Soluble NSF Attachment Receptors (SNAREs) in RBL-2H3 Mast Cells: Functional Role of 4 in Exocytosis and Identification of a Vesicle-Associated This information is current as 8-Containing Secretory of September 25, 2021. Compartment Fabienne Paumet, Joëlle Le Mao, Sophie Martin, Thierry Galli, Bernard David, Ulrich Blank and Michèle Roa Downloaded from J Immunol 2000; 164:5850-5857; ; doi: 10.4049/jimmunol.164.11.5850 http://www.jimmunol.org/content/164/11/5850 http://www.jimmunol.org/ References This article cites 60 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/164/11/5850.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Soluble NSF Attachment Protein Receptors (SNAREs) in RBL-2H3 Mast Cells: Functional Role of Syntaxin 4 in Exocytosis and Identification of a Vesicle-Associated Membrane Protein 8-Containing Secretory Compartment1

Fabienne Paumet,* Joe¨lle Le Mao,* Sophie Martin,* Thierry Galli,† Bernard David,* Ulrich Blank,* and Miche`le Roa2*

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of spe- cialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investi-

gated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor Downloaded from (SNARE) machinery. Several isoforms of each family of SNARE were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI- VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor , caused inhibition of Fc⑀RI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with

VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be http://www.jimmunol.org/ involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment. The Journal of Immunology, 2000, 164: 5850–5857.

ast cells are of central importance in allergic and in- neuronal cells, the SNARE complex is composed of a vesicle- flammatory reactions (1). After IgE-dependent stimu- bound v-SNARE protein, namely VAMP or , and M lation, they secrete a variety of pharmacologically ac- two target organelle t-SNARE proteins, called SNAP25/23/29 and tive products that are stored in secretory granules (2). Recent syntaxin (9). These cognate SNARE proteins assemble in a ternary studies have demonstrated the intimate connection between endo- complex through coiled-coil domains forming a parallel four-helix by guest on September 25, 2021 cytic and exocytic pathways in these cells (3, 4), leading to con- bundle structure (13–15). Dissociation of the complex with soluble sider secretory granules, like all granules of the hemopoietic cell NSF and ␣-SNAP, a process requiring ATP hydrolysis, is neces- lineage, as secretory lysosomes (5–7). Mast cells may thus use sary to allow the recycling of SNARE components (16). In agree- specialized mechanisms to enable lysosomes to function as regu- ment with the multiple isoforms identified to date, at least part of lated secretory granules. The initial molecular events leading to the specificity of vesicle trafficking is ensured by a unique set of secretion are now well characterized (8). However, the final steps SNAREs in the different membrane compartments (9, 17). Among that control fusion of secretory granules remain largely unknown. t-SNAREs of the SNAP25/23/29 family, the ubiquitously ex- All vesicular traffic requires close apposition between mem- pressed isoform, SNAP23 (18, 19), is involved in the regulation of branes before fusion. It has been established that this function is exocytosis of nonneuronal cells (20–22). The second group of t- partly fulfilled by SNARE3 proteins (9–12). Initially discovered in SNAREs comprises isoforms of the syntaxin family displaying differential localizations and functions. To date, 1, 2, 3, and 4 are the only isoforms found on the plasma membrane and *Unite´d’Immuno-Allergie, Institut Pasteur, Paris, France; and †Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 144, Institut Curie, Paris, France reported to regulate exocytosis (21–25). Within the v-SNARE Received for publication December 27, 1999. Accepted for publication March family, some are involved in regulated exocytosis, such as 16, 2000. VAMP1 and VAMP2, also called 1 and 2 (26–28), The costs of publication of this article were defrayed in part by the payment of page and others in constitutive vesicle recycling (endocytosis/exocyto- charges. This article must therefore be hereby marked advertisement in accordance sis) such as cellubrevin/VAMP3 (29–31). TI-VAMP/VAMP7 has with 18 U.S.C. Section 1734 solely to indicate this fact. recently been implicated in apical transport in epithelial cells (22, 1 This work was supported by grants from l’Association pour la Recherche sur le Cancer (no. 1100) and Institut Pasteur. 32), and in endosome to lysosome transport (33). As for endobre- 2 Address correspondence and reprint requests to Dr. Miche`le Roa, Unite´d’Immuno- vin/VAMP8, it has been localized to endosomes (34, 35). Recon- Allergie, Institut Pasteur, 28 rue du Dr. Roux 75724 Paris Cedex 15, France. E-mail stitution experiments have shown that the SNARE complex min- address: [email protected] imal structure (composed of two t-SNAREs and one v-SNARE) 3 Abbreviations used in this paper: SNARE, SNAP receptor; SNAP, soluble NSF can induce membrane fusion in vitro (12), albeit the slow kinetics attachment protein; GFP, green fluorescent protein; NEM, N-ethylmaleimide; NSF, NEM-sensitive factor; PE-SA, phycoerythrin-streptavidin; RBL-2H3, rat basophilic suggests that additional mechanisms and regulatory proteins are leukemia cell line; SNAP23, 25, 29, synaptosome-associated protein of 23 kDa, 25 operative (36). In RBL-2H3 mast cells, recent data have shown kDa, 29 kDa; t-SNARE, target-SNARE; TI-VAMP, tetanus neurotoxin insensitive VAMP; v-SNARE, vesicle-SNARE; TRSC, Texas Red sulfonide chloride; VAMP, rab3d and II as regulators of IgE-mediated exocy- vesicle-associated membrane protein. tosis (37, 38).

