degree of similarity with those of other eukaryotes. However, a larger number of SNARE homologs in plants could account for a specialized diversity in their cellular functions (Sanderfoot et al., 2000; Pratelli et al., 2004; Surpin and Raikhel, 2004).

In their systematic study devoted to SNARE locations in Arabidopsis suspension-cultured cells, Uemura et al. (2004) have shown that Sec22 (R-SNARE) and Memb11 (a Qb-SNARE membrin, alias Bos1 in yeast and GS27 or membrin in mammals; Hong, 2005) have a subcellular distribution compatible with a role at the ER-Golgi interface. In addition, based on the sequence similarities with their animal and yeast counterparts, it can be expected that the v-SNAREs Sec22 and Memb11 and the t-SNARE Sed5 (Qa-SNARE, alias SYP31 in the plant nomenclature; Sanderfoot et al., 2000) are putative SNARE candidates of the anterograde ER-Golgi pathway. However, to date, the role of these SNAREs has not been studied in plants. To investigate the putative role of these proteins, we have overexpressed these SNAREs in tobacco leaf epidermal cells, and looked at the effect of their expression on various well known markers of the secretory pathway.

In this study, we have first confirmed the subcellular distribution of SNAREs with fluorescent protein constructs in tobacco leaf epidermal cells, and we have then analyzed their role in the early secretory pathway by coexpression with membrane and soluble markers of the plant secretory pathway. We have also looked at the effect of the Qc-SNARE Bet11 that has potentially a different subcellular distribution (a trans-Golgi location was observed in protoplasts from Arabidopsis suspension-cultured cells; Uemura et al., 2004) and that will serve as a control for the SNAREs of the ER-Golgi interface in the coexpression studies.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

SNARE Location in the Early Secretory Pathway

To establish the subcellular distribution of the SNAREs Sec22, Memb11, Sed5, and Bet11 in tobacco leaf epidermal cells, we generated fluorescent protein fusions of these proteins for confocal microscopy. It is known that these SNARE proteins have a very short C terminus in the lumen of the corresponding membrane and an N terminus in the cytoplasm that can have regulatory functions (Hong, 2005). Being aware not to disturb the respective N terminus of the SNAREs, we produced cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) constructs with the fluorescent protein attached to the C terminus of the SNARE fusion proteins. For each SNARE, similar subcellular locations were obtained whether the fluorescent-attached protein was CFP or YFP. Therefore, we used both constructs according to the needs of the coexpressions in the following experiments.

As shown with a fluorescent CFP fusion of Sec22 (Sec22-CFP), we found the fusion protein to label a polygonal and tubular network characteristic of ER (Fig. 1A), and punctate structures (Fig. 1A) that were mobile in the cytoplasm and resembled the plant Golgi apparatus (Boevink et al., 1998). However, we could not use Golgi fusion protein markers (the K/HDEL receptor ERD2 and the sialyltransferase ST) to identify the punctate structures because these markers were redistributed (entirely or partly) to the ER (see Figs. 2 and 4A, and the other "Results" sections). On another hand, it has been shown that in tobacco leaf epidermal cells, ER export sites (ERES) and Golgi bodies are in close vicinity (daSilva et al., 2004). Sar1 is a GTP-binding protein that is involved in the recruitment of COPII proteins and that labels the ERES (daSilva et al., 2004). To confirm that Sec22-CFP was located near the ER-Golgi interface, we coexpressed Sec22-CFP with a constitutive GTP-bound form of Sar1-YFP, and observed a colocalization of both constructs (Fig. 1D). Although this coexpression does not allow distinguishing between a Golgi and an ERES location, the colocalization of Sar1-YFP and Sec22-CFP demonstrates that Sec22-CFP is present in the vicinity of the ER-Golgi interface. In the following experiments, except in the case of a Golgi marker alone for which the Golgi location has been demonstrated earlier, we will use the term "punctate structures" to describe any labeling appearing as dots that could be either Golgi bodies and/or ERES.

