Article |
Address correspondence to Ludger Johannes, Traffic and Signaling Laboratory, UMR144 Institut Curie/CNRS, 26 rue d'Ulm, F-75248 Paris Cedex 05, France. Tel.: 33-1-42346351. Fax: 33-1-42346507. E-mail: johannes{at}curie.fr
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Abstract |
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Key Words: shiga toxin; early/recycling endosomes; SNARE; Rab protein; retrograde transport
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Introduction |
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Morphological and biochemical evidence indicates that the receptor-binding, nontoxic B-subunit of Shiga toxin (STxB) traffics via an alternative pathway directly from the early endosome (EE) to the TGN, bypassing the late endocytic pathway (Mallard et al., 1998). This newly described pathway may also involve the recycling endosome (RE) and the GTPase Rab11 (Wilcke et al., 2000). Similar observations were made studying TGN38, a cellular protein known to recycle between the TGN and the plasma membrane (Ghosh et al., 1998). The molecular mechanisms underlying EE/RE-to-TGN transport are still largely unexplored. Our observation that internalized STxB colocalized with -adaptin and clathrin on EE/RE implied that AP1/clathrin coats might regulate membrane dynamics at the EE/RE-TGN interface (Mallard et al., 1998). Based on functional studies, a role for AP1 and the AP1 interactor PACS-1 was revealed in retrograde transport to the TGN of furin and MPR (Meyer et al., 2000; Crump et al., 2001; Fölsch et al., 2001), suggesting that these molecules may at least in part also use the EE/RE-to-TGN pathway. Importantly, this direct pathway may allow signaling molecules such as CD19 (Khine et al., 1998), interferon
/ß receptors (Khine and Lingwood, 2000), or GPI-anchored receptors, e.g., CD14 (Thieblemont and Wright, 1999), to escape the degradative environment of late endosomes to reach internal sites where they may interact with their intracellular targets.
Another group of proteins with key functions in membrane transport are small N-ethylmaleimide (NEM)-sensitive fusion factor (NSF) attachment protein (SNAP) receptors (SNAREs) (Rothman and Wieland, 1996; Hay and Scheller, 1997; Johannes and Galli, 1998). These tail-anchored molecules are located on specific membranes inside the cell where they form stable complexes that can be dissociated by the ATPase NSF/SNAP (Söllner et al., 1993). The current model suggests that t-SNAREs on target membranes, and v-SNAREs on transport vesicles cooperate with other proteins to form trans-complexes that bring vesicle- and target-membranes in close proximity to promote membrane fusion (Weber et al., 1998). However, the details of the fusion reaction and the exact role of the SNAREs remain to be established (Mayer, 1999). After fusion, the cis-SNARE complexes are dissociated by NSF/SNAP activity and SNAREs can reenter a new functional cycle.
Here, we identified the central elements of the molecular machinery of post-Golgi retrograde transport, thus establishing, by functional means, the existence of a regulated and efficient transport connection between the early endocytic pathway and the TGN.
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Results |
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To determine whether STxB transport to the TGN was energy dependent, we examined both complete and ATP-depleted cytosol (Fig. 1 E). These experiments were done with [35S]-labeled PAPS to render the sulfation reaction itself ATP independent. Under these conditions, TGN-localized STxB-Sulf2 was still efficiently sulfated, independent of the addition of an ATP regeneration system (Fig. 1 E Golgi, black bars). However, STxB transport to the TGN from the EE was strongly inhibited in the absence of ATP (Fig. 1 E, EE, white bars).
STxB-Sulf2 was transported to the TGN with comparable kinetics in permeabilized and intact cells. In fact, maximal sulfation was reached after 45 min in permeabilized cells (Fig. 1 F), as in intact cells (Mallard et al., 1998). Furthermore, we found that the efficiency of transport in permeabilized cells was 25% of that in intact cells (Fig. 1 A, insert), comparable to other in vitro systems that reconstitute coupled budding and fusion reactions. Throughout this manuscript, this percentage was set to 100% for comparison purposes. Finally, electron microscopical studies established that in SLO-permeabilized cells, a significant part of internalized STxB (Fig. 1, GH, 15 nm) gained access to structures labeled by the TGN markers TGN46 (Fig. 1 G, 10-nm gold particles, arrows) and galactosyl-transferase (Fig. 1 H, 10-nm particles, arrows), as previously described in intact cells (Johannes et al., 1997; Mallard et al., 1998). Morphologically identifiable Golgi stacks were also marked under these conditions (Fig. 1 H). In the absence of cytosol, no STxB transport to the Golgi could be detected (unpublished data).
