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*
Unité d’Immuno-Allergie, Institut Pasteur, Paris, France; and
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, Institut Curie, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All vesicular traffic requires close apposition between membranes
before fusion. It has been established that this function is partly
fulfilled by SNARE3
proteins (9, 10, 11, 12). Initially discovered in neuronal cells,
the SNARE complex is composed of a vesicle-bound v-SNARE protein,
namely VAMP or synaptobrevin, and two target organelle t-SNARE
proteins, called SNAP25/23/29 and syntaxin (9). These
cognate SNARE proteins assemble in a ternary complex through
coiled-coil domains forming a parallel four-helix bundle structure
(13, 14, 15). Dissociation of the complex with soluble NSF and
-SNAP, a process requiring ATP hydrolysis, is necessary to allow the
recycling of SNARE components (16). In agreement with the
multiple isoforms identified to date, at least part of the specificity
of vesicle trafficking is ensured by a unique set of SNAREs in the
different membrane compartments (9, 17). Among t-SNAREs of
the SNAP25/23/29 family, the ubiquitously expressed isoform, SNAP23
(18, 19), is involved in the regulation of exocytosis of
nonneuronal cells (20, 21, 22). The second group of t-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 reported to regulate
exocytosis (21, 22, 23, 24, 25). Within the v-SNARE family, some are
involved in regulated exocytosis, such as VAMP1 and VAMP2, also called
synaptobrevins 1 and 2 (26, 27, 28), and others in
constitutive vesicle recycling (endocytosis/exocytosis) such as
cellubrevin/VAMP3 (29, 30, 31). TI-VAMP/VAMP7 has recently
been implicated in apical transport in epithelial cells (22, 32), and in endosome to lysosome transport (33). As
for endobrevin/VAMP8, it has been localized to endosomes (34, 35). Reconstitution experiments have shown that the SNARE
complex minimal structure (composed of two t-SNAREs and one v-SNARE)
can induce membrane fusion in vitro (12), albeit the slow
kinetics suggests that additional mechanisms and regulatory proteins
are operative (36). In RBL-2H3 mast cells, recent data
have shown rab3d and synaptotagmin II as regulators of
IgE-mediated exocytosis (37, 38).
Although the bulk of available information on SNARE proteins concerns
neuronal and neuroendocrine cells, these proteins are also implicated
in the regulated exocytosis of cells of the hemopoietic lineage
(39, 40). In mast cells, the expression of several SNARE
proteins has been reported, and SNAP23 relocation was shown to be
coupled to secretion in permeabilized cells (41, 42). The
aim of our study was to characterize additional SNARE proteins
expressed in RBL-2H3 mast cells and to investigate their role in
FcRI-triggered exocytosis. We show that besides SNAP23, syntaxin 4
is the only t-SNARE that interacts with the identified v-SNAREs in
intact cells. We provide evidence for the function of syntaxin 4 in
degranulation. Furthermore, we observe that VAMP8 is tetanus toxin
insensitive and colocalizes with a part of the secretory granules,
suggesting this v-SNARE as the potential partner of syntaxin 4 and
SNAP23 in the regulated secretion of mast cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RBL-2H3, COS-7, and transiently transfected cells were maintained as monolayer cultures, as previously described (37). For stimulation, RBL-2H3 cells were seeded in 175-mm2 flasks (70% confluency) or used in suspension (1 x 106 cells/ml), sensitized with IgE anti-DNP, and stimulated with Ag, as described (37).
RT-PCR and PCR
Total RNA was isolated as described (37). For RT-PCR, 2 µg of total RNA of RBL-2H3 cells or brain was reverse transcribed using Superscript reverse transcriptase (Life Technologies, Eragny, France). The cDNAs were amplified between bp 13 and 544 for SNAP25a/b (GenBank database accession number U56261/U56262), 1 and 633 for SNAP23/syndet (U73143), 245/162 and 404/321 for VAMP1/VAMP2 (M24104/M24105), 104 and 415 for cellubrevin (S63830), 67 and 290 for VAMP8 (AA049140), 1 and 874 and 1 and 794 for, respectively, full-length and truncated syntaxin 2 (L20823), and 32 and 928, and 32 and 841 for, respectively, full-length and truncated syntaxin 4 (L20821). The cDNAs for syntaxin 3 were obtained from Dr. R. H. Scheller (Stanford University, School of Medicine, Stanford, CA) and were amplified between bp 78 and 947 and 78 and 866 to generate, respectively, full-length and truncated cDNAs (L20820).
cDNA constructs and recombinant proteins
Truncated and full-length cDNAs for syntaxin 2, 3, and 4 were
cloned in the eukaryotic expression vector pSRpuro
(37). pEGFP-C1 vector was purchased from Clontech (Palo
Alto, CA). The cDNA for RFc in pSrneo was a gift of Dr. R.
