From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, April 20, 2001, and in revised form, May 11, 2001
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ABSTRACT |
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SNAP-25 and its ubiquitous homolog SNAP-23 are
members of the SNARE family of proteins that regulate membrane fusion
during exocytosis. Although SNAP-23 has been shown to participate in a
variety of intracellular transport processes, the structural domains of
SNAP-23 that are required for its interaction with other SNAREs have
not been determined. By employing deletion mutagenesis we found that
deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in
the first exon) dramatically inhibited binding of SNAP-23 to both the
target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane
protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino
acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23
binding to syntaxin but profoundly inhibits its binding to VAMP. To
determine the functional relevance of the modular structure of SNAP-23,
we overexpressed SNAP-23 in cells possessing the capacity to undergo
regulated exocytosis. Expression of human SNAP-23 in a rat mast cell
line significantly enhanced exocytosis, and this effect was not
observed in transfectants expressing the carboxyl-terminal VAMP-binding
mutant of SNAP-23. Despite considerable amino acid identity, we found
that human SNAP-23 bound to SNAREs more efficiently than did rat
SNAP-23. These data demonstrate that the introduction of a "better"
SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in
mast cells.
Characterization of the molecular mechanism of membrane-membrane
fusion in living cells has revealed that the proteins regulating this
process are conserved in systems such as protein secretion in yeast,
synaptic vesicle exocytosis, and intracellular vesicle fusion during
membrane traffic in mammalian cells (1, 2). One key set of proteins
that regulate these diverse biological processes are called
SNAREs.1 SNARE proteins are
present on both vesicle membranes (vesicle SNAREs, or v-SNAREs) and on
target membranes (target SNAREs, or t-SNAREs). The v-SNAREs include
proteins of the VAMP family, whereas t-SNAREs include proteins of the
syntaxin and SNAP-25 families. The neuronal SNAREs include syntaxin 1A,
SNAP-25 and VAMP 2, and these proteins readily associate with one
another to form a stable ternary complex that is essential for synaptic
vesicle exocytosis.
The neuronal SNARE complex is composed of a parallel four-helical
bundle of coiled-coils (3, 4). Syntaxin 1A and VAMP 2 (tethered to
opposing membranes) each contain one coiled-coil domain, and SNAP-25
contains two coiled-coil domains, one at its amino terminus and one at
its carboxyl terminus (5). By employing deletion analysis, several
groups have analyzed the domains of the neuronal SNAREs that are
essential for efficient SNARE complex assembly (5-7). Both
coiled-coils of SNAP-25 are important for its interaction with VAMP 2 (5) while the major syntaxin binding region is present in the first
coiled-coil (5, 6). In agreement with these data, proteolysis of
SNAP-25 by Botulinum neurotoxins that remove the carboxyl terminus of
SNAP-25 does not alter its binding to syntaxin but dramatically
inhibits its binding to VAMP 2 (6). Under conditions of neurotoxin
poisoning exocytosis is abolished (8), highlighting the functional
importance of the carboxyl-terminal region of SNAP-25 to
VAMP binding.
Two non-neuronal homologs of SNAP-25 have been identified, and they
have been named SNAP-23 (Refs. 9-11) and SNAP-29 (Refs. 12 and 13).
These isoforms exhibit a significant similarity at the protein level
with SNAP-25, and each possesses the two coiled-coils necessary for the
formation of SNARE complexes. The role of SNAP-29 is still unresolved,
with one study suggesting that it plays a role primarily in intra-Golgi
traffic (13) and another suggesting that SNAP-29 regulates a variety of
intracellular traffic events (12). On the other hand, SNAP-23 is
thought to play a role both in regulated and constitutive protein
trafficking pathways in non-neuronal cells. For example, SNAP-23 is
involved in diverse protein trafficking events such as GLUT4
trafficking in adipose cells (14, 15), compound exocytosis in mast
cells (16), polarized protein traffic (17), platelet dense core granule
release (18), and transferrin receptor recycling (19).
Because of its wide tissue distribution, its ability to interact with
several syntaxin and VAMP isoforms, and the aforementioned functional
data, SNAP-23 is likely to be a key player in many distinct protein
trafficking events in non-neuronal cells. Both SNAP-23 and SNAP-25 are
thought to function primarily as t-SNARE heterodimers together with
syntaxin (20, 21); however, the ability of SNAP-23 to replace SNAP-25
in regulated exocytosis is limited (22). A systematic analysis of the
domains of SNAP-23 that are required for its binding to syntaxin and
VAMP could allow the identification of SNAP-23 mutants that could alter
intracellular granule-plasma membrane fusion events in distinct cell
types in vivo.