Taken together, these results show that STxB transport from EE/RE to the TGN was efficiently reconstituted in SLO-permeabilized cells. The process exhibited the hallmarks characteristics of in vivo transport, and revealed canonical biochemical requirements observed for other in vitro reconstituted transport steps.
t-SNARE proteins in EE/RE-to-TGN transport
SNAREs are key regulators of vesicular membrane traffic. To test whether EE/RE-to-TGN transport was SNARE dependent, SNARE activity was inhibited using the dominant-negative -SNAP mutant L294A that is unable to stimulate the ATPase activity of NSF (Barnard et al., 1997). When added to permeabilized cells, recombinant
-SNAP(L294A) inhibited STxB transport in a dose-dependent manner (Fig. 2 A). Transport could also be slightly stimulated by the addition of low concentrations of wild-type
-SNAP (Fig. 2 A). These data strongly indicated a role for SNARE proteins in EE/RE-to-TGN transport.
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Interestingly, we observed that the inhibitory effects of anti-Syn6 and anti-Syn16 antibodies were not additive (Fig. 2 E), indicating that their respective target molecules function in the same transport pathway, possibly within the same molecular complex. The latter indication was tested by coimmunoprecipitation. Syn16 was immunoprecipitated from Triton X-100 lysates of HeLa cells. The precipitate was then probed by immunoblot for the presence of a cis-Golgi SNARE, Syn5 (Dascher et al., 1994; Rowe et al., 1998), the Golgi/TGN and endosomal SNAREs Syn6, Vti1a, and Vti1b (Bock et al., 1997; Advani et al., 1998; Xu et al., 1998), and the endosomal SNAREs Syn7 and Syn12 (Tang et al., 1998c; Nakamura et al., 2000). Syn6 was coimmunoprecipitated with Syn16 (Fig. 2 F), confirming their physical association. Vti1a was also found in the Syn16 immunoprecipitate (Fig. 2 F), an observation that is consistent with the inhibitory effect of the anti-Vti1a antibody on transport to the TGN (Fig. 2 B). In a separate experiment, both Vti1a and Syn16 were also coimmunoprecipitated with Syn6 (unpublished data).
We used an overexpression approach to further confirm the role of Syn6 and Syn16 in retrograde transport to the TGN in intact cells. Previous studies had indicated that the cellular TGN marker protein TGN38, like STxB, cycles from EE/RE to the TGN (Ghosh et al., 1998; Mallard et al., 1998). As syntaxins are tail-anchored proteins with a majority of the polypeptide exposed to the cytosol, the cytosolic domains (cyto) of these molecules are expected to function as dominant negative mutants. We therefore tested whether expression of Syn6-cyto and Syn16-cyto would have an effect on the trafficking of TGN38 in CHO cells stably expressing a Tac epitopetagged version of the protein (Ghosh et al., 1998). In most untransfected cells or cells expressing Syn7-cyto, anti-Tac antibody, added from the outside of the cell, was found to accumulate in the perinuclear Golgi area (Fig. 2 G). In contrast, >50% of cells expressing Syn16-cyto or Syn6-cyto did not display Golgi-accumulation (Fig. 2 G), indicating that the soluble cytosolic domains of Syn6 and Syn16 blocked TGN38 transport to the TGN. Similar results were obtained when STxB EE/RE-to-TGN transport was followed in cytosolic domain expressing cells (unpublished data). In conclusion, the same Golgi/TGN-localized t-SNAREs appeared to regulate the retrograde transport of two markers, STxB and TGN38, to the TGN.