Monteiro (Hopital Necker, Paris, France). The cytoplasmic domains of
syntaxin 24–265 (syn2
C), syntaxin
34–263 (syn3C), and syntaxin
49–270 (syn4C) were also expressed as GST
fusion proteins using the pGEX-2TK vector (Amersham Pharmacia Biotech,
Uppsala, Sweden). Fusion proteins were purified as described in the
supplier’s protocols. Thrombin (Boehringer Mannheim, Indianapolis, IN)
was used to release the syntaxins from GST. All cDNAs concerning
syntaxin isoforms were sequenced and compared with the corresponding
data deposited in GenBank.
Antibodies
New Zealand white rabbits were immunized with the cytoplasmic
domains of rat syntaxin 2, syntaxin 3, and syntaxin 4 proteins. Abs
were affinity purified from sera on Sepharose 4B (Amersham Pharmacia
Biotech) coupled to GST-syn2C, GST-syn3C, and GST-syn4C fusion
proteins, respectively. Each batch was further depleted of common
syntaxin epitopes using serial Sepharose 4B columns coupled to the two
other cross-reacting isoforms. Affinity-purified anti-GST Abs (our
laboratory) have been used. Anti-DNP-specific IgE and FcRI ß-chain
mAb, JRK, provided by Dr. J. Rivera (National Institute of Arthritis
and Musculoskeletal and Skin Diseases, National Institutes of Health,
Bethesda, MD), have been described (37). Rabbit
anti-SNAP23 (TG8), anti-VAMP1 (MC9), anti-cellubrevin
(MC16) sera, as well as affinity-purified anti-TI-VAMP (TG11), and
anti-VAMP8 (TG15) Abs have been used (22, 30, 32).
Anti-syntaxin 1 mouse mAb HCP-1 was from Sigma (St. Louis, MO). Rabbit
anti-VAMP2 Abs (43) were provided by A. Klip and B.
Trimble (The Hospital for Sick Children, Toronto, Canada). The mouse
anti-serotonin mAb (clone 5HT-H209) was from Dako (Trappes,
France). Mouse mAbs against rat p80 Ag and against human CD8 were
produced respectively from the 5G10 clone provided by Dr. J.
Bonifacino (44) and from the 10D11.5 clone. Both Abs were
biotinylated with the Enzotin Biotinylation Kit, according to the
manufacturer’s instructions (Enzo Biochem, Syosset, NY). Biotinylated
mouse mAb A59 directed against RFc was provided by Dr. R. Monteiro.
Peroxidase-coupled goat anti-rabbit and goat anti-mouse IgGs
and Texas Red sulfonide chloride (TRSC)-conjugated goat anti-mouse
IgG, all specific for the Fc
fragment, were purchased at Jackson
ImmunoResearch (West Grove, PA). FITC-conjugated goat anti-rabbit
IgGs (H+L) was from Biosys S.A. (Compiègne, France).
Confocal immunofluorescence microscopy
RBL-2H3 cells were seeded on 10-mm-diameter glass coverslips (2 x 105 cells/coverslip) 16 h before analysis. For indirect immunofluorescence analysis, cells were fixed in 3% paraformaldehyde for 10 min, followed by treatment with 50 mM NH4Cl for 15 min. After washing, cells were permeabilized in 0.05% saponin and saturated in the presence of 0.25% BSA and 5% goat serum (MedGene Science, Pantin, France). Primary Abs were incubated 1 h in the same buffer and then revealed with FITC- and/or TRSC-conjugated secondary Abs. Finally, the coverslips were mounted in Mowiol and viewed with a Leica TCS confocal laser-scanning microscope.
NEM treatment
Adherent RBL-2H3 cells were briefly washed with PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2 (D-PBS) and incubated with D-PBS containing 1 mM NEM (Sigma) for 15 min on ice, followed by 15 min in the presence of 2 mM DTT to quench NEM. Control cells were incubated in 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 subsequently used for immunoprecipitation (32).