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5851

Although the bulk of available information on SNARE proteins and against human CD8 were produced respectively from the 5G10 clone concerns neuronal and neuroendocrine cells, these proteins are also provided by Dr. J. Bonifacino (44) and from the 10D11.5 clone. Both Abs implicated in the regulated exocytosis of cells of the hemopoietic were biotinylated with the Enzotin Biotinylation Kit, according to the man- ufacturer’s instructions (Enzo Biochem, Syosset, NY). Biotinylated mouse lineage (39, 40). In mast cells, the expression of several SNARE mAb A59 directed against RFc␣ was provided by Dr. R. Monteiro. Per- proteins has been reported, and SNAP23 relocation was shown to oxidase-coupled goat anti-rabbit and goat anti-mouse IgGs and Texas Red be coupled to secretion in permeabilized cells (41, 42). The aim of sulfonide chloride (TRSC)-conjugated goat anti-mouse IgG, all specific for our study was to characterize additional SNARE proteins ex- the Fc␥ fragment, were purchased at Jackson ImmunoResearch (West Grove, PA). FITC-conjugated goat anti-rabbit IgGs (HϩL) was from Bio- pressed in RBL-2H3 mast cells and to investigate their role in sys S.A. (Compie`gne, France). Fc⑀RI-triggered exocytosis. We show that besides SNAP23, syn- taxin 4 is the only t-SNARE that interacts with the identified v- Confocal immunofluorescence microscopy SNAREs in intact cells. We provide evidence for the function of RBL-2H3 cells were seeded on 10-mm-diameter glass coverslips (2 ϫ 105 syntaxin 4 in degranulation. Furthermore, we observe that VAMP8 cells/coverslip) 16 h before analysis. For indirect immunofluorescence is tetanus toxin insensitive and colocalizes with a part of the se- analysis, cells were fixed in 3% paraformaldehyde for 10 min, followed by treatment with 50 mM NH Cl for 15 min. After washing, cells were per- cretory granules, suggesting this v-SNARE as the potential partner 4 meabilized in 0.05% saponin and saturated in the presence of 0.25% BSA of syntaxin 4 and SNAP23 in the regulated secretion of mast cells. and 5% goat serum (MedGene Science, Pantin, France). Primary Abs were incubated1hinthesame buffer and then revealed with FITC- and/or Materials and Methods TRSC-conjugated secondary Abs. Finally, the coverslips were mounted in Cell cultures, stimulation, and secretion assay Mowiol and viewed with a Leica TCS confocal laser-scanning microscope. Downloaded from RBL-2H3, COS-7, and transiently transfected cells were maintained as NEM treatment monolayer cultures, as previously described (37). For stimulation, RBL- 2 Adherent RBL-2H3 cells were briefly washed with PBS supplemented with 2H3 cells were seeded in 175-mm flasks (70% confluency) or used in 1 mM MgCl and 1 mM CaCl (D-PBS) and incubated with D-PBS con- ϫ 6 2 2 suspension (1 10 cells/ml), sensitized with IgE anti-DNP, and stimu- taining 1 mM NEM (Sigma) for 15 min on ice, followed by 15 min in the lated with Ag, as described (37). presence of 2 mM DTT to quench NEM. Control cells were incubated in RT-PCR and PCR D-PBS with 1 mM NEM plus 2 mM DTT for 30 min on ice. After rinsing, cells were incubated for 30 min at 37°C in fresh complete medium and Total RNA was isolated as described (37). For RT-PCR, 2 ␮g of total RNA subsequently used for immunoprecipitation (32). http://www.jimmunol.org/ of RBL-2H3 cells or brain was reverse transcribed using Superscript re- verse transcriptase (Life Technologies, Eragny, France). The cDNAs were Immunoprecipitation and immunoblotting amplified between bp 13 and 544 for SNAP25a/b (GenBank database ac- For some experiments, rat brain and RBL-2H3 homogenates were ex- cession number U56261/U56262), 1 and 633 for SNAP23/syndet tracted with TCA, as described (45). For immunoprecipitation, RBL-2H3 (U73143), 245/162 and 404/321 for VAMP1/VAMP2 (M24104/M24105), cells (5 ϫ 107 cells/ml) were lysed on ice for1hinbufferA(50mM 104 and 415 for cellubrevin (S63830), 67 and 290 for VAMP8 Tris-HCl, pH 8, 150 mM NaCl, 10 mM EDTA, and 1% Triton X-100) (AA049140), 1 and 874 and 1 and 794 for, respectively, full-length and containing a mixture of protease inhibitors. After centrifugation for 30 min truncated syntaxin 2 (L20823), and 32 and 928, and 32 and 841 for, re- at 14,000 ϫ g, soluble lysates were incubated overnight at 4°C with Abs spectively, full-length and truncated syntaxin 4 (L20821). The cDNAs for prebound to protein A-Sepharose beads (Amersham Pharmacia Biotech). syntaxin 3 were obtained from Dr. R. H. Scheller (Stanford University, Beads were pelleted and washed three times