View larger version (79K): [in this window] [in a new window]   Figure 1. SNARE location in the early secretory pathway. A, In Sec22-CFP-transformed epidermal tobacco cells, fluorescent punctate structures and the ER network are visible. Bar = 5 µm. B, A strong ER labeling is observed after incubation of Sec22-YFP-expressing cells with BFA (50 µg/mL) for 30 min. Bar = 5 µm. C, An ER colabeling is observed after incubation of Sec22-CFP (blue, left section)/ST-YFP (yellow, middle section)-expressing cells with BFA (50 µg/mL) for 30 min, as shown in the merged image (right section). A typical Golgi labeling is obtained with ST-YFP-expressing cells without BFA (see Fig.4C). Bar = 5 µm. D, A colabeling of punctate structures by Sec22-CFP (left section, blue) and Sar1-YFP (middle section, yellow) is shown in the merged image (right section). Bar = 10 µm. E, Only punctate structures are labeled with Memb11-YFP. Bar = 10 µm. F and G, Sed5-CFP and Bet11-CFP are also located in punctate structures. Bars = 10 µm.

View larger version (88K): [in this window] [in a new window]   Figure 4. Effects of the expression of the SNAREs on ST-YFP distribution. A, A colabeling of the ER by ST-YFP (middle section, yellow) and Sec22-CFP (left section, blue) is observed in the right section. However, the two proteins are also colocated in punctate structures. Bar = 10 µm. B, ST-YFP (yellow, middle section) is found in both the ER and Golgi bodies/punctate structures when coexpressed with Memb11-CFP (blue, left section), and some colocation is found in punctate structures (right section). Bar = 10 µm. C, ST-YFP is, as expected, in the Golgi bodies when it is expressed alone. Bar = 10 µm. D, Sed5-CFP coexpressed with ST-YFP shows a colocation in punctate structures and no labeling of the ER by ST-YFP. Bar = 5 µm. E, Bet11-CFP coexpressed with ST-YFP shows a colocation in punctate structures and no labeling of the ER by ST-YFP. Bar = 10 µm.

View larger version (95K): [in this window] [in a new window]   Figure 2. Expressions of Sec22 and Memb11 redistribute ERD2 to the ER. A, In ERD2-YFP-expressing cells, ERD2-YFP is found predominantly in the Golgi bodies as expected. The ER network is only weakly labeled. Bar = 10 µm. B, A redistribution of ERD2-YFP (red) in the ER network is observed when coexpressed with Sec22-CFP (green) as shown by a yellow ER network, and punctate structures are only labeled by Sec22-CFP and appear green (arrows). Bar = 5 µm. C, The same result as in B except that the ER is mainly labeled by ERD2-YFP (red) and less by Sec22-CFP (green). Arrows indicate punctate structures only labeled by Sec22-CFP (green). Bar = 10 µm. D, Redistribution of ERD2-CFP (blue) in the ER network is observed when coexpressed with Sec22-YFP (yellow). The ER network appears green-white due to a colabeling by both Sec22-YFP and ERD2-CFP; punctate structures are only labeled by Sec22-YFP (yellow, see arrows). Bar = 5 µm. E, A redistribution of ERD2-YFP in the ER network is also observed after coexpression with an untagged version of Sec22. Bar = 5 µm. F, When ERD2-CFP (blue) is coexpressed with Memb11-YFP (yellow), a stronger labeling of the ER network by ERD2-YFP is observed; punctate structures are only labeled by Memb11-YFP (yellow dots). Bar = 10 µm. G, Coexpression of untagged Memb11 with ERD2-YFP led also to a redistribution of the KDEL receptor to the ER network. Bar = 10 µm.

We also determined the subcellular localization of the other SNAREs in tobacco leaf epidermal cells. We found that these SNAREs labeled punctate structures (Fig. 1, E–G). In addition, Sed5-CFP and Bet11-CFP colocalized with ERD2-YFP and ST-YFP as Golgi markers (Figs. 3, A and B, and 4, D and E, respectively), which could indicate in this case that Sed5-CFP and Bet11-CFP were associated with the Golgi apparatus. On the contrary, the v-SNARE Memb11 had a similar effect as Sec22 on those markers (Figs. 2F and 4B), and coexpressions could not be used to confirm Golgi location of this SNARE. For this reason, we looked at the effect of BFA on the subcellular location of Memb11-YFP, and we observed a redistribution of the protein from mobile dots into the polygonal ER network as for Sec22-CFP and Sec22-YFP (data not shown). Therefore, these results suggest that Memb11 is also located near the ER-Golgi interface.

View larger version (63K): [in this window] [in a new window]   Figure 3. Effects of the expression of Sed5 and Bet11 on ERD2 distribution. A, A high colocalization of Sed5-CFP (green, left section) and ERD2-YFP (red, middle section) is found in punctate structures (right section). Bar = 10 µm. B, As for Sed5, a colocation of Bet11-CFP (green, left section) and ERD2-YFP (red, middle section) in punctate structures is observed (right section). Bar = 10 µm. C and E, Expressions of untagged versions of Sed5 and Bet11, respectively, did not redistribute ERD2 to the ER, and a typical Golgi pattern was observed. Bar = 10 µm. D, As in Figure 2A, ERD2-YFP is found predominantly in Golgi bodies as expected.