Immunoprecipitation and immunoblotting
For some experiments, rat brain and RBL-2H3 homogenates were extracted with TCA, as described (45). For immunoprecipitation, RBL-2H3 cells (5 x 107 cells/ml) were lysed on ice for 1 h in buffer A (50 mM Tris-HCl, pH 8, 150 mM NaCl, 10 mM EDTA, and 1% Triton X-100) containing a mixture of protease inhibitors. After centrifugation for 30 min at 14,000 x g, soluble lysates were incubated overnight at 4°C with Abs prebound to protein A-Sepharose beads (Amersham Pharmacia Biotech). Beads were pelleted and washed three times with buffer A and once with buffer A 0.5% Triton X-100. Immunoprecipitated complexes were resolved on SDS-PAGE gels (46). Experimental conditions for transfer to nitrocellulose membrane, Ab incubations, and enhanced chemiluminescent (ECL) revelation were as described (37).
Transfections
Transient transfections into COS-7 cells were as reported
(37). For functional secretion assays, a transient
cotransfection procedure was optimized for RBL-2H3 cells. Briefly,
4 x 106 cells kept at room temperature were
electroporated using 5 µg of vector pEGFP-C1 (transfection marker)
and 10 µg of respectively pSrpuro-syntaxin 2, pSrpuro-syntaxin
3, pSrpuro-syntaxin 4, or pSrpuro (empty vector) in complete DMEM
medium. The cells received a single electrical pulse of 250 V, 1500
microfarads, using an Eurobio EasyJect electroporation apparatus
(Eurogentec, Angers, France). Cells were then plated in complete
medium, which was replaced after 24 h. Exocytosis was determined
48 h after transfection using 5G10 mAb (see below). Using these
conditions, the efficiency of transfection (GFP-positive cells) was
routinely in the range of 30% of total population, as determined by
flow cytofluorometry (see below). The cotransfection efficiency was
estimated to be over 80%, as tested by cotransfection of GFP and
RFc cDNAs, the latter being detected at the surface of cells using
biotinylated A59 mAb and PE-SA (Dako).
Exocytosis measurement by flow cytofluorometry
Transfected RBL-2H3 cells in suspension were sensitized with IgE for 2 h and stimulated for 20 min. After washing in cold PBS-0.05% BSA (buffer B), all pellets were incubated on ice for 1 h with 20 µg/ml biotinylated 5G10 or 10D11.5 mAb. After washing, the cells were reincubated for 1 h at 4°C with PE-SA in buffer B, washed again, and resuspended in 0.5 ml cold buffer B for analysis in a FACScan cytofluorograph (Becton Dickinson, San Jose, CA). Fluorescence of 50,000 viable cells was analyzed, and GFP-positive cells were selected within a gate on the FL1 channel (A530/30 band path filter). Exocytosis of single cells was measured by p80 exposure at cell surface. Exposure of this Ag and secretion are closely correlated in time and Ag dose response (Ref. 44 and our unpublished results). In our conditions, maximal values of degranulation were obtained after 20 min and were in the range of 30–50% of total ß-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 used as an irrelevant Ab to detect nonspecific binding. Data were analyzed using CellQuest software, and expression levels were quantified by calculating 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 <0.05.
Tetanus toxin proteolysis
RBL-2H3 cells (1.6 x 107 cells/ml) were lysed at 4°C in 150 mM NaCl, 8 mM MgCl2, 300 mM glycine, and 20 mM HEPES, pH 7.4, containing a mixture of protease inhibitors and sonicated three times 10 s in ice. After removing the debris at 14,000 x g for 1 min, 30-µl aliquots were incubated for 2.5 h at 37°C with various concentrations of light chain tetanus toxin (a gift of Dr. M. Popoff, Institut Pasteur, Paris, France). This chain bears the catalytic activity of the toxin. Proteolysis was stopped by addition of sample buffer under reducing conditions and boiling for 3.5 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, mRNA expression in RBL-2H3 for different t- and v-SNAREs was assessed by RT-PCR, taking rat brain mRNA as a control. In the case of syntaxins, in which multiple isoforms have been characterized, we particularly searched for those that have been described to be present on the plasma membrane. We identified a large number of expressed SNARE mRNAs. They included the t-SNAREs syntaxin 2, syntaxin 3, syntaxin 4, and SNAP23, as well as the v-SNAREs VAMP1/2, cellubrevin, and VAMP8. No SNAP25 mRNA was found (data not shown). All fragments amplified by RT-PCR corresponded to the expected size; in addition, the DNA sequences for syntaxins 2, 3, and 4 were verified.