with buffer A and once with by guest on September 25, 2021 School of Medicine, Stanford, CA) and were amplified between bp 78 and buffer A 0.5% Triton X-100. Immunoprecipitated complexes were resolved 947 and 78 and 866 to generate, respectively, full-length and truncated on SDS-PAGE gels (46). Experimental conditions for transfer to nitrocel- cDNAs (L20820). lulose membrane, Ab incubations, and enhanced chemiluminescent (ECL) revelation were as described (37). cDNA constructs and recombinant proteins Transfections Truncated and full-length cDNAs for syntaxin 2, 3, and 4 were cloned in the eukaryotic expression vector pSR␣puro (37). pEGFP-C1 vector was Transient transfections into COS-7 cells were as reported (37). For func- purchased from Clontech (Palo Alto, CA). The cDNA for RFc␣ in pSr␣neo tional secretion assays, a transient cotransfection procedure was optimized was a gift of Dr. R. Monteiro (Hopital Necker, Paris, France). The cyto- for RBL-2H3 cells. Briefly, 4 ϫ 106 cells kept at room temperature were ⌬ ⌬ plasmic domains of syntaxin 24–265 (syn2 C), syntaxin 34–263 (syn3 C), electroporated using 5 ␮g of vector pEGFP-C1 (transfection marker) and ⌬ and syntaxin 49–270 (syn4 C) were also expressed as GST fusion proteins 10 ␮g of respectively pSr␣puro-syntaxin 2, pSr␣puro-syntaxin 3, using the pGEX-2TK vector (Amersham Pharmacia Biotech, Uppsala, pSr␣puro-syntaxin 4, or pSr␣puro (empty vector) in complete DMEM me- Sweden). Fusion proteins were purified as described in the supplier’s pro- dium. The cells received a single electrical pulse of 250 V, 1500 micro- tocols. Thrombin (Boehringer Mannheim, Indianapolis, IN) was used to farads, using an Eurobio EasyJect electroporation apparatus (Eurogentec, release the syntaxins from GST. All cDNAs concerning syntaxin isoforms Angers, France). Cells were then plated in complete medium, which was were sequenced and compared with the corresponding data deposited in replaced after 24 h. Exocytosis was determined 48 h after transfection GenBank. using 5G10 mAb (see below). Using these conditions, the efficiency of transfection (GFP-positive cells) was routinely in the range of 30% of total Antibodies population, as determined by flow cytofluorometry (see below). The co- transfection efficiency was estimated to be over 80%, as tested by cotrans- New Zealand white rabbits were immunized with the cytoplasmic domains ␣ of rat syntaxin 2, syntaxin 3, and syntaxin 4 proteins. Abs were affinity fection of GFP and RFc cDNAs, the latter being detected at the surface purified from sera on Sepharose 4B (Amersham Pharmacia Biotech) cou- of cells using biotinylated A59 mAb and PE-SA (Dako). pled to GST-syn2⌬C, GST-syn3⌬C, and GST-syn4⌬C fusion proteins, Exocytosis measurement by flow cytofluorometry respectively. Each batch was further depleted of common syntaxin epitopes using serial Sepharose 4B columns coupled to the two other cross-reacting Transfected RBL-2H3 cells in suspension were sensitized with IgE for 2 h isoforms. Affinity-purified anti-GST Abs (our laboratory) have been used. and stimulated for 20 min. After washing in cold PBS-0.05% BSA (buffer Anti-DNP-specific IgE and Fc⑀RI ␤-chain mAb, JRK, provided by Dr. J. B), all pellets were incubated on ice for 1 h with 20 ␮g/ml biotinylated Rivera (National Institute of Arthritis and Musculoskeletal and Skin Dis- 5G10 or 10D11.5 mAb. After washing, the cells were reincubated for 1 h eases, National Institutes of Health, Bethesda, MD), have been described at 4°C with PE-SA in buffer B, washed again, and resuspended in 0.5 ml (37). Rabbit anti-SNAP23 (TG8), anti-VAMP1 (MC9), anti-cellubrevin cold buffer B for analysis in a FACScan cytofluorograph (Becton Dickin- (MC16) sera, as well as affinity-purified anti-TI-VAMP (TG11), and anti- son, San Jose, CA). Fluorescence of 50,000 viable cells was analyzed, and VAMP8 (TG15) Abs have been used (22, 30, 32). Anti-syntaxin 1 mouse GFP-positive cells were selected within a gate on the FL1 channel mAb HCP-1 was from Sigma (St. Louis, MO). Rabbit anti-VAMP2 Abs (A530/30 band path filter). Exocytosis of single cells was measured by p80 (43) were provided by A. Klip and B. Trimble (The Hospital for Sick exposure at cell surface. Exposure of this Ag and secretion are closely Children, Toronto, Canada). The mouse anti-serotonin mAb (clone 5HT- correlated in time and Ag dose response (Ref. 44 and our unpublished H209) was from Dako (Trappes, France). Mouse mAbs against rat p80 Ag results). In our conditions, maximal values of degranulation were obtained 5852 SNARE PROTEINS IN RBL-2H3 MAST CELLS