In this study, we have characterized the interaction of SNAP-23 with
syntaxin 4 and VAMP 2. These SNARE isoforms were chosen because
complexes containing these proteins with SNAP-23 are common in exocytic
events in non-neuronal cells (23, 24). Truncation analyses revealed
that while the carboxyl-terminal 23 amino acids of SNAP-23
are not required for SNAP-23 binding to syntaxin, they are essential
for SNAP-23 binding to VAMP in vitro and in vivo.
Overexpression of wild-type human SNAP-23 in rat mast cells resulted in
enhanced exocytosis from the cells, and this effect was completely
abolished in mast cells expressing a human SNAP-23 carboxyl-terminal
VAMP-binding mutant. Thus, these data demonstrate that SNAP-23 mutants
that bind to syntaxin but not to VAMP are unable to support exocytosis
from rat mast cells and point to the SNAP-23 carboxyl terminus as an
important regulator of exocytosis.
Plasmids and Recombinant Proteins--
cDNAs encoding
full-length human SNAP-23 (9), mouse SNAP-25b (25), rat syntaxin 1A
(21), and human syntaxin 4 (26) were subcloned into pcDNA3
(Invitrogen). FLAG-tagged rat VAMP 2 in pRc-CMV was the gift
of Dr. Richard Scheller (Stanford University). GST-syntaxin and
GST-VAMP fusion proteins were generated by standard cloning and
expression strategies in Escherichia coli (9). The
recombinant proteins were affinity-purified using glutathione-Sepharose
beads according to the manufacturers instructions (Amersham Pharmacia
Biotech). Deletion mutants of SNAP-23 in pcDNA3 were
generated by the polymerase chain reaction using mutant oligonucleotide primers encoding stop codons. The sequence of all mutants was verified
by automated DNA sequence analysis.
In Vitro Binding Studies--
Radiolabeled proteins were
generated by in vitro translation reactions using rabbit
reticulocyte lysate in the presence of [35S]methionine
using the T7 Quick in vitro transcription/translation kit
(Promega). For in vitro binding studies, GST alone,
GST-syntaxin 4, or GST-VAMP 2 (10 µg each) were immobilized onto
glutathione-Sepharose beads and were mixed with in vitro
translated wild-type or mutant proteins in a final volume of 200 µl
of binding buffer (4 mM HEPES, 100 mM NaCl, 3.5 mM CaCl2, 3.5 mM MgCl2,
1 mM EDTA, 0.1% Nonidet P-40, pH 7.4) for 2 h at
4 °C. All incubations contained nearly identical amounts of in
vitro translated material, and total amount of rabbit reticulocyte
lysate in all tubes was kept constant by adding aliquots from a mock
translation mixture. After incubation, the beads were washed three
times with 1 ml of washing buffer (50 mM Tris, 100 mM NaCl, 2.5 mM MgCl2, 0.1%
Nonidet P-40, pH 8.0) and the resulting pellet was resuspended in
SDS-PAGE sample buffer containing 0.1% Antibodies, Immunoprecipitation, and
Immunoblotting--
Anti-peptide rabbit antisera recognizing the amino
terminus of human SNAP-23 have been described previously (25). A
broadly reactive SNAP-23 antisera was generated by immunizing rabbits with purified, recombinant human His6-SNAP-23 fusion
protein. To ensure that these antibodies reacted equally well with
SNAP-23 truncation mutants, we analyzed in vitro translated
wild-type or mutant proteins by immunoprecipitation and immunoblot
analyses. These studies confirmed that the introduction of these
mutations did not alter the immunogenicity of the various constructs
used in this study. The SNAP-25 monoclonal antibody was from
Sternberger Monoclonals, Inc. (Baltimore, MD), the syntaxin 4 monoclonal antibody was from Transduction Labs, and the FLAG-epitope
monoclonal antibody M5 was from Sigma.
