Soluble NSF Attachment Protein Receptors (SNAREs) in RBL-2H3 Mast Cells: Functional Role of Syntaxin 4 in Exocytosis and Identification of a Vesicle-Associated This information is current as Membrane Protein 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 proteins 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 syntaxin 3, 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 synaptobrevin, 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, syntaxins 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 synaptobrevins 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 synaptotagmin 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