FIGURE 1. Isoform specificity of anti-syntaxin Abs. Lysates from COS-7 cells (8.5 ϫ 105 cells/lane) transfected with cDNAs coding for either syn2⌬C, syn3⌬C, syn4⌬C, or empty pSR␣puro vector (mock) were resolved on SDS-PAGE and immunoblotted with anti-syntaxin 2, 3, and 4 Downloaded from Abs. Bands were visualized using ECL for detection.

after 20 min and were in the range of 30–50% of total ␤-hexosaminidase content. Expression of p80 Ag was measured using biotin-5G10 plus

PE-SA in the FL2 channel (585/42 band path filter). Biotin-10D11.5 was http://www.jimmunol.org/ used as an irrelevant Ab to detect nonspecific binding. Data were analyzed using CellQuest software, and expression levels were quantified by calcu- lating the ratio of the mean fluorescence intensities of specific (5G10) vs irrelevant (10D11.5) mAb staining. All data were analyzed by using the Mann-Whitney U test. Significance was assumed at p values Ͻ0.05. FIGURE 2. Characterization of SNARE proteins in RBL-2H3 lysates. TCA-precipitated total homogenates (5 and 20 ␮g of proteins from rat Tetanus toxin proteolysis brain and RBL-2H3 cells, respectively) were resolved on SDS-PAGE and RBL-2H3 cells (1.6 ϫ 107 cells/ml) were lysed at 4°C in 150 mM NaCl, immunoblotted with Abs specific for the various t-SNARE (A) and

8 mM MgCl2, 300 mM glycine, and 20 mM HEPES, pH 7.4, containing a v-SNARE (B) proteins. Bands were visualized using ECL for detection.

mixture of protease inhibitors and sonicated three times 10 s in ice. After The relevant section of the different blots is shown. The relative m.w. for by guest on September 25, 2021 removing the debris at 14,000 ϫ g for 1 min, 30-␮l aliquots were incubated each immunoreactive band is indicated by arrows. Shown is one of three for 2.5 h at 37°C with various concentrations of light chain tetanus toxin (a representative experiments. gift of Dr. M. Popoff, Institut Pasteur, Paris, France). This chain bears the catalytic activity of the toxin. Proteolysis was stopped by addition of sam- ple buffer under reducing conditions and boiling for 3.5 min. SNAP23 and detected a band migrating at about 29 kDa in RBL- Results 2H3 extracts and in the brain. Concerning v-SNAREs, VAMP2 SNARE proteins are endogenously expressed in RBL-2H3 cells (18 kDa) and cellubrevin (14 kDa), but not VAMP1, were ex- In initial experiments, mRNA expression in RBL-2H3 for different pressed in RBL-2H3 cells (Fig. 2B). In addition, we revealed t- and v-SNAREs was assessed by RT-PCR, taking rat brain VAMP8, a newly described isoform of 12 kDa, and a faint band of mRNA as a control. In the case of syntaxins, in which multiple 25 kDa corresponding to TI-VAMP. The above results show that isoforms have been characterized, we particularly searched for isoforms of each protein family composing the SNARE core com- those that have been described to be present on the plasma mem- plex are expressed in RBL-2H3 mast cells, thus confirming and brane. We identified a large number of expressed SNARE extending recent data obtained in rat peritoneal mast cells (42). To mRNAs. They included the t-SNAREs syntaxin 2, syntaxin 3, syn- further explore these findings, we investigated their interactions in taxin 4, and SNAP23, as well as the v-SNAREs VAMP1/2, cel- intact cells and their possible involvement in degranulation. lubrevin, and VAMP8. No SNAP25 mRNA was found (data not shown). All fragments amplified by RT-PCR corresponded to the Plasma membrane SNAP23 is central in SNARE complexes in expected size; in addition, the DNA sequences for syntaxins 2, 3, RBL-2H3 cells and 4 were verified. Because the t-SNARE SNAP23 has been shown to play a role in To characterize t- and v-SNAREs, we examined expressed pro- exocytosis of rat peritoneal mast cells (42), we analyzed its sub- teins by immunoblot analysis. We produced Abs against the three cellular location in RBL-2H3 cells and tested its capacity to form syntaxin isoforms identified above. Their isoform specificity was SNARE complexes. The staining pattern observed by confocal im- tested by immunoblot analysis of COS-7 cells transfected with munofluorescence microscopy was shown in Fig. 3A. A bright each syntaxin isoform. Fig. 1 shows that anti-syntaxin 2, 3, and 4 staining was uniformly distributed on the plasma membrane, no Abs did not react with unrelated syntaxins or mock-transfected signal being visible in intracellular organelles. To characterize the controls. Examination of t-SNAREs in RBL-2H3 cell extracts potential partners associated with SNAP23, a coimmunoprecipita- (Fig. 2A) shows that syntaxin 2, syntaxin 3, and syntaxin 4 migrate tion approach was undertaken. As protein interactions were very as molecular species of 36 kDa comparable with those present in faint in resting cells, we made use of NEM, a sulfhydryl-alkylating a crude rat brain homogenate. No band corresponding to syntaxin agent known to inactivate NSF (47) and induce SNARE complex 1 was revealed. As we did not detect SNAP25, we focused on accumulation (32, 48). After NEM treatment of RBL-2H3 cells, The Journal of Immunology 5853