We next wanted to analyze the role of Sec22 and Memb11 in protein transport at the ER-Golgi interface. To do so, we aimed to investigate the effect of overexpression of these SNAREs on a Golgi marker. If these v-SNAREs are involved in ER-to-Golgi protein transport, their overexpression would affect the distribution of a Golgi marker. For this purpose, we used ERD2, the H/KDEL receptor (Boevink et al., 1998). At steady state, the receptor is known to be predominantly located at the Golgi, where it captures soluble proteins to move them back to the ER and then recycles to the Golgi (Lee et al., 1993). Consistent with the role of this protein, a fluorescent fusion of ERD2 has been suggested to cycle between the Golgi and the ER membranes (Brandizzi et al., 2002; daSilva et al., 2004). Therefore, this marker was used to analyze whether overexpression of Sec22 and Memb11 could alter the dynamic equilibrium of ERD2 distribution between the ER and Golgi. Consistent with previous findings, a yellow fluorescent fusion (ERD2-YFP) was found predominantly in the Golgi bodies and to a far lesser extent in the ER (Fig. 2A; Brandizzi et al., 2002; daSilva et al., 2004). However, when ERD2-YFP was coexpressed with Sec22-CFP, we systematically observed a shift in the distribution of ERD2-YFP to the ER (Fig. 2, B and C). Sec22-CFP either colocalized (Fig. 2B) or not (Fig. 2C) with ERD2-YFP in the ER but still labeled punctate structures that were not labeled by ERD2-YFP (Fig. 2, B and C). To control that the redistribution of ERD2 was not due to the nature of the fusion protein but to the SNARE, we first exchanged the CFP and the YFP between ERD2 and Sec22. The same results were obtained by coexpressing ERD2-CFP with Sec22-YFP (Fig. 2D), i.e. a redistribution of ERD2 into the ER (as shown by the labeling by ERD2-CFP of a polygonal ER network), and the labeling of punctuate structures only by Sec22-YFP. Then, to test if such an effect was attributable to the SNARE Sec22 and not to loss or gained function of the SNARE due to tagging, we expressed the untagged SNARE with ERD2-YFP. Coexpressing an untagged version of Sec22 gave a similar redistribution of ERD2-YFP into the ER (Fig. 2E), clearly demonstrating that the effect was due to the expression of the SNARE protein. These data reveal that Sec22 has a profound effect on the dynamic distribution of a Golgi protein that cycles continuously between the ER and the Golgi apparatus. Therefore, it is likely that Sec22 is involved in any process related to the transport of proteins in the early secretory pathway.

Similarly, when we coexpressed Memb11-YFP with ERD2-CFP, we observed a stronger labeling of the ER network due to ERD2-CFP and Memb11-YFP still labeled punctate structures (Fig. 2F). In addition, we also tested an untagged version of Memb11 on the distribution of ERD2-YFP (Fig. 2G) and found very similar results as those obtained for the untagged Sec22, i.e. an ER redistribution (Fig. 2E). As a consequence, these data also suggest that Memb11, as Sec22, is engaged in some processes related to membrane dynamics and protein transport at the ER-Golgi interface.

Effect of Other SNAREs on ERD2 Distribution

To determine the specificity of the effect on the redistribution of ERD2 by the v-SNAREs Sec22 and Memb11, we analyzed the role of another v-SNARE and a t-SNARE from distinct families (i.e. the Qc-SNARE Bet11 and the Qa-SNARE Sed5, respectively). The Qc-SNARE Bet11 has been shown to have a different location than Sec22 and Memb11, i.e. a trans-Golgi location in protoplasts from Arabidopsis suspension-cultured cells (Uemura et al., 2004), and could represent a control for the v-SNAREs Sec22 and Memb11 of the ER-Golgi interface. In addition, it has been shown in yeast that Sed5 is a Golgi syntaxin involved in ER-to-Golgi transport (Sacher et al., 1997).

Coexpression of Sed5-CFP with ERD2-YFP showed a colocation of the two proteins in punctuate structures that could correspond to Golgi bodies, and no equivalent redistribution of the marker proteins into the ER was observed (Fig. 3A), as found with the fusion proteins of Sec22 and Memb11. Figure 3D shows the expected punctate labeling by ERD2-YFP of Golgi bodies.