To characterize t- and v-SNAREs, we examined expressed proteins by
immunoblot analysis. We produced Abs against the three syntaxin
isoforms identified above. Their isoform specificity was tested by
immunoblot analysis of COS-7 cells transfected with each syntaxin
isoform. Fig. 1
shows that
anti-syntaxin 2, 3, and 4 Abs did not react with unrelated
syntaxins or mock-transfected controls. Examination of t-SNAREs in
RBL-2H3 cell extracts (Fig. 2A) shows that syntaxin 2,
syntaxin 3, and syntaxin 4 migrate as molecular species of 36 kDa
comparable with those present in a crude rat brain homogenate. No band
corresponding to syntaxin 1 was revealed. As we did not detect SNAP25,
we focused on SNAP23 and detected a band migrating at about 29 kDa in
RBL-2H3 extracts and in the brain. Concerning v-SNAREs, VAMP2 (18 kDa)
and cellubrevin (14 kDa), but not VAMP1, were expressed in RBL-2H3
cells (Fig. 2B). In addition, we revealed VAMP8, a newly
described isoform of 12 kDa, and a faint band of 25 kDa corresponding
to TI-VAMP. The above results show that isoforms of each protein family
composing the SNARE core complex are expressed in RBL-2H3 mast cells,
thus confirming and extending recent data obtained in rat peritoneal
mast cells (42). To further explore these findings, we
investigated their interactions in intact cells and their possible
involvement in degranulation.
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Because the t-SNARE SNAP23 has been shown to play a role in
exocytosis of rat peritoneal mast cells (42), we analyzed
its subcellular location in RBL-2H3 cells and tested its capacity to
form SNARE complexes. The staining pattern observed by confocal
immunofluorescence microscopy was shown in Fig. 3A. A bright staining was
uniformly distributed on the plasma membrane, no signal being visible
in intracellular organelles. To characterize the potential partners
associated with SNAP23, a coimmunoprecipitation approach was
undertaken. As protein interactions were very faint in resting cells,
we made use of NEM, a sulfhydryl-alkylating agent known to inactivate
NSF (47) and induce SNARE complex accumulation (32, 48). After NEM treatment of RBL-2H3 cells, we immunoprecipitated
SNAP23 and screened for the interactions with syntaxin and VAMP
proteins using immunoblotting (Fig. 3B). As expected, all
interactions were increased after NEM treatment. SNAP23 was able to
form multiple interactions with syntaxin 2, syntaxin 3, and syntaxin 4,
although coimmunoprecipitation of syntaxin 3 was very faint even in the
presence of NEM. Interactions were also detectable with VAMP isoforms
VAMP2, cellubrevin, and VAMP8, but not TI-VAMP (data not shown). Taken
together, these results show that SNAP23 is capable of forming
different sets of SNARE complexes in RBL-2H3 cells.
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As SNAP23 seems to be present in multiple complexes with different
syntaxin isoforms, we examined more precisely the role of this second
group of t-SNARE proteins. Syntaxins 2, 3, and 4 were
immunoprecipitated from cell lysates before and after NEM treatment,
and the presence of associated proteins was investigated by
immunoblotting (Fig. 4). In syntaxin 2
and 3 immunoprecipitates, SNAP23 was only seen after treatment, and
none of the examined v-SNAREs was revealed. In contrast, syntaxin 4
immunoprecipitates contained SNAP23 as well as VAMP2, cellubrevin, and
VAMP8, whereas TI-VAMP was undetectable (not shown). Thus, in addition
to SNAP23, syntaxin 4 may be a good candidate for the formation of
SNARE complexes, ensuring the traffic between granules and the plasma
membrane.
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RI-stimulated
degranulation
We investigated in a next step whether the overexpression of
syntaxin 4 could affect FcRI-mediated degranulation compared with
overexpression of syntaxins 2 and 3. For this purpose, we developed an
assay that allows the quantitative analysis of exocytosis of single
cells using flow cytofluorometry. This assay took advantage of the
stimulation-dependent surface expression of the intragranular protein
p80 to measure exocytosis. The change of localization has been shown to
correlate with the release of granular content (44).
RBL-2H3 cells were transiently transfected with the cDNA coding for
each syntaxin or empty vector together with GFP as a marker for the
transfected population. We verified that each syntaxin was
overexpressed using isoform-specific syntaxin Abs (Fig. 5A). Cells were then
stimulated with Ag, and p80 expression was measured by cytofluorometry
after gating GFP-positive cells. Fig. 5B illustrates the
distribution of fluorescence intensity for each type of transfectant in
a typical experiment. The histogram for syntaxin 4 tranfectants shifted
to lower levels as compared with those for syntaxin 2, 3, or
mock-transfected cells, indicating a decrease in p80 levels at the
surface after overexpression of syntaxin 4. The mean inhibition of p80
expression for each type of transfectant is presented in Fig. 5C. Overexpression of syntaxin 4 significantly inhibited p80
expression by 31% (p = 0.0039). In contrast,
overexpression of syntaxin 2 and syntaxin 3 did not affect p80
expression when compared with mock-transfected cells
(p > 0.5). In conclusion, our results strongly
support a role for syntaxin 4 in regulated exocytosis of RBL-2H3
cells.