FIGURE 4. Analysis of syntaxin 2-, 3-, and 4-associated SNAREs in FIGURE 3. SNAP23 is part of SNARE complexes present in RBL-2H3 resting RBL-2H3 cells. Cells were treated on ice with NEM plus 2 mM Downloaded from cells. A, Resting cells grown on coverslips were fixed, permeabilized, DTT for 30 min (Ϫ) or with 1 mM NEM for 15 min, followed by 2 mM stained with anti-SNAP23 Abs, revealed with goat anti-rabbit Abs coupled DTT for 15 min (ϩ). After lysis, immunoprecipitations were performed ␮ to FITC, and viewed by confocal microscopy. The bar represents 10 m. with anti-syntaxin 2, anti-syntaxin 3, and anti-syntaxin 4 Abs, or anti-GST Ϫ B, Cells were treated on ice with NEM plus 2 mM DTT for 30 min ( )or Abs as a control. Immunoprecipitated complexes (107 cell equivalents per ϩ with 1 mM NEM for 15 min, followed by 2 mM DTT for 15 min ( ). After lane) were resolved on SDS-PAGE and processed for immunoblotting with cell lysis, immunoprecipitations were performed with anti-SNAP23 Abs or specific Abs for t- and v-SNARE proteins. Bands were visualized by ECL, 7 http://www.jimmunol.org/ anti-GST Abs. Immunoprecipitated complexes (10 cell equivalents per and the relative m.w. for each immunoreactive band is indicated by arrows. lane) were resolved on SDS-PAGE and processed for immunoblotting with Shown is one representative experiment of three. GST immunoprecipitates specific Abs for t- and v-SNARE proteins. Bands were visualized using blotted with anti-syntaxin 2 and anti-syntaxin 3 Abs were similarly nega- ECL for detection, and the relative m.w. for each immunoreactive band is tive (not shown). indicated by arrows. Shown is one of three representative experiments.

single cells using flow cytofluorometry. This assay took advantage we immunoprecipitated SNAP23 and screened for the interactions of the stimulation-dependent surface expression of the intragranu-