Immunoblot analysis of cell lysates or immunoprecipitation from cell
lysates was performed as previously described (27). For
immunoprecipitations from cell extracts, cells were briefly washed with
Hanks' balanced salt solution and lysed at 4 °C with 1 ml of lysis
buffer (1% Triton X-100, 10 mM Tris, 150 mM
NaCl, 0.02% NaN3, pH 7.5) containing 1 mg/ml bovine serum
albumin and protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM tosyl-lysine
chloromethyl ketone, 5 mM iodoacetamide). The lysates were
subjected to centrifugation at 14,000 rpm at 4 °C to remove cell
debris, and the cleared lysates were used for immunoprecipitation reactions. Immunoprecipitated proteins were analyzed on 10.5% SDS-PAGE
gels and immunoblotting or fluorography (as indicated). In some cases
the gels were stained with Coomassie Brilliant Blue R-250 to confirm
that samples contained similar amounts of GST fusion proteins.
Transfection of HeLa and RBL Cells--
Subconfluent HeLa cells
were transfected using LipofectAmine (Life Technologies, Inc.)
according to the manufacturer's specifications. Transfected HeLa cells
were generally analyzed 18-24 h after transfection. The rat mast cell
line RBL-2H3 was maintained as adherent cultures in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum in a
humidified atmosphere of 5% CO2 at 37 °C. RBL cells
(5 × 106/ml) in Dulbecco's modified Eagle's medium
without serum were transfected by electroporation (250 V, 960 microfarad) using 10 µg of empty vector, 10 µg of wild-type human
SNAP-23 in pcDNA3, or 10 µg of human SNAP-23-(1-188) in
pcDNA3. Dilutions of cells were immediately replated in Dulbecco's
modified Eagle's medium/fetal bovine serum and allowed to recover
overnight. G418 (0.5 mg/ml) was added to the cultures, and stable
transfectants were selected within 7 days by picking individual
colonies with a sterile pipette tip. Stable transfectants were
maintained in Dulbecco's modified Eagle's medium/fetal bovine serum
containing 0.25 mg/ml G418.
Stimulation of RBL Cell Exocytosis--
Exocytosis in the
RBL-2H3 mast cells was triggered using phorbol 12-myristate 13-acetate
and ionomycin. RBL cells (1 × 106) were washed with
RPMI (without phenol red), and degranulation was induced by adding
phorbol 12-myristate 13-acetate (10 nM) and ionomycin (1 µM) in a final volume of 1.5 ml. Plates were incubated at
37 °C, aliquots of the medium were withdrawn at various times, and
Identification of SNAP-23/SNARE Interacting Domains in
Vitro--
SNAP-23 is known to be an important participant in the
formation of the SNARE complex that leads to membrane-membrane fusion. In our attempt to identify the regions of SNAP-23 that are essential for its function, we set out to identify the domains of SNAP-23 that
are essential for its ability to form binary SNARE complexes. We have
recently cloned the entire SNAP-23 gene (28), and because distinct
exons often encode functional domains in proteins, we generated a
series of amino- and carboxyl-terminal truncation mutants of human
SNAP-23 based on the exon structure of SNAP-23 (Fig.
1A). These mutants correspond
to deletions of the first coding exon (SNAP-23-(19-211)), the first
and second coding exon (SNAP-23-(33-211)), the first four coding exons
(including the first coiled-coil domain, SNAP-23-(76-211)), the last
coding exon (SNAP-23-(1-188)), and the last two coding exons
(SNAP-23-(1-140)).
In vitro binding assays revealed that deletion of 18 amino
acids from the amino terminus of SNAP-23 (SNAP-23-(19-211)) reduced the ability of SNAP-23 to bind to GST-VAMP 2 by 60% (Fig.
1B). Deletion of an additional 14 residues from the amino
terminus (SNAP-23-(33-211)) eliminated binding of SNAP-23 to VAMP 2, establishing the presence of a VAMP 2 binding region at the extreme
amino terminus of SNAP-23. We also examined carboxyl-terminal
truncation mutants of SNAP-23 using in vitro binding assays.
A deletion mutant lacking the last 23 amino acids of SNAP-23
(SNAP-23-(1-188)) bound very poorly to GST-VAMP 2 (12% binding as
compared with wild-type SNAP-23), and further truncations that removed
the entire second coiled-coil domain (SNAP-23-(1-140)) completely
eliminated the binding of SNAP-23 to VAMP 2.
After observing that the integrity of the extreme amino-and
carboxyl-terminal regions of SNAP-23 contributed to its binding to VAMP
2, we examined the importance of these regions in the interaction of
SNAP-23 with syntaxin 4. As was seen in the binding assays with VAMP 2, deletion of 18 amino acids from the amino terminus of SNAP-23 almost
completely eliminated binding to GST-syntaxin 4 (Fig. 1C).