Identification of v-SNAREs in EE-to-TGN transport
A SNARE-mediated EE/RE-to-TGN transport model would implicate an interaction between the TGN-localized Syn6/Syn16/Vti1a t-SNAREs and putative early endosomal v-SNAREs. Therefore, anti-Syn6 or anti-Syn16 immunoprecipitates were analyzed for the presence of four VAMP family proteins known to be localized to endosomes: VAMP3/cellubrevin (McMahon et al., 1993; Galli et al., 1994), VAMP4 (Steegmaier et al., 1999), VAMP7/TI-VAMP (Galli et al., 1998), and VAMP8/endobrevin (Wong et al., 1998). Intact cells were treated before lysis either with NEM to accumulate cognate SNARE complexes, or with DTT-inactivated NEM as a control. An increase of SNARE complex recovery after NEM treatment indicates that the complexes did not form after lysis and dilution (Galli et al., 1998). VAMP3/cellubrevin, VAMP4, and VAMP8/endobrevin coimmunoprecipitated with Syn6 (Fig. 3 A). Of these, only VAMP4, and to a lesser extent VAMP3/cellubrevin coimmunoprecipitated with Syn16 (Fig. 3 B). Association of VAMP3/cellubrevin and VAMP4 with Syn6 and Syn16 implied that they were candidate v-SNAREs for the regulation of EE-to-TGN transport.
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To study the function of VAMP4, an antibody was made against a peptide from the NH2 terminus of the protein (residues 115) with maximal sequence divergence compared with other VAMPs. Anti-VAMP4 inhibited EE/RE-to-TGN transport in a dose dependent manner, and inhibition was lost when the antibody was prebound to its antigen (Fig. 4 A). We furthermore observed that purified VAMP4-cyto inhibited STxB transport to the TGN on permeabilized HeLa cells, whereas VAMP8-cyto had no effect (Fig. 4 A). As shown for Syn6 and Syn16 (Fig. 2 G), overexpression of VAMP4-cyto also inhibited TGN38 cycling to the TGN, whereas VAMP7-cyto was without effect (Fig. 4 B). Thus, VAMP4 function was necessary not only for retrograde transport of STxB, but also for that of TGN38.
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Our anti-peptide antibody against the NH2 terminus of VAMP3/cellubrevin was without effect, even at high concentrations (unpublished data). Because it has been reported that VAMP3/cellubrevin is cleaved by tetanus neurotoxin (TeNT) (Galli et al., 1994), we tested whether TeNT would modulate transport. As shown in Fig. 5 A, TeNT cleaved VAMP3/cellubrevin (Galli et al., 1994), but not VAMP7/TI-VAMP (Galli et al., 1998), VAMP4, or VAMP8/endobrevin. Furthermore, an antibody that recognizes all synaptobrevin-like and TeNT-sensitive VAMPs (clone 10.1) (McMahon et al., 1993) revealed that VAMP3/cellubrevin was the only member of this group to be expressed in HeLa cells (Fig. 5 B). At TeNT concentrations above 100 nM, a condition under which most VAMP3/cellubrevin was degraded (Fig. 5 A), EE/RE-to-TGN transport was inhibited by 25%, whereas an inactive mutant of TeNT, TeNT (E234Q), was without effect (Fig. 5 C). As a whole, these data suggest that the TeNT effect was due to modulation of VAMP3/cellubrevin activity.
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To further investigate the potential role of VAMP3/cellubrevin and VAMP4 in EE/RE-to-TGN transport, the distribution of both proteins was compared with that of STxB internalized at low temperatures into the EE (Fig. 6). Many of the STxB containing early endosomal structures were also labeled with VAMP3/cellubrevin. Using anti-VAMP4 (unpublished data) or GFP-coupled VAMP4 (Fig. 6), similar observations were made.
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All Rab6 mutants that were used in this study were expressed to similar levels (Fig. 8 B). The specificity of our observations is illustrated by the fact that overexpression under identical conditions as those described above of a dominant negative Rab4 mutant did not affect post-Golgi retrograde transport (unpublished data). Furthermore, recently published data suggests that dominant negative Rab5 does not interfere with STxB transport to the Golgi apparatus (Nichols et al., 2001). In view of the potent and specific inhibition of retrograde transport by Rab6a'T27N, we therefore conclude that Rab6a' function is required in this transport step.
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Discussion |
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Specific SNAREs regulate EE/RE-to-TGN transport
In recent years, it has become clear that SNARE proteins are key regulators of membrane fusion (Rothman and Wieland, 1996; Hay and Scheller, 1997; Johannes and Galli, 1998). As each SNARE has a precise subcellular distribution, it has been suggested that selective interactions between SNAREs contribute to the specificity of membrane exchanges between intracellular compartments. This concept has recently been further documented by in vitro (McNew et al., 2000) and permeabilized cell (Scales et al., 2000) studies. Our data is in accordance with this concept of specific SNARE interactions. In fact, we show by coimmunoprecipitation that Syn16 interacts with Syn6 and Vti1a, but not Syn5, Syn7, Syn12, or Vti1b, and Syn6/Syn16/Vti1a interacts with VAMP3/cellubrevin and VAMP4, but not with VAMP7/TI-VAMP or VAMP8/endobrevin.