Coexpression of Bet11-CFP with ERD2-YFP (Fig. 3B) gave similar results with a colabeling of punctate structures that could correspond to Golgi bodies, and a very weak labeling of the ER membrane network.

Controls were also made with untagged SNAREs. When ERD2-YFP was coexpressed with untagged versions of Sed5 (Fig. 3C) or Bet11 (Fig. 3E), no redistribution of the Golgi reporter protein was observed in any cells. These data indicate that the effect of Sec22 and Memb11 on the redistribution of a Golgi marker into the ER is not a general feature of SNAREs, but it appears to be more specific to the v-SNAREs Sec22 and Memb11 being located at or near the ER-Golgi interface.

Effect of the Expression of the Different SNAREs on ST Distribution

The results obtained with the Golgi marker ERD2 led us to check another Golgi reporter protein (ST). Expression of a fusion ST-YFP protein confirmed a punctate labeling likely to correspond to Golgi bodies as expected (Fig. 4C; Boevink et al., 1998; Saint-Jore et al., 2002).

Coexpression of Sec22-CFP with ST-YFP led to a shift in the location of ST-YFP to the ER (Fig. 4A). However, we did not observe the same efficiency in the redistribution of ST-YFP to the ER that we observed with ERD2-YFP. Effectively, a higher punctate labeling was observed with ST-YFP (Fig. 4A) than when ERD2-YFP was used (see Fig. 2), and also as compared to a BFA treatment (see Fig. 1C). In the latter case, it was expected that BFA redistributed the majority of the proteins to the ER.

The coexpression of ST-YFP with Memb11-CFP also increased the labeling of the ER by ST-YFP (Fig. 4B), and as for Sec22-CFP, a higher punctate labeling was observed (Fig. 4B).

On the contrary, no redistribution at all of ST-YFP was observed when either Sed5-CFP or Bet11-CFP was used in coexpression studies (Fig. 4, D and E).

Although the redistribution of ST-YFP to the ER by expressing Sec22-CFP and Memb11-CFP was less efficient than on ERD2-YFP, we confirmed with ST-YFP that there is a particular effect of the v-SNARES Sec22 and Memb11 on Golgi proteins in the ER-Golgi pathway.

Sec22 and Memb11 Reduce the Secretion of a Soluble Marker at Early Stages in the Secretory Pathway

The redistribution of Golgi membrane proteins to the ER induced by expression of Sec22 and Memb11 prompted us to check the effects of these proteins on the secretion of a soluble cargo, an ER targeted YFP (secYFP). In accordance with previous findings (Batoko et al., 2000; Kotzer et al., 2004), secYFP was found at the cell surface of tobacco leaf epidermal cells, indicating that the protein is transported through the ER and released into the apoplast (Fig. 5, A and B). Z-confocal scanning of cells did not reveal any significant labeling of the intracellular organelles. Coexpression of Sec22-CFP with secYFP resulted in most of the secretory protein being retained into the ER and in small punctate structures where Sec22 is also labeled (Fig. 5, C and D). We also found that in cells coexpressing the two proteins Memb11-CFP and secYFP, the secretion of secYFP was strongly affected as the YFP fluorescence was both retained into the ER and in small punctate structures (Fig. 5, E and F). For both Sec22- and Memb11-expressing cells, Z-confocal scanning of cells did not reveal any significant labeling of the apoplast.

View larger version (80K): [in this window] [in a new window]   Figure 5. Sec22 and Memb11 reduce the secretion of secYFP at early stages in the secretory pathway. A and B, In secYFP-expressing cells, the apoplast is highly labeled. Bar = 10 µm. C and D, When Sec22-CFP (green) is coexpressed with secYFP (red), secYFP and Sec22-CFP label the ER and punctate structures. Bar = 10 µm. E and F, secYFP (red) is also found in the ER and some dots when coexpressed with Memb11-CFP. Bar = 10 µm. G, secYFP (red) is found in punctate structures and also in the ER when expressed together with Sed5-CFP (green). Bar = 10 µm. H, For Bet11-CFP (green) and secYFP (red) coexpressions, secYFP could be retained in punctate structures and the ER. Bar = 10 µm.

Our data indicated that all the SNAREs can have an effect on the transport of secYFP, and Sec22 and Memb11 had a marked effect on the transport of secYFP since Z-confocal scanning of cells did not reveal any labeling of the apoplast.