However, unlike the results obtained with VAMP 2, deletion of 23 amino
acids from the carboxyl terminus of SNAP-23 did not inhibit its ability
to bind syntaxin 4. Removal of the entire second coiled-coil domain of
SNAP-23 (SNAP-23-(1-140)) completely prevented the binding of SNAP-23
to syntaxin. Control experiments confirmed that each reaction condition
contained similar amounts of SNAP-23 protein (Fig. 1D).
These data reveal that the first 18 amino acids of SNAP-23 harbor a
major binding site for both syntaxin and VAMP, that the presence of the
first coiled-coil alone is insufficient to allow SNAP-23 binding to
syntaxin and VAMP, and that the extreme carboxyl terminus of SNAP-23 is
not required for the binding of SNAP-23 to syntaxin 4 but is essential for the binding of SNAP-23 to VAMP 2.
Identification of SNAP-23/SNARE Interacting Domains in
Vivo--
To confirm and extend the results obtained from our in
vitro binding studies, we examined the binding characteristics of
wild-type SNAP-23 or SNAP-23-(1-188) to syntaxin and VAMP in
transfected HeLa cells. In agreement with results obtained in our
in vitro binding assays, the association of VAMP 2 with
SNAP-23-(1-188) was significantly reduced as compared with the
association of VAMP 2 with wild-type SNAP-23 (Fig.
2A). Immunoblotting of the cell extracts confirmed that the expression of wild-type
SNAP-23-(1-211) and SNAP-23-(1-188) was similar in the various
transfectants and that transfection did not alter the amount of VAMP 2 or syntaxin 4 expressed in the cells. Quantitation of various
immunoblots revealed that truncation of the carboxyl-terminal 23 amino
acids of SNAP-23 inhibited its binding to VAMP 2 by 65%.
Cotransfection experiments examining SNAP-23/syntaxin 4 interactions
demonstrated that the binding of syntaxin 4 to SNAP-23-(1-188) was as
good as that observed with wild-type SNAP-23 (Fig. 2B). When
the binding properties of the other SNAP-23 truncation mutants were
analyzed in vivo the results were similar to those obtained
in our in vitro binding studies (data not shown). Therefore,
our binding data obtained using recombinant SNARE proteins is in
excellent agreement with our in vivo binding data in
transfected cells and clearly demonstrate that the carboxyl-terminal
domain of SNAP-23 is important for binding to VAMP but not to
syntaxin.
The VAMP-binding Domain of SNAP-23 Is Required for Regulated
Exocytosis from Mast Cells--
In an attempt to confirm the
importance of the carboxyl-terminal domain of SNAP-23 in a functional
exocytosis assay, we overexpressed wild-type SNAP-23-(1-211) and the
SNAP-23-(1-188) mutant in the RBL mast cell line and isolated stable
transfectants. We chose to express human SNAP-23 in these cells since
we have an antibody that specifically recognizes the human SNAP-23
amino terminus, and immunoblot analysis of cell lysates confirmed that
wild-type SNAP-23-(1-211) and the SNAP-23-(1-188) mutant were
expressed at similar levels in these clones (Fig.
3A). Immunoblotting with an
antibody that recognizes both human and rat SNAP-23 revealed that the
amount of human SNAP-23 expressed in these cells was low because the
total amounts of SNAP-23 present in mock-transfected and human
SNAP-23-transfected RBL cells were similar, and the expression of human
SNAP-23-(1-188) can only be seen in long exposures of the immunoblot
(Fig. 3A). Confocal immunofluorescence microscopy also
demonstrated that wild-type SNAP-23 and SNAP-23-(1-188) were expressed
primarily on the plasma membrane of these RBL clones (Fig.
3B). This agrees with the localization of endogenous SNAP-23 in rat peritoneal mast cells (16) and in RBL cells (our data not shown
and Ref. 29). The low level of SNAP-23 expression was observed in
several independent clones, and we never obtained clones expressing
large amounts of human SNAP-23.
RBL mast cell exocytosis can be triggered by cross-linking high
affinity receptors for IgE or by direct stimulation with phorbol esters
and ionomycin. Despite the low level of expression of human SNAP-23 in
the RBL transfectants, cells expressing wild-type human SNAP-23-(1-211) were capable of increased exocytosis when stimulated with phorbol myristate acetate and ionomycin as compared with mock-transfected cells (Fig.