with syntaxin and VAMP proteins using immunoblotting (Fig. lar protein p80 to measure exocytosis. The change of localization by guest on September 25, 2021 3B). As expected, all interactions were increased after NEM treat- has been shown to correlate with the release of granular content ment. SNAP23 was able to form multiple interactions with syn- (44). RBL-2H3 cells were transiently transfected with the cDNA taxin 2, syntaxin 3, and syntaxin 4, although coimmunoprecipita- coding for each syntaxin or empty vector together with GFP as a tion of syntaxin 3 was very faint even in the presence of NEM. marker for the transfected population. We verified that each syn- Interactions were also detectable with VAMP isoforms VAMP2, taxin was overexpressed using isoform-specific syntaxin Abs (Fig. cellubrevin, and VAMP8, but not TI-VAMP (data not shown). 5A). Cells were then stimulated with Ag, and p80 expression was Taken together, these results show that SNAP23 is capable of measured by cytofluorometry after gating GFP-positive cells. Fig. forming different sets of SNARE complexes in RBL-2H3 cells. 5B illustrates the distribution of fluorescence intensity for each type of transfectant in a typical experiment. The histogram for Only syntaxin 4 interacts with a specific set of SNARE proteins syntaxin 4 tranfectants shifted to lower levels as compared with As SNAP23 seems to be present in multiple complexes with dif- those for syntaxin 2, 3, or mock-transfected cells, indicating a de- ferent syntaxin isoforms, we examined more precisely the role of crease in p80 levels at the surface after overexpression of syntaxin this second group of t-SNARE proteins. Syntaxins 2, 3, and 4 were 4. The mean inhibition of p80 expression for each type of trans- immunoprecipitated from cell lysates before and after NEM treat- fectant is presented in Fig. 5C. Overexpression of syntaxin 4 sig- ment, and the presence of associated proteins was investigated by nificantly inhibited p80 expression by 31% ( p ϭ 0.0039). In con- immunoblotting (Fig. 4). In syntaxin 2 and 3 immunoprecipitates, trast, overexpression of syntaxin 2 and syntaxin 3 did not affect SNAP23 was only seen after treatment, and none of the examined p80 expression when compared with mock-transfected cells ( p Ͼ v-SNAREs was revealed. In contrast, syntaxin 4 immunoprecipi- 0.5). In conclusion, our results strongly support a role for syntaxin tates contained SNAP23 as well as VAMP2, cellubrevin, and 4 in regulated exocytosis of RBL-2H3 cells. VAMP8, whereas TI-VAMP was undetectable (not shown). Thus, in addition to SNAP23, syntaxin 4 may be a good candidate for the VAMP8 is insensitive to tetanus toxin formation of SNARE complexes, ensuring the traffic between Because SNAP23 and syntaxin 4 appear to be part of the SNARE granules and the plasma membrane. core complex involved in RBL-2H3 exocytosis, we investigated which VAMP isoform(s) could constitute the third member of this ⑀ Syntaxin 4 is functionally involved in Fc RI-stimulated complex. A particular feature of VAMP2 and cellubrevin is their degranulation sensitivity to tetanus toxin (29, 49). Previous functional studies We investigated in a next step whether the overexpression of syn- have shown that RBL-2H3 degranulation is resistant to this toxin, taxin 4 could affect Fc⑀RI-mediated degranulation compared with suggestive of a role for a tetanus toxin-insensitive VAMP isoform overexpression of syntaxins 2 and 3. For this purpose, we devel- in that process (50). Fig. 6 confirms that VAMP2 and cellubrevin oped an assay that allows the quantitative analysis of exocytosis of were sensitive to tetanus toxin in a dose-dependent manner, while 5854 SNARE PROTEINS IN RBL-2H3 MAST CELLS Downloaded from http://www.jimmunol.org/

FIGURE 5. Overexpression of syntaxin 4 inhibits degranulation of RBL-2H3 cells. A, Syntaxin 2, syntaxin 3, and syntaxin 4 were overexpressed by transient transfection. Cells were transfected with full-length syntaxin cDNAs or pSR␣puro empty vector (mock) together with GFP cDNA. Total lysates (2.5 ϫ 105 equivalent cells per lane) were migrated on SDS-PAGE and probed with Abs directed either against syntaxin 2, syntaxin 3, or syntaxin 4. The loading of equivalent amounts of proteins in each lane was verified after reprobing the blots with the anti-Fc⑀RI ␤-chain mAbs. B, Decrease of stimulation- dependent p80 surface expression in syntaxin 4-overexpressing cells. Cells were transfected with full-length syntaxin cDNAs or pSR␣puro empty vector (mock) together with GFP cDNA. After 48 h, cells were stimulated with DNP-human serum albumin for 20 min, and p80 expression was assayed by incubating cells with biotinylated 5G10 plus PE-SA. After washing in PBS containing 0.05% BSA, cells were analyzed by cytofluorometry after gating GFP-positive cells. Histograms represent the distribution of p80 fluorescence intensities for syntaxin 2-, 3-, 4-, and mock-transfected cells for a particular by guest on September 25, 2021 experiment. The control histogram was obtained for each transfected cell population by incubation with irrelevant biotinylated anti-CD8 mAb, and the histogram shown is representative of the unspecific fluorescence distribution. C, Inhibition of p80 expression in transfected cells. Data are expressed as percentage of inhibition of p80 expression for transfected cells as compared with mock-transfected cells. Error bars represent the mean Ϯ SEM of six independent transfection experiments. The asterisks denote a significant difference (p Ͻ 0.02).