Taken together, our results show that the v-SNAREs Sec22 and Memb11 have a critical effect on the distribution of Golgi reporter proteins and on the secretion of a soluble marker, suggesting a role in the early secretory pathway. Instead, the other v-SNARE Bet11 does not affect the distribution of the Golgi markers but can have an effect on secYFP transport. Finally, Sed5 had also a less effect on Golgi reporter proteins but led to retention of secYFP in early membranes of the secretory pathway.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
SNAREs are critical proteins as they are required in a number of fusion processes involved in different trafficking pathways in eukaryotic cells. In plants, the role of these proteins has only recently been studied in vacuolar transport, cell-surface assembly, and cell-plate formation during cytokinesis (Pratelli et al., 2004; Surpin and Raikhel, 2004; Jürgens, 2005). However, the role of SNAREs in the early secretory pathway of plants has yet to be established. The homology of the SNAREs Sec22, Memb11, and Sed5 from Arabidopsis with their animal and yeast counterparts was found to be relatively high and therefore it was expected that these proteins were involved at the early ER-Golgi step. It was also interesting to study these proteins in a plant model (tobacco leaf epidermal cells) that has a very different organization of the ER-Golgi interface to other eukaryotic cells such as mammal and yeast cells, and may be of other plant cell types (Ward and Brandizzi, 2004; Hawes and Satiat-Jeunemaitre, 2005). This model is particular due to the fact that ER-Golgi transport is not cytoskeleton dependent (Brandizzi et al., 2002) and that ER export sites and Golgi bodies are tightly associated (daSilva et al., 2004).

We have first confirmed the subcellular distribution of the SNAREs with fluorescent protein constructs, and we have then analyzed their role in the early secretory pathway by coexpression with membrane and soluble markers of the plant secretory pathway. We have also looked at the effect of the SNARE Bet11, which had a different subcellular distribution (a putative trans-Golgi location), to compare with the SNAREs preferentially located at the ER-Golgi interface.

Location of the SNARE Constructs in the Early Secretory Pathway

To gather information on the distribution of different SNAREs in tobacco leaf epidermal cells, we have expressed fluorescent fusions of these SNAREs and analyzed their location at subcellular level by confocal laser scanning microscopy. Sec22 is present in both the ER and mobile punctate structures, while the other SNAREs are only found in mobile punctate structures.

The colabeling of Bet11 and Sed5 with ERD2 and ST in punctate structures supports the location of these proteins in the Golgi bodies. In protoplasts from Arabidopsis suspension-cultured cells, Uemura et al. (2004) found both Bet11 and Sed5 in the Golgi bodies. In addition, BFA treatment led to a distribution of Bet11 in clumped Golgi structures compatible with a more distal location at the trans-Golgi (Uemura et al., 2004). We have also observed a pattern of clumped Golgi after BFA treatment of Bet11-YFP-expressing cells (data not shown). Therefore, it is likely that Bet11 is chiefly in more distal cisternae in the Golgi bodies of tobacco leaf epidermal cells. The situation was different for Sed5 when comparing tobacco leaf epidermal cells and Arabidopsis protoplasts. BFA treatment was found to change the labeling of Sed5 to a more typical ER pattern in Arabidopsis (Uemura et al., 2004). However, in tobacco leaf epidermal cells, BFA treatment led only to a partial redistribution of Sed5 to the ER with the formation of clumped Golgi structures (data not shown). Therefore, some discrepancy appears between the two systems that could be either due to different levels of expression and/or different distributions of the expressed proteins between the two systems. Whatever the differences, we can suggest that Sed5 is located in the Golgi bodies perhaps with a wider distribution in tobacco leaf epidermal cells than in Arabidopsis cultured cells.

Memb11 was found to be located in small dots that were mobile as are Golgi bodies. In addition, we observed a total redistribution of Memb11 to an ER pattern after BFA treatment of Memb11-YFP-expressing cells (data not shown). The effect of BFA on the distribution of Memb11 may support the contention that Memb11 is located toward the cis-Golgi membranes, as also observed in Arabidopsis protoplasts (Uemura et al., 2004), but some ERES location cannot be excluded (see below).