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Because SNAP23 and syntaxin 4 appear to be part of the SNARE core
complex involved in RBL-2H3 exocytosis, we investigated which VAMP
isoform(s) could constitute the third member of this complex. A
particular feature of VAMP2 and cellubrevin is their sensitivity to
tetanus toxin (29, 49). Previous functional studies have
shown that RBL-2H3 degranulation is resistant to this toxin, suggestive
of a role for a tetanus toxin-insensitive VAMP isoform in that process
(50). Fig. 6 confirms that
VAMP2 and cellubrevin were sensitive to tetanus toxin in a
dose-dependent manner, while TI-VAMP was resistant in RBL-2H3 lysates.
Interestingly, VAMP8 was also insensitive to this neurotoxin even at
concentrations as high as 0.45 µM. These results point to TI-VAMP and
VAMP8 as candidates for being part of the exocytotic process in RBL-2H3
cells.
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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 using a mAb directed against
serotonin, which specifically labels mast cell secretory granules. In
agreement with their earlier described localization (51),
serotonin-containing compartments appeared as large granular organelles
(Fig. 7E–H). It should be noted that not all
cells are serotonin positive, possibly reflecting a difference in their
stage of maturation. There was no significant colocalization of VAMP2
or TI-VAMP with serotonin-containing structures (compare A
and E, C and G, respectively, and
overlay images I and K), and only a limited
colocalization of cellubrevin with these serotonin-containing
structures. On the contrary, VAMP8 colocalized with a larger set of the
serotonin-containing vesicles, suggesting that VAMP8 is present on a
subset of secretory granules (D, H, and overlay
in L). Altogether, these results could support the
involvement of VAMP8 in exocytosis of these mediator-containing
granules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-stimulated secretion and for the localization of its
binding partner, the v-SNARE VAMP8, on secretory granules. Identification of t-SNARE proteins SNAP23, syntaxin 3, and syntaxin 4 as well as the v-SNARE VAMP2 corroborates previous observations in rat peritoneal mast cells (42). However, in adherent RBL-2H3 cells, SNAP23 is linearly localized along the plasma membrane at discrepancy with the patchy appearance of SNAP23 reported in suspended peritoneal mast cells (42). A distinct functional state of the cellular cytoskeleton according to the status of adherence of the mast cell could explain this difference (52). Indeed, SNAP23 may interact with the cytoskeleton, possibly actin microfilaments, as reported in suspended peritoneal mast cells (42). The characterization of syntaxin 2, cellubrevin, TI-VAMP, and VAMP8 reported in this work further extends the number of SNARE proteins potentially involved in regulation of intracellular traffic in mast cells.
SNARE proteins function in membrane fusion by cognate pairing to form a stable receptor for SNAP and NSF proteins. In resting RBL-2H3 cells, we detected a small amount of SNAP23, VAMP2, or cellubrevin in interaction with syntaxin 4. Following NEM treatment, which is known to block NSF and accumulate SNARE complexes, all of these interactions were increased, and those between syntaxin 4 and VAMP8 could be detected. On the contrary, neither syntaxin 2 nor syntaxin 3 was able to bind these different SNAREs, except a faint binding with SNAP23. Interestingly, both syntaxins are found in intracellular organelles in RBL-2H3 cells (Ref. 42 and our unpublished results), suggesting that they are implicated in other trafficking pathways remaining to be defined, possibly related to the various types of granules present in these cells. Altogether, our coimmunoprecipitation results support the idea that syntaxin 4 as well as SNAP23 preferentially interact with VAMP2, cellubrevin, and VAMP8 in intact cells.
The implication of syntaxin 4 in degranulation is confirmed by
functional studies. Indeed, overexpression of syntaxin 4, but not of
syntaxin 2 nor 3, significantly inhibited FcRI-mediated exocytosis.