TI-VAMP was resistant in RBL-2H3 lysates. Interestingly, VAMP8 as candidates for being part of the exocytotic process in VAMP8 was also insensitive to this neurotoxin even at concen- RBL-2H3 cells. trations as high as 0.45 ␮M. These results point to TI-VAMP and

VAMP8 is localized on secretory granules To further investigate the role of these v-SNAREs, we examined the subcellular localization of VAMP proteins in RBL-2H3 resting cells. VAMP2 and cellubrevin both stained diffuse small vesicular structures (Fig. 7, A and B), whereas TI-VAMP and VAMP8 stained larger organelles distributed throughout the cytoplasm (Fig. 7, C and D). Colocalization experiments were performed us- ing a mAb directed against serotonin, which specifically labels mast cell secretory granules. In agreement with their earlier de- scribed localization (51), serotonin-containing compartments ap- peared as large granular organelles (Fig. 7E–H). It should be noted FIGURE 6. VAMP8 is insensitive to tetanus toxin. Cell lysates were that not all cells are serotonin positive, possibly reflecting a dif- subjected to digestion with various concentrations of tetanus toxin light ference in their stage of maturation. There was no significant co- chain: lane 1, no toxin; lane 2, 0.12 ␮M; lane 3, 0.30 ␮M; lane 4, 0.45 ␮M. localization of VAMP2 or TI-VAMP with serotonin-containing ϫ 5 Equal aliquots of the lysates (2.4 10 cell equivalents per lane) were structures (compare A and E, C and G, respectively, and overlay resolved by SDS-PAGE and immunoblotted with Abs directed against images I and K), and only a limited colocalization of cellubrevin VAMP2, cellubrevin, TI-VAMP, or VAMP8, and revealed by ECL. In sensitive proteins, the immunoreactivity is lost due to the proteolysis of with these serotonin-containing structures. On the contrary, epitope-containing fragments (aa 1–76 in VAMP2 and cellubrevin), which VAMP8 colocalized with a larger set of the serotonin-containing run out of the SDS gel. The same blots were reprobed with anti-Fc⑀RI ␤-chain vesicles, suggesting that VAMP8 is present on a subset of secre- to control that the same quantities of protein were deposited (not shown). tory granules (D, H, and overlay in L). Altogether, these results The Journal of Immunology 5855

FIGURE 7. VAMP8 is colocalized on secretory granules with serotonin. Resting cells grown on coverslips were fixed, permeabilized, and dou- blelabeledwithanti-VAMP2(A),anti- Downloaded from cellubrevin (B), anti-TI-VAMP (C), or anti-VAMP8 Abs (D) together with anti-serotonin mAb (E, F, G, and H). They were revealed with goat anti-rabbit Abs coupled to FITC and goat anti-mouse Abs coupled to TRSC and viewed by confocal mi- http://www.jimmunol.org/ croscopy. In the two-color overlay image, granules containing both se- rotonin and the VAMP8 Ag have yel- low to orange tints (I, J, K, and L). No significant staining was seen in the absence of first Abs or in the pres- ence of irrelevant Abs (not shown). The bar represents 10 ␮m. by guest on September 25, 2021

could support the involvement of VAMP8 in exocytosis of these role of syntaxin 4 in Fc⑀RI-stimulated secretion and for the local- mediator-containing granules. ization of its binding partner, the v-SNARE VAMP8, on secretory granules. Discussion Identification of t-SNARE proteins SNAP23, syntaxin 3, and Although many unique SNAREs with specific locations have been syntaxin 4 as well as the v-SNARE VAMP2 corroborates previous identified, we are only beginning to understand how these proteins observations in rat peritoneal mast cells (42). However, in adherent regulate the specificity of particular vesicle-mediated transport RBL-2H3 cells, SNAP23 is linearly localized along the plasma pathways. The hemopoietic cell lineage, with the rather peculiar membrane at discrepancy with the patchy appearance of SNAP23 feature of secretory lysosomes, may use specialized mechanisms reported in suspended peritoneal mast cells (42). A distinct func- for sorting and secretion, which differ from those found in con- tional state of the cellular cytoskeleton according to the status of ventional secretory cells (7). Taking RBL-2H3 cells as a model for adherence of the mast cell could explain this difference (52). In- mast cells, we show that several isoforms of the three SNARE deed, SNAP23 may interact with the cytoskeleton, possibly actin protein families are expressed and that they form different sets of microfilaments, as reported in suspended peritoneal mast cells complexes in intact cells. We provide evidence for a functional (42). The characterization of syntaxin 2, cellubrevin, TI-VAMP, 5856 SNARE PROTEINS IN RBL-2H3 MAST CELLS and VAMP8 reported in this work further extends the number of Concerning v-SNAREs involved in exocytosis, two independent SNARE proteins potentially involved in regulation of intracellular findings could support for a role for VAMP8. On the one hand, we traffic in mast cells. have demonstrated that VAMP8 was colocalized, at least in large SNARE proteins function in membrane fusion by cognate pair- part, with serotonin-containing granules, in contrast to the other ing to form a stable receptor for SNAP and NSF proteins. In rest- VAMPs examined (cellubrevin was found to be superimposed ing RBL-2H3 cells, we detected a small amount of SNAP23, with serotonin in a small subset of structures). On the other hand, VAMP2, or cellubrevin in interaction with syntaxin 4. Following we have found that VAMP8 was insensitive to tetanus toxin treat- NEM treatment, which is known to block NSF and accumulate ment, the latter result being consistent with the data showing that SNARE complexes, all of these interactions were increased, and RBL-2H3 cells pretreated with this neurotoxin are still able to those between syntaxin 4 and VAMP8 could be detected. On the secrete serotonin (50). contrary, neither syntaxin 2 nor syntaxin 3 was able to bind these The localization of VAMP8 to a subset of secretory granules is different SNAREs, except a faint binding with SNAP23. Interest- a new finding in light of previous data that have shown its enrich- ingly, both syntaxins are found in intracellular organelles in RBL- ment in the later compartment of early endosomes for a number of 2H3 cells (Ref. 42 and our unpublished results), suggesting that cell types (34). This particular staining pattern could be character- they are implicated in other trafficking pathways remaining to be istic for the lysosomal nature of these organelles in mast cells (4, defined, possibly related to the various types of granules present in 5, 7). Indeed, recent data have demonstrated the existence of at these cells. Altogether, our coimmunoprecipitation results support least three types of secretory granules, all of them containing the the idea that syntaxin 4 as well as SNAP23 preferentially interact lysosomal enzyme ␤ hexosaminidase (3, 38). Type I and type II with VAMP2, cellubrevin, and VAMP8 in intact cells. granules contained MHC class II molecules and were labeled by Downloaded from The implication of syntaxin 4 in degranulation is confirmed by fluid endocytic markers, whereas serotonin was only observed in functional studies. Indeed, overexpression of syntaxin 4, but not of type II and type III granules. Given its endocytic nature, an attrac- syntaxin 2 nor 3, significantly inhibited Fc⑀RI-mediated exocyto- tive hypothesis would be to consider VAMP8 as a marker for type sis. A role for syntaxin 4 in regulating vesicular docking and fusion I and type II granules. The latter would be consistent with the events has also been reported in ␣-granule secretion of platelets observed staining pattern, because VAMP8 labels both serotonin-