4A) and untransfected RBL
cells (data not shown). In the absence of these agents there was no
detectable degranulation from these cells (data not shown). The
increase in exocytosis was apparent even at the earliest time points
examined, demonstrating that in addition to increasing the absolute
amount of exocytosis, overexpression of human SNAP-23 led to an
increase in the rate of exocytosis of rat mast cells. Unlike cells
expressing wild-type human SNAP-23, cells expressing the human SNAP-23
carboxyl-terminal truncation mutant SNAP-23-(1-188) showed no increase
in exocytosis as compared with mock-transfected cells (Fig.
4B). These data demonstrate that the elevated levels of
exocytosis from mast cells overexpressing human SNAP-23 were indeed a
consequence of expression of wild-type human SNAP-23 protein and
demonstrate that the carboxyl-terminal VAMP-binding domain
of human SNAP-23 is important for its function in regulated exocytosis
in mast cells.
Human SNAP-23 Binds Syntaxin and VAMP More Efficiently than Does
Rat SNAP-23--
We were intrigued by our data showing that expression
of small amounts of human SNAP-23 in mast cells that already express significant amounts of SNAP-23 was able to significantly augment exocytosis, and we set out to identify a molecular mechanism for this
effect. Because SNARE complex assembly is essential for exocytosis, we
compared the ability of rat SNAP-23 and human SNAP-23 to bind to
syntaxin 4 and VAMP 2 using in vitro binding assays. Rat and mouse SNAP-23 (which are 98% identical) bound syntaxin 4 poorly and
their binding to VAMP 2 was almost undetectable (Fig.
5). The binding of human SNAP-23 to these
SNAREs was considerably better, and the binding of SNAP-25 to each of
these GST-SNARE fusion proteins was very efficient. It is important to
note that in these binding assays nearly identical amounts of in
vitro translated SNAP-23 or SNAP-25 were used, allowing a direct
comparison between the binding of these different SNAP-23 family
members to GST-SNARE fusion proteins. Data from multiple different
in vitro binding assays were compared and show
quantitatively that human SNAP-23 binds to syntaxin 4 and VAMP 2 much
better than does rat SNAP-23 (Fig. 6).
This is in good agreement with a yeast two-hybrid analysis showing that
human SNAP-23 bound to syntaxin 4 ~10 times better than did mouse
SNAP-23 and that SNAP-25 bound to many different syntaxins very well
whereas SNAP-23 did not (11). Thus, the increase in exocytosis from rat
mast cells can be explained in part by the expression of a
better SNARE binding form of SNAP-23 in these cells.
Assembly and disassembly of SNARE complexes and their role in
exocytosis has been studied in several systems (1). Formation of a core
complex consisting of SNAP-25, syntaxin, and VAMP is a central event in
steps leading to membrane fusion, and the biogenesis of the neuronal
SNARE complex has been studied in detail (2). SNAP-23 is considered to
be the functional homolog of SNAP-25 in non-neuronal cells and
participates in a variety of constitutive and regulated exocytotic
events (14, 16-19). Furthermore, these two proteins are the products
of an ancestral gene duplication event and share significant amino acid
identity (9, 10, 28). Despite this, sequence determinants that regulate
the assembly of non-neuronal SNARE complexes are poorly understood.
In this study we have investigated the interactions of the ubiquitously
expressed SNAP-25 homolog SNAP-23 with both syntaxin 4 and VAMP 2. We
have chosen these particular SNAREs for analysis because they have been
shown to be physiologically relevant partners for SNAP-23 in living
cells (23, 24). By truncation analysis we have identified a major
syntaxin and VAMP binding domain within the first coding exon of
SNAP-23 (amino acids 1-19). It is unlikely that the failure of
in vitro translated SNAP-23-(19-211) to bind to GST-SNARE
fusion proteins is due to a misfolding of the protein since even
GST-SNAP-23-(19-211) fusion proteins did not bind to in
vitro translated SNAREs and SNAP-23-(19-211) expressed in HeLa cells did not bind to SNAREs in
vivo2.
Furthermore, even full-length SNAP-25 does not have appreciable secondary structure until it binds to syntaxin (7), suggesting that
syntaxin binding actually assists in the folding of a relatively unfolded SNAP-25 molecule.