A role for syntaxin 4 in regulating vesicular docking and fusion events
has also been reported in -granule secretion of platelets
(39) and in adipocytes (53), suggesting that
syntaxin 4 is an important t-SNARE in regulated exocytosis of
nonneuronal cells. The mechanisms by which overexpression of wild-type
syntaxin 4 inhibits regulated exocytosis pathways is not completely
clear. Overproduction of this syntaxin protein may perturb the steady
state between the three SNARE, components leading to aberrant
interactions and blockade of recycling by "titrating" general
factors like NSF or SNAP proteins. Alternatively, overexpression of
syntaxin 4 could disturb regulatory factors implicated in the
specificity of SNARE complexes such as munc18 isoforms. Two lines of
evidence argue in favor of that hypothesis: 1) overexpression of a
munc18 homologue involved in endoplasmic reticulum to Golgi transport
was able to overcome the dominant negative effects of overexpressed
syntaxin 5 (54); and 2) munc18 expression together with
syntaxin 1A partially relieves the blockage of secretion in a storage
compartment observed when expressing syntaxin 1A alone
(55). A third possibility relates to rab
GTPases, which, among the proteins playing a key role in the secretory
pathway, could facilitate SNARE complex formation (56, 57). Rab3d, involved in mast cell exocytosis, (37, 58), may be a good candidate for direct or indirect interactions
with syntaxin 4.
The function of this t-SNARE in exocytosis implies the mobilization of
syntaxin 4-containing complexes during stimulation. However, we were
not able to show that any of the proteins interacting with syntaxin 4,
i.e., SNAP23, VAMP2, cellubrevin, or VAMP8, were modulated in response
to FcRI-mediated stimulation (data not shown). These observations
either suggest that there is no change or that this change is too
subtle to be revealed by coimmunoprecipitation experiments. In favor of
the latter hypothesis, a kinetic analysis of SNARE complexes in
chromaffin cells has revealed a short lifetime of complexes, suggesting
that they cannot be stabilized physiologically in sufficient amounts
(59, 60). In addition, mast cells possess a particular
mechanism of granule fusion known as compound exocytosis, which is
characterized by the fusion of adjacent granules with each other before
fusion with plasma membrane (61, 62). This peculiar
feature may explain the very weak interaction between syntaxin 4 and
other SNARE proteins even after Ag-dependent stimulation, because in
this process only few interactions between the plasma membrane and
prefused granules are necessary to allow massive granular
discharge.
Concerning v-SNAREs involved in exocytosis, two independent findings could support for a role for VAMP8. On the one hand, we have demonstrated that VAMP8 was colocalized, at least in large part, with serotonin-containing granules, in contrast to the other VAMPs examined (cellubrevin was found to be superimposed with serotonin in a small subset of structures). On the other hand, we have found that VAMP8 was insensitive to tetanus toxin treatment, the latter result being consistent with the data showing that RBL-2H3 cells pretreated with this neurotoxin are still able to secrete serotonin (50).
The localization of VAMP8 to a subset of secretory granules is a new finding in light of previous data that have shown its enrichment in the later compartment of early endosomes for a number of cell types (34). This particular staining pattern could be characteristic for the lysosomal nature of these organelles in mast cells (4, 5, 7). Indeed, recent data have demonstrated the existence of at least three types of secretory granules, all of them containing the lysosomal enzyme ß hexosaminidase (3, 38). Type I and type II granules contained MHC class II molecules and were labeled by fluid endocytic markers, whereas serotonin was only observed in type II and type III granules. Given its endocytic nature, an attractive hypothesis would be to consider VAMP8 as a marker for type I and type II granules. The latter would be consistent with the observed staining pattern, because VAMP8 labels both serotonin-negative (which would correspond to type I) and part of the serotonin-positive (which would correspond to type II) granules. VAMP8 localization on serotonin-containing granules could thus represent a specific feature of mast cells, or more widely, of secretory cells within the hemopoietic cell lineage.
In conclusion, the present study demonstrates that the SNARE molecular machinery is involved in RBL-2H3 degranulation, and suggests that it depends on an exocytotic complex composed of syntaxin 4, SNAP23, and VAMP8. The presence of VAMP8 in this complex opens the possibility that a v-SNARE, originally localized on an endosomal compartment, functions in exocytosis. In this context, secretory granules in mast cells could be considered as a specialized compartment at the intersection between exocytotic and endocytic pathways.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michèle Roa, Unité d’Immuno-Allergie, Institut Pasteur, 28 rue du Dr. Roux 75724 Paris Cedex 15, France.
3 Abbreviations used in this paper: SNARE, SNAP receptor; SNAP, soluble NSF attachment protein; GFP, green fluorescent protein; NEM, N-ethylmaleimide; NSF, NEM-sensitive factor; PE-SA, phycoerythrin-streptavidin; RBL-2H3, rat basophilic leukemia cell line; SNAP23, 25, 29, synaptosome-associated protein of 23 kDa, 25 kDa, 29 kDa; t-SNARE, target-SNARE; TI-VAMP, tetanus neurotoxin insensitive VAMP; v-SNARE, vesicle-SNARE; TRSC, Texas Red sulfonide chloride; VAMP, vesicle-associated membrane protein.