In addition to the ER, Sec22 was also found to be located in punctate structures that appeared to be rapidly redistributed to the ER by BFA. Moreover, a colocation with the GTP-bound mutant of Sar1p that labels ERES was observed. These results suggest that the fraction of Sec22 that is found in punctate structures may reside in the ERES and/or cis-Golgi. Sec22 was found to be distributed in the ER in Arabidopsis protoplasts (Uemura et al., 2004), but we do not know how the ER-Golgi interface is organized in these cells and whether ERES can be visualized. However, some labeled dots were apparently present in Sec22-expressing cells (Fig. 3; Uemura et al., 2004). Unfortunately, BFA treatment was also found to redistribute Sar1-YFP to a reticular ER-like pattern (daSilva et al., 2004). It was not possible to distinguish between a cis-Golgi location and an association with ERES for Sec22, a situation that is also true for Memb11. Therefore, we can conclude that Sec22 is located in the ER and punctate structures corresponding either to ERES and/or cis-Golgi cisternae, and that Memb11 is distributed either in cis-Golgi cisternae and/or ERES. To get information on the more precise location of the endogenous proteins, antibodies are being produced from recombinant proteins.

Whatever their precise subcellular location, our results demonstrate clearly that the fluorescent fusion proteins of the v-SNAREs Sec22 and Memb11 are located near the ER-Golgi interface, and the corresponding endogenous proteins can be predicted to play a role in ER-Golgi traffic in tobacco leaf epidermal cells.

Expressions of SNAREs and Particularly of Sec22 and Memb11 Alter the Transport of Cargo Proteins

Visualization of different cargo proteins in coexpression with different SNAREs has provided useful insights on the distribution and putative function of these proteins. We have found that fluorescent fusion proteins of Sec22 and Memb11 had similar effects on the dynamics of the H/KDEL receptor (Fig. 2) and on the transport of other cargo proteins such as ST (Fig. 4) and secYFP (Fig. 5). We systematically observed a higher redistribution of ERD2 than ST from the Golgi bodies to the ER when Sec22 or Memb11 were expressed, and this could be due to the fact that ERD2 (the H/KDEL receptor) normally cycles between the Golgi and the ER and could be more sensitive to the overexpression of these v-SNAREs. That we did not find an effect of Bet11 on the distribution of both ERD2 and ST was not surprising since Bet11 is likely to be located in the trans-Golgi (no redistribution of Bet11 to the ER by BFA; Uemura et al., 2004). On the contrary, it was more surprising that Sed5 did not have a strong effect on the distribution of the Golgi reporter proteins (Figs. 3 and 4). Effectively, it has been shown in yeast that an overexpression of Sed5 can rescue an ERD2 null mutant (Hardwick and Pelham, 1992) by blocking the anterograde pathway, and therefore compensating for the block in the retrograde pathway and allowing yeast to survive. In addition, Sed5 being the t-SNARE syntaxin involved in the SNARE complex active in ER-to-Golgi transport (Sacher et al., 1997), an effect on the transport of Golgi reporter proteins could be expected. However, it was found that only the overexpression in tobacco of the cytosolic domain of NtSyr1 (a syntaxin-related protein) but not the entire protein blocked growth and resulted in an altered morphology of leaves and roots (Geelen et al., 2002). It must also be noted that in these experiments, membrane fusion at the plasma membrane was considered, and that SNARE requirement could be different to the situation of the ER-Golgi interface that has a very particular organization. Finally, it has recently been shown in yeast that Sed5 interaction with Sly1 is dispensable for ER-to-Golgi transport and that Sed5 may be, under certain conditions, bypassed in the activation of SNARE pairing for the formation of fusogenic SNARE complexes (Peng and Gallwitz, 2004). In an attempt to explain the different behavior of Sed5 as compared to Sec22 and Memb11 on Golgi reporter proteins, we could consider at least two possibilities.

First, ERD2 is essentially retained in the ER by overexpression of Sec22 and Memb11, and some proteins are retained in punctate structures that could correspond to a few ERES or Golgi dots. On the contrary, we can imagine that with the overexpression of the syntaxin Sed5, ERD2 is less retained into the ER but is more concentrated at the level of the ERES that cannot be distinguished from the Golgi bodies. In this case, ERD2 would have been blocked by Sed5 overexpression as it was observed for the coexpression with secYFP that was partly blocked to the ER and punctuate structures (Fig. 5G). Such a block in the transport of secYFP to the cell surface was also observed for Sec22 and Memb11 (Fig. 5, C–F).