In agreement with the interaction studies, we found that EE/RE-to-TGN transport depends on two endosomal v-SNAREs. Indeed, anti-VAMP4 antibody and TeNT, which does not cleave VAMP4, both inhibited STxB and TGN38 transport to the TGN. The only TeNT-sensitive v-SNARE that we could detect in HeLa cells was VAMP3/cellubrevin, thus identifying this protein as a second candidate v-SNARE. Importantly, TeNT and anti-VAMP4 antibody had additive inhibitory effects, supporting the function of these v-SNAREs in separate molecular complexes. This conclusion is given further credence by the observation that VAMP3/cellubrevin did not coimmunoprecipitate with VAMP4, and vice versa.
The above-mentioned interaction data also suggests the formation of a specific t-SNARE complex between Syn6, Syn16, and Vti1a, and we indeed observed that Syn6, but not Syn5, stimulated binding of Syn16 to GST-VAMP4 (unpublished data). However, due to the low solubility of Syn16, binding was inefficient and final evidence for the existence of a stable and functional Syn6/Syn16/Vti1a t-SNARE complex will require in vitro fusion studies with VAMP3/cellubrevin and VAMP4 containing liposomes, as described by McNew and colleagues (2000).
In this context it appears noteworthy that Syn6 is not only found on the TGN, but also on the EE (Bock et al., 1996, 1997), and the protein interacts with a number of early endosomal v-SNAREs, such as VAMP3/cellubrevin (Bock et al., 1997), VAMP4 (Steegmaier et al., 1999), and VAMP8/endobrevin (this study). Furthermore, Syn6 is the most distant member of the syntaxin family, and its sequence is similar to the SN2 domain of SNAP23/25/29, much as Vti1a is similar to the SN1 domain (Bock et al., 2001). Therefore, it appears likely that Syn6 and Vti1a may serve as light chains of t-SNARE complexes with a more general function at the EE/RE-TGN interface (Bock et al., 2001). In contrast, Syn16 localization on the TGN appears to be very restrictive (this study), suggesting that this molecule is of primary importance for the definition of specificity in EE/RE-to-TGN transport.
The yeast SNARE proteins Tlg1p and Tlg2p show sequence similarity with Syn6 and Syn16, respectively. Conflicting results have been obtained on the function(s) of these proteins. They have been implicated in several pathways, such as cytosol-to-vacuole (Tlg2p) (Abeliovich et al., 1999), EE-to-TGN (Tlg1p and Tlg2p) (Lewis et al., 2000), endocytosis (Tlg2p) (Seron et al., 1998), and intra-Golgi transport (Tlg1p) (Coe et al., 1999). Assuming that Tlg1p and Tlg2p are in fact functional homologues of Syn6 and Syn16, respectively, our results establish their role in EE/RE-to-TGN transport. Indeed, in addition to the approach of expression of dominant negative mutants in whole cells, we have also used a permeabilized cell approach that allows the reconstitution of the key retrograde transport steps within the complex array of post-Golgi traffic. Similarly, the role of Vti1 proteins in yeast (Lupashin et al., 1997; Fischer von Mollard and Stevens, 1999; Ungermann et al., 1999; Sato et al., 2000), plants (Zheng et al., 1999), and mammals (Xu et al., 1998) has remained a matter of debate, particularly in the light of their multiple interactions with other SNAREs. Using in vitro assays, the mammalian Vti1b was found to function in homotypic fusion of late endosomes, and Vti1a in that of the early endosome (Antonin et al., 2000). In conjunction with our data, the latter observation indicates a more generalized role for Vti1a in membrane fusion on the EE.