Received for publication December 27, 1999. Accepted for publication March 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI): from physiology to pathology. Annu. Rev. Immunol. 17:931.[Medline]
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-granule secretion. J. Biol. Chem. 274:2492.This article has been cited by other articles:
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  M. Chaineau, L. Danglot, V. Proux-Gillardeaux, and T. Galli Role of HRB in Clathrin-dependent Endocytosis J. Biol. Chem., December 5, 2008; 283(49): 34365 - 34373. [Abstract] [Full Text] [PDF]
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 A.-A. Raymond, A. G. de Peredo, A. Stella, A. Ishida-Yamamoto, D. Bouyssie, G. Serre, B. Monsarrat, and M. Simon Lamellar Bodies of Human Epidermis: Proteomics Characterization by High Throughput Mass Spectrometry and Possible Involvement of CLIP-170 in their Trafficking/Secretion Mol. Cell. Proteomics, November 1, 2008; 7(11): 2151 - 2175. [Abstract] [Full Text] [PDF]
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 J.-i. Kashiwakura, W. Xiao, J. Kitaura, Y. Kawakami, M. Maeda-Yamamoto, J. R. Pfeiffer, B. S. Wilson, U. Blank, and T. Kawakami Pivotal Advance: IgE accelerates in vitro development of mast cells and modifies their phenotype J. Leukoc. Biol., August 1, 2008; 84(2): 357 - 367. [Abstract] [Full Text] [PDF]
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 H. Higashio, N. Nishimura, H. Ishizaki, J. Miyoshi, S. Orita, A. Sakane, and T. Sasaki Doc2{alpha} and Munc13-4 Regulate Ca2+-Dependent Secretory Lysosome Exocytosis in Mast Cells J. Immunol., April 1, 2008; 180(7): 4774 - 4784. [Abstract] [Full Text] [PDF]
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 N. Tiwari, C.-C. Wang, C. Brochetta, G. Ke, F. Vita, Z. Qi, J. Rivera, M. R. Soranzo, G. Zabucchi, W. Hong, et al. VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways Blood, April 1, 2008; 111(7): 3665 - 3674. [Abstract] [Full Text] [PDF]
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Y. H. S. Ho, D. T. Cai, C.-C. Wang, D. Huang, and S. H. Wong Vesicle-Associated Membrane Protein-8/Endobrevin Negatively Regulates Phagocytosis of Bacteria in Dendritic Cells J. Immunol., March 1, 2008; 180(5): 3148 - 3157. [Abstract] [Full Text] [PDF]
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 N. Puri and P. A. Roche Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms PNAS, February 19, 2008; 105(7): 2580 - 2585. [Abstract] [Full Text] [PDF]
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B. Textor, A. H. Licht, J. P. Tuckermann, R. Jessberger, E. Razin, P. Angel, M. Schorpp-Kistner, and B. Hartenstein JunB Is Required for IgE-Mediated Degranulation and Cytokine Release of Mast Cells J. Immunol., November 15, 2007; 179(10): 6873 - 6880. [Abstract] [Full Text] [PDF]
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N. Tiwari, N. Garbi, T. Reinheckel, G. Moldenhauer, G. J. Hammerling, and F. Momburg A Transporter Associated with Antigen-Processing Independent Vacuolar Pathway for the MHC Class I-Mediated Presentation of Endogenous Transmembrane Proteins J. Immunol., June 15, 2007; 178(12): 7932 - 7942. [Abstract] [Full Text] [PDF]
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M. Nanamori, J. Chen, X. Du, and R. D. Ye Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells J. Immunol., January 1, 2007; 178(1): 416 - 427. [Abstract] [Full Text] [PDF]
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 Q. Ren, H. K. Barber, G. L. Crawford, Z. A. Karim, C. Zhao, W. Choi, C.-C. Wang, W. Hong, and S. W. Whiteheart Endobrevin/VAMP-8 Is the Primary v-SNARE for the Platelet Release Reaction Mol. Biol. Cell, January 1, 2007; 18(1): 24 - 33. [Abstract] [Full Text] [PDF]
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 S. Tadokoro, M. Nakanishi, and N. Hirashima Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process J. Cell Sci., May 15, 2005; 118(10): 2239 - 2246. [Abstract] [Full Text] [PDF]
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R. Hepp, N. Puri, A. C. Hohenstein, G. L. Crawford, S. W. Whiteheart, and P. A. Roche Phosphorylation of SNAP-23 Regulates Exocytosis from Mast Cells J. Biol. Chem., February 25, 2005; 280(8): 6610 - 6620. [Abstract] [Full Text] [PDF]