Second, we could also consider that competition with the endogenous proteins was more critical for the v-SNAREs Sec22 and Memb11 than for Sed5, and/or that replacing the endogenous proteins takes more time in the case of Sed5. On another hand, overexpression of Sed5 did have some effect on secYFP secretion. This could be due to a higher sensitivity of secYFP transport since overexpression of Bet11 could also affect secYFP secretion with some retention into the ER (Fig. 5H).

Sec22 and Memb11 Are v-SNAREs Involved in the Dynamics of the ER-Golgi Interface

Our overall results can be summarized as follows. (1) The v-SNARE Sec22 is located in the ER and punctate structures corresponding either to ERES and/or cis-Golgi cisternae and the v-SNARE Memb11 is most likely distributed in the cis-Golgi. (2) Both SNAREs are rapidly relocated to the ER by BFA treatment. (3) Overexpression of both SNAREs affects the dynamics of ERD2 and ST, with consequent retention of these markers into the ER. (4) The trafficking of a secretory marker (secYFP) was mainly blocked at the level of the ER by the expression of Sec22 and Memb11. (5) Another v-SNARE Bet11 with a different subcellular location (i.e. trans-Golgi) did not have the same effects on the Golgi reporter proteins, indicating the specificity of the effects induced by Sec22 and Memb11. As a consequence, our data demonstrate that Sec22 and Memb11 are critical v-SNAREs of the ER-Golgi interface in tobacco leaf epidermal cells.

Since ERES can be visualized in the ER membranes and are mobile together with the Golgi bodies (daSilva et al., 2004), we can speculate as to whether SNAREs such as Sec22 and Memb11 can, at least in part, be located to these exchange/transport zones between the ERES and the Golgi bodies. As a consequence of the data obtained on the location of the SNAREs and according to the theory on the formation of SNARE complexes (insofar as they need to be formed), we can speculate that part of Sec22 could be present both on the ERES and on the interacting Golgi cisternae. At the same time, Memb11 could be partly associated with Golgi cisternae engaged in interactions with ERES. We would also expect that Sed5 (as the syntaxin) would, at least in part, be present in the cis-Golgi cisternae interacting with ERES.

The fact that Sec22 labels the entire ER network (Fig. 1A) may be in contradiction with a location in the ERES. This may be easily explained if we consider that the association of this SNARE with the ERES is only transient, as may happen for the Sar1p exchange factor Sec12 (daSilva et al., 2004). We can also consider that overexpression of Sec22 may indeed alter the normal functioning of the ERES since we have shown that it leads to a shift of cargo proteins into the ER network. In addition, we have observed that coexpression of cargo proteins may also modulate the ER labeling of Sec22 (no reticulate labeling being observed in Fig. 1D). Thus, the block of normal trafficking of proteins between the ER and the Golgi may determine either a redistribution of Sec22 and/or factors that interact with this SNARE to the ER and/or an accumulation at the ERES.

To investigate further these SNAREs and their functions, we will determine their distribution between ER, ERES, and Golgi bodies by electron microscope immunocytochemistry, and by studying SNARE dynamics and interactions with proteins of the transport machinery in vitro and in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

SNARE Cloning

Standard molecular techniques were used as described by Sambrook et al. (1989).

Total RNAs were extracted from young Arabidopsis (Arabidopsis thaliana) leaves and were purified with a Promega kit SV Total RNA Isolation system. Total RNAs were submitted to reverse transcriptase (Moloney murine leukemia virus-reverse transcriptase; Stratagene) and PCR (Sed5-5': 5'-atgggctcgacgttcagag; AtSed5-3': 5'-ttaagccacaaagaagaggaaaac; AtSec22-5': 5'-atggtgaaaatgacattgatag; AtSec22-3': 5'-ttaccatagcttgttcttgac; AtMemb11-5': 5'-atggcgtctggtatcgtc; AtMemb11-3': 5'-ttagcgtgtccatcttatgaac; AtBet11-5': 5'-atgaatcctagaagggagcc; and AtBet11-3': 5'-ttaccgagtaagatagtatatgac). cDNAs were controlled by systematic sequencing.