A Rab6 isoform regulates EE/RE-to-TGN transport
In addition to the previously suggested role of Rab11 in EE/RE-to-TGN transport (Wilcke et al., 2000), we have now provided evidence that another Rab protein, Rab6, is involved in this transport step. Lower eukaryotes, such as the yeast Saccharomyces cerevisiae and the parasite Plasmodium falciparum express only one Rab6 protein (Echard et al., 2000). In mammalian cells, an exon duplication within the Rab6a gene has given rise to two different isoforms, Rab6a and Rab6a', of which the latter appears to represent the ancestral form of Rab6 (Echard et al., 2000). In agreement with the hypothesis that Ypt6p and Rab6a' are evolutionarily related, we found here that the Rab6a' isoform specifically regulates retrograde transport to the TGN, as suggested for Ypt6p in yeast (Tsukada and Gallwitz, 1996; Bensen et al., 2001; Siniossoglou and Pelham, 2001). Because Ypt6p/Rab6a' are located on TGN membranes, their function could be to regulate targeting/docking of EE/RE-derived vesicles with the TGN.
Without ruling out that also Rab6a may play a role in docking of EE/RE-derived vesicles at the TGN, Rab6a was previously shown to function in Golgi-to-ER retrograde transport (Martinez et al., 1997; Girod et al., 1999; White et al., 1999). It appears therefore that in mammalian cells, two isoforms of the same Rab protein control successive steps in retrograde transport. In addition, through an interaction with Rabkinesin-6 (Echard et al., 2000), Rab6a may participate in the coordination between retrograde membrane transport and progression through the cell cycle (Hill et al., 2000; Fontijn et al., 2001).
The complexity of the EE/RE-to-Golgi retrograde transport route in mammalian cells
The fact that EE/RE-to-TGN transport is regulated by specific SNAREs and Rab proteins suggests that it is a physiological, preexisting cellular pathway. The question arises as to whether transport from the EE, which is accessible during low temperature incubations used as a starting point in this study, to the TGN necessarily transits via the RE. That such a passage exists appears likely considering the accumulation of TGN38 (Ghosh et al., 1998) and STxB (Mallard et al., 1998) in the RE. Furthermore, modulating the activity of Rab11, a marker of the RE (Sheff et al., 1999), affects post-Golgi retrograde transport to the TGN (this study and Wilcke et al., 2000). However, both in permeabilized and intact cells, the Rab11 effect is weak as compared with the modulation of Rab6a' activity. One interpretation of this data is that both GTPases act on two sequential steps of retrograde transport to the TGN, a hypothesis that is in agreement with the observation that modulating the activities of Rab6 and Rab11 in the same system does not result in additive inhibition of retrograde transport. Rab6a' would also control an additional, Rab11-independent transport reaction to the TGN. VAMP3/cellubrevin- and VAMP4-dependent transport inhibition is additive, but not complete. Whether this reflects technical limitations or a high degree of versatility in membrane exchange between the early endocytic pathway and the TGN, as reported by Lippincott-Schwartz and colleagues (Nichols et al., 2001), remains to be determined.
In this study, we have provided the first molecular dissection of the EE/RE-to-TGN retrograde transport step in mammalian cells. The observed transport may correspond to the one required in S. cerevisiae for proper localization and/or recycling of TGN proteins. Indeed, the same molecules appear to be involved: Rab6a' may be the functional homologue of Ypt6p, and Tlg1p and Tlg2p, recently found to interact with Ypt6p via the Vps52/53/54p complex (Siniossoglou and Pelham, 2001), show sequence similarity with Syn6 and Syn16, respectively. However, the fact that in the mammalian system two v-SNAREs appear to functionally interact with the same t-SNAREs suggests that EE/RE-to-TGN transport has gained in complexity during evolution, with the appearance of the RE and the involvement of Rab11 in higher eucaryotes.
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Materials and methods |
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HeLa S3 cytosol
6 x 109 HeLa S3 cells were resuspended in 20 ml (final volume) ice-cold HK buffer (90 mM KCl, 50 mM Hepes, pH 7.2) supplemented with protease inhibitor cocktail. Cells were then homogenized in a potter. The supernatant of low-speed (10 min, 6,000 g, 4°C) and high-speed centrifugation (45 min, 105 g, 4°C) was dialyzed against ICT/DTT buffer (78 mM KCl, 4 mM MgCl2, 8.4 mM CaCl2, 10 mM EGTA, 1 mM DTT, 50 mM Hepes, pH 7.2). Protein concentration was around 10 mg/ml.
Immunofluorescence, immunoelectron microscopy, and immunoprecipitation.