(39) and in adipocytes (53), suggesting that syntaxin 4 is an im- negative (which would correspond to type I) and part of the sero- http://www.jimmunol.org/ portant t-SNARE in regulated exocytosis of nonneuronal cells. The tonin-positive (which would correspond to type II) granules. mechanisms by which overexpression of wild-type syntaxin 4 in- VAMP8 localization on serotonin-containing granules could thus hibits regulated exocytosis pathways is not completely clear. Over- represent a specific feature of mast cells, or more widely, of se- production of this syntaxin protein may perturb the steady state cretory cells within the hemopoietic cell lineage. between the three SNARE, components leading to aberrant inter- In conclusion, the present study demonstrates that the SNARE actions and blockade of recycling by “titrating” general factors like molecular machinery is involved in RBL-2H3 degranulation, and NSF or SNAP proteins. Alternatively, overexpression of syntaxin suggests that it depends on an exocytotic complex composed of 4 could disturb regulatory factors implicated in the specificity of syntaxin 4, SNAP23, and VAMP8. The presence of VAMP8 in SNARE complexes such as munc18 isoforms. Two lines of evi- this complex opens the possibility that a v-SNARE, originally lo- by guest on September 25, 2021 dence argue in favor of that hypothesis: 1) overexpression of a calized on an endosomal compartment, functions in exocytosis. In munc18 homologue involved in endoplasmic reticulum to Golgi this context, secretory granules in mast cells could be considered transport was able to overcome the dominant negative effects of as a specialized compartment at the intersection between exocy- overexpressed syntaxin 5 (54); and 2) munc18 expression together totic and endocytic pathways. with syntaxin 1A partially relieves the blockage of secretion in a storage compartment observed when expressing syntaxin 1A alone Acknowledgments (55). A third possibility relates to GTPases, which, among the proteins playing a key role in the secretory pathway, could facil- We thank Drs. J. Bonifacino, A. Klip, R. Monteiro, M. Popoff, J. Rivera, itate SNARE complex formation (56, 57). Rab3d, involved in mast and R. Scheller for reagents. We are grateful to Drs. M. Benhamou, S. Mecheri, H. Metzger, and A. Weyer for critical reading of the manu- cell exocytosis, (37, 58), may be a good candidate for direct or script and helpful discussions. We thank R. Hellio for excellent confocal indirect interactions with syntaxin 4. microscopy assistance, B. Iannascoli for technical help, and R. Peronet for The function of this t-SNARE in exocytosis implies the mobi- help in preparing polyclonal Abs. The confocal microscope was purchased lization of syntaxin 4-containing complexes during stimulation. with a donation from Marcel and Liliane Pollack. However, we were not able to show that any of the proteins in- teracting with syntaxin 4, i.e., SNAP23, VAMP2, cellubrevin, or References VAMP8, were modulated in response to Fc⑀RI-mediated stimula- tion (data not shown). These observations either suggest that there 1. Abraham, S. N., and R. Malaviya. 1997. Mast cells in infection and immunity. Infect. Immun. 65:3501. is no change or that this change is too subtle to be revealed by 2. Galli, S. J., and C. 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