Construction of SNAREs-XFP Fusion Proteins and Untagged SNAREs

We used the binary vector pVKH18-En6 for expressions in tobacco (Nicotiana tabacum) leaf epidermal cells (Batoko et al., 2000). SNAREs sequences were amplified by PCR (AtSed5ox-5': 5'-gctctagaccatgggctcgacgttcag; AtSed5ox-3': 5'-acgcgtcgacatggcagccacaaagaagagg; AtSec22-5': 5'-gctctagaccatggtgaaaatgacattg; AtSec22-3': 5'-acgcgtcgacatggcccatagcttgttcttg; AtMemb11-5': 5'-gctctagaccatggcgtctggtatcgtc; AtMemb11-3': 5'-acgcgtcgacatggcgcgtgtccatcttatg; AtBet11-5': 5'-gctctagaccatgaatcctagaagggag; and AtBet11-3': 5'-acgcgtcgacatggcccgagtaagatagtattg) and fused to the 5' end of YFP or CFP in place of ERD2 in pVKH-ERD2-YFP or pVKH-ERD2-CFP using SalI and XbaI restriction sites, in pVKHEn6ERD2 plasmid.

For untagged SNARE proteins, we amplified SNARE cDNAs by PCR with specific primers and we inserted the DNA coding sequence of these proteins within the unique XbaI and SacI sites of the vector.

The constructs for ERD2-YFP and ST-YFP fusion proteins were as described earlier (Brandizzi et al., 2002).

Plant Material and Transient Expression Systems

Four-week-old tobacco (N. tabacum cv Petit Havana) greenhouse plants grown at 22°C to 24°C were used for Agrobacterium tumefaciens (strain GV3101)-mediated transient expression (Batoko et al., 2000). PVKH18En6YFP- or pVKH18En6CFP-transformed A. tumefaciens were cultured at 28°C, until the stationary phase (approximately 24 h), washed, and resuspended in infiltration medium (MES 50 mM pH 5.6, Glc 0.5% [w/v], Na₃Po₄ 2 mM, acetosyringone [Aldrich] 100 µM from 200 mM stock in dimethyl sulfoxide). The bacterial suspension was inoculated using a 1-mL syringe without a needle by gentle pressure through the stomata on the lower epidermal surface (Brandizzi et al., 2002). Transformed plants were then incubated under normal growth conditions for 2 d at 22°C to 24°C.

BFA Treatments

Segments (roughly 5 mm²) of transformed leaves were used for drug treatment, confocal imaging, and analysis. BFA (stock solution: 5 mg/mL in dimethyl sulfoxide; Sigma-Aldrich) was used at a concentration of 10 µg/mL and 50 µg/mL as described by Brandizzi et al. (2002). Stock solution was kept at –20°C, and working solutions were prepared fresh just before use.

Confocal Microscopy

Transformed leaves were analyzed 48 h after infection of the lower epidermis. Confocal imaging was performed using either an inverted Zeiss 510 laser scanning microscope or a Leica TCS SP2 confocal microscope with a 63x oil immersion objective. For imaging expression of GFP constructs, excitation lines of an argon ion laser of 488 nm were used with a 505/530-nm bandpass filter in the single-track facility of the microscopes. For imaging CFP and YFP constructs, excitation lines of an argon ion laser of 458 nm for CFP and 514 nm for YFP were used alternately with line switching using the multi-track facilities of the microscopes. Imaging settings were as described by Brandizzi et al. (2002), and appropriate controls were done to exclude any cross talk and bleed through of fluorescence. Time-lapse scanning was performed with Zeiss LSM 510 or Leica TCS SP2 imaging system software. Postacquisition image processing was with the LSM 5 image browser (Zeiss), the Leica software, and Adobe Photoshop 5.0 software.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At1g11890 (Sec22), At2g36900 (Memb11), At5g05760 (Sed5), and At3g58170 (Bet11).

	ACKNOWLEDGMENTS

 
We thank Christina Calmels and the sequencing facilities of the IFR 66 of the University of Bordeaux II for sequencing of the cDNAs and the constructs, and the confocal microscope facilities of the IFR 103 (IBVM, Institut National de la Recherche Agronomique-Bordeaux). Ian Moore (Oxford University) kindly gave us secYFP. We thank John Runions (Oxford Brookes University) for his help with confocal microscopy.

Received June 19, 2005; returned for revision September 21, 2005; accepted September 21, 2005.

	FOOTNOTES

 
¹ This work was supported by the Centre National de la Recherche Scientifique and the University Victor Segalen Bordeaux 2. P.M. and C.H. were the recipients of a Franco-British Research Partnership Program Alliance (Egide/British Council).

The authors 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.plantphysiol.org) are: Patrick Moreau (pmoreau{at}biomemb.u-bordeaux2.fr) and Chris Hawes (chawes{at}brookes.ac.uk).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.067447.

^* Corresponding author; e-mail pmoreau{at}biomemb.u-bordeaux2.fr; fax 33–5–56–51–83–61.

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