Immunofluorescence and immuno-EM were performed essentially as described (Johannes et al., 1997). Coimmunoprecipitations: NRK or HeLa cells were lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1 mM PMSF and a protease inhibitor cocktail, and lysates were incubated with affinity-purified antibodies and protein A-Sepharose beads or IgG-coupled Dynabeads (Dynal). Immunoprecipitates were tested by Western blotting for the indicated proteins. In some cases, cells on ice were treated with either 1 mM NEM for 15 min followed by 15 min with 2 mM DTT, or for 30 min with 1 mM NEM quenched with 2 mM DTT, followed by incubated for 30 min at 37°C before lysis and immunoprecipitation.
STxB transport assay in SLO-permeabilized HeLa cells.
105 HeLa cells were incubated for 90 min in MEM without sulfate at 37°C and for 45 min at 19.5°C in the presence of 1 µM STxB-Sulf2. Cells were then placed on ice and incubated for 10 min in the presence of 1 µg/ml of SLO in ICT/DTT. Unbound SLO was removed by washes and cells were transferred to 37°C for 10 min in ICT/DTT. Transport of STxB-Sulf2 to the TGN was assayed by incubating the permeabilized cells for 30 min at 37°C in the presence of cytosol mix (3 mg/ml cytosol and ATP regenerating system in ICT/DTT, except when otherwise stated), supplemented with 1 µCi/ml [35S]sulfate (or 10 µCi/ml [35S]-PAPS where indicated). After incubation, the cells were lysed in 1 ml RIPA buffer containing a protease inhibitor cocktail and subjected to quantitative immunoprecipitation with 13C4 antibody. Immunoprecipitated STxB-Sulf2 was separated from radioactive contaminants by migration on a Tris-Tricine gel, and the amount of sulfated STxB-Sulf2 for each condition was quantified using a PhosphorImager and the ImageQuant software (Molecular Dynamics). Sulfation of endogenous proteins and proteoglycans was quantified by scintillation counting of TCA-precipitable material. The corresponding results were used to normalize the amount of sulfated STxB-Sulf2 for each condition. Only experiments were included where endogenous sulfation was within a 10% range in all conditions.
Antibodies were added from the permeabilization step on, whereas recombinant (His)6--SNAP or (His)6-
-SNAP(L294A) were only added to the transport mix. For GDI or TeNT treatment, SLO-permeabilized cells were incubated at 37°C for 15 min in the presence of, respectively, 1 µM GDIß, 100 nM TeNT or TeNT(E234Q).
STxB retrograde transport assay on intact cells
The indicated Rab proteins or cytosolic domains of syntaxins (residues 1234 of Syn6, residues 1237 of Syn7, and residues 1284 of Syn16) were overexpressed for, respectively, 6 or 10 h in intact cells using the vaccinia virus expression system, as described (Martinez et al., 1997). Transfection efficiencies were routinely >70% in all conditions. Cy3-coupled STxB and STxB-Sulf2 transport to the TGN was assayed as described (Mallard et al., 1998). After STxB-Sulf2 binding, HeLa cells were incubated for 20 min in the presence of radioactive sulfate, followed by immunoprecipitation and gel electrophoresis.
Anti-Tac uptake experiments
CHO cells expressing a chimeric TGN38 protein with an extracellular Tac domain (Ghosh et al., 1998) were transfected with expression constructs for the cytoplasmic domains of Syn6, Syn7 or Syn16. 24 h after transfection, the cells were incubated for 1 h with culture supernatant containing Tac monoclonal IgGs, washed extensively, fixed and labeled with the indicated antibodies.
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Footnotes |
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W. Hong, B. Goud, and L. Johannes were principal investigators.
* Abbreviations used in this paper: EE, early endosome; NEM, N-ethylmaleimide; NSF, NEM-sensitive soluble factor; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; RE, recycling endosome; SLO, streptolysin O; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; STxB, Shiga toxin; Syn, syntaxin; TeNT, tetanus neurotoxin; TGN, trans-Golgi network; VAMP, vesicle associated membrane protein.
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Acknowledgments |
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This work was supported by grants from the Ligue Nationale contre le Cancer and the Association pour la Recherche sur le Cancer (grants 9028 and 9254) to L. Johannes, B. Goud, and C. Antony, and by Action concertée incitative Jeune chercheurs (grants 5254 and 5233) to T. Galli and L. Johannes. W. Hong and B.L. Tang are supported by research grants from the Singapore National Science and Technology Board.
Submitted: 16 October 2001
Revised: 16 January 2002
Accepted: 16 January 2002
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