In addition to identifying a SNARE-binding motif in the extreme amino
terminus of SNAP-23, a carboxyl-terminal truncation mutant that
possessed only the first coiled-coil domain of SNAP-23 did not bind to
either syntaxin or VAMP, demonstrating that although the extreme amino
terminus of SNAP-23 is necessary for SNARE binding it is not
sufficient. The amino terminus of SNAP-25 is known to possess a
syntaxin binding site; however, carboxyl-terminal deletion mutants of
SNAP-25 that contain only the amino-terminal coiled domain of the
protein (e.g. SNAP-25-(2-141) and SNAP-25-(1-100)) still
retain syntaxin binding activity (5, 6). This could reflect the fact
that there are 12 heptad repeats in the amino-terminal coiled-coil
domain of SNAP-25 and only six in the corresponding region of SNAP-23,
thereby limiting the affinity of the SNAP-23 amino-terminal coiled-coil
for syntaxin.
We have also identified the carboxyl-terminal 23 amino acids of SNAP-23
as important determinants for binding to VAMP but not to syntaxin. This
is in very good agreement with previous studies examining the behavior
of SNAP-25 deletion mutants (5-7) and the effects of Botulinum
neurotoxin cleavage of SNAP-25 on its SNARE-binding properties (6). To
test the functional importance of the SNAP-23 carboxyl-terminal
VAMP-binding domain in vivo, we generated rat mast cell
lines overexpressing wild-type human SNAP-23 and the human
SNAP-23-(1-188) carboxyl-terminal deletion mutant. Despite the fact
that the total amount of SNAP-23 was minimally altered in these
transfectants, expression of human SNAP-23 significantly enhanced
exocytosis from these cells. Furthermore, the observed increase in
exocytosis required the presence of the carboxyl-terminal 23 amino
acids of SNAP-23 as the human SNAP-23-(1-188) mutant was unable to
support enhanced exocytosis in rat mast cells. This finding is in very
good agreement with data showing that treatment of permeabilized
Madin-Darby canine kidney cells with Botulinum neurotoxin E, which
cleaves ~20 amino acids from the carboxyl terminus of canine SNAP-23,
dramatically inhibits transferrin receptor recycling (19) and apical
and basolateral membrane traffic (17). These data highlight the
importance of the carboxyl-terminal domain of SNAP-23 (encoded by the
last exon) in the binding to VAMP 2 and regulating exocytosis.
Despite the similarity in structure and proposed similarity in function
for SNAP-23 and SNAP-25, we and others (11) found that SNAP-23 bound to
various syntaxins and VAMP 2 much more poorly than did SNAP-25 in
vitro. This result is unlikely to be due to our assay system as
similar results were obtained in experiments examining the binding of
in vitro translated SNAP-23 or SNAP-25 to GST-syntaxin and
GST-VAMP fusion proteins and in experiments examining the binding of
in vitro translated syntaxins to GST-SNAP-23 and GST-SNAP-25
fusion proteins.3 In
addition, these observations are in excellent agreement with our
earlier in vivo experiments demonstrating that SNAP-25 was able to bind to syntaxin more effectively than was SNAP-23 in transfected HeLa cells (30). On the other hand, we also found that
human SNAP-23 bound to syntaxin and VAMP 2 better than did rat or mouse
SNAP-23, demonstrating that among the SNAP-23 family of proteins there
are dramatic differences in SNARE binding ability. It is currently
unknown if these differences alone can account for enhanced exocytosis
in human SNAP-23-expressing rat mast cells or whether additional
features of human SNAP-23 can augment exocytosis in rat cells.
Pancreatic The carboxyl terminus of SNAP-23 has previously been implicated in
GLUT4 trafficking in 3T3-L1 adipocytes. Microinjection of a
carboxyl-terminal peptide of SNAP-23 into 3T3-L1 cells inhibited GLUT4
trafficking (14), although the mechanism by which this peptide
inhibited translocation was not explored. In a related study, infection
of 3T3-L1 cells with a recombinant virus encoding mouse
SNAP-23-(1-202) also inhibited GLUT4 trafficking (15), yet in this
study even wild-type SNAP-23 inhibited GLUT4 trafficking. Our study is
the first to reveal that the introduction of human SNAP-23 into
regulated secretory cells actually augments exocytosis, that this form
of (human) SNAP-23 binds to SNARE proteins better than endogenous (rat)
SNAP-23, and that the increase in exocytosis can be directly attributed
to the integrity of the carboxyl-terminal VAMP-binding domain of
SNAP-23. These data also highlight the similarities between the
mechanism of GLUT4 translocation in fat cells and granule exocytosis in
mast cells.
Besides the current work, several studies have shown a direct
relationship between SNARE complex assembly and fusion. In the neuroendocrine PC12