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M. Neeft, M. Wieffer, A. S. de Jong, G. Negroiu, C. H.G. Metz, A. van Loon, J. Griffith, J. Krijgsveld, N. Wulffraat, H. Koch, et al. Munc13-4 Is an Effector of Rab27a and Controls Secretion of Lysosomes in Hematopoietic Cells Mol. Biol. Cell, February 1, 2005; 16(2): 731 - 741. [Abstract] [Full Text] [PDF]
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 J. Stinchcombe, G. Bossi, and G. M. Griffiths Linking Albinism and Immunity: The Secrets of Secretory Lysosomes Science, July 2, 2004; 305(5680): 55 - 59. [Abstract] [Full Text] [PDF]
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S. K. Rao, C. Huynh, V. Proux-Gillardeaux, T. Galli, and N. W. Andrews Identification of SNAREs Involved in Synaptotagmin VII-regulated Lysosomal Exocytosis J. Biol. Chem., May 7, 2004; 279(19): 20471 - 20479. [Abstract] [Full Text] [PDF]
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N. Puri, M. J. Kruhlak, S. W. Whiteheart, and P. A. Roche Mast Cell Degranulation Requires N-Ethylmaleimide-Sensitive Factor-Mediated SNARE Disassembly J. Immunol., November 15, 2003; 171(10): 5345 - 5352. [Abstract] [Full Text] [PDF]
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T. D. Schraw, T. W. Rutledge, G. L. Crawford, A. M. Bernstein, A. L. Kalen, J. E. Pessin, and S. W. Whiteheart Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release Blood, September 1, 2003; 102(5): 1716 - 1722. [Abstract] [Full Text] [PDF]
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S. Martinez-Arca, R. Rudge, M. Vacca, G. Raposo, J. Camonis, V. Proux-Gillardeaux, L. Daviet, E. Formstecher, A. Hamburger, F. Filippini, et al. A dual mechanism controlling the localization and function of exocytic v-SNAREs PNAS, July 22, 2003; 100(15): 9011 - 9016. [Abstract] [Full Text] [PDF]
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S. Martinez-Arca, V. Proux-Gillardeaux, P. Alberts, D. Louvard, and T. Galli Ectopic expression of syntaxin 1 in the ER redirects TI-VAMP- and cellubrevin-containing vesicles J. Cell Sci., July 1, 2003; 116(13): 2805 - 2816. [Abstract] [Full Text] [PDF]
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S. Martin-Verdeaux, I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, J. Rivera, and U. Blank Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis J. Cell Sci., January 15, 2003; 116(2): 325 - 334. [Abstract] [Full Text] [PDF]
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 X. Li, S. H. Low, M. Miura, and T. Weimbs SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes Am J Physiol Renal Physiol, November 1, 2002; 283(5): F1111 - F1122. [Abstract] [Full Text] [PDF]
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Z. Guo, L. Liu, D. Cafiso, and D. Castle Perturbation of a Very Late Step of Regulated Exocytosis by a Secretory Carrier Membrane Protein (SCAMP2)-derived Peptide J. Biol. Chem., September 13, 2002; 277(38): 35357 - 35363. [Abstract] [Full Text] [PDF]
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G. Way, N. Morrice, C. Smythe, and A. J. O'Sullivan Purification and Identification of Secernin, a Novel Cytosolic Protein that Regulates Exocytosis in Mast Cells Mol. Biol. Cell, September 1, 2002; 13(9): 3344 - 3354. [Abstract] [Full Text] [PDF]
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I. Pombo, S. Martin-Verdeaux, B. Iannascoli, J. Le Mao, L. Deriano, J. Rivera, and U. Blank IgE Receptor Type I-dependent Regulation of a Rab3D-associated Kinase. A POSSIBLE LINK IN THE CALCIUM-DEPENDENT ASSEMBLY OF SNARE COMPLEXES J. Biol. Chem., November 9, 2001; 276(46): 42893 - 42900. [Abstract] [Full Text] [PDF]
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V. V. Vaidyanathan, N. Puri, and P. A. Roche The Last Exon of SNAP-23 Regulates Granule Exocytosis from Mast Cells J. Biol. Chem., June 29, 2001; 276(27): 25101 - 25106. [Abstract] [Full Text] [PDF]
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K. M. S. Misura, L. C. Gonzalez Jr., A. P. May, R. H. Scheller, and W. I. Weis Crystal Structure and Biophysical Properties of a Complex between the N-terminal SNARE Region of SNAP25 and Syntaxin 1a J. Biol. Chem., October 26, 2001; 276(44): 41301 - 41309. [Abstract] [Full Text] [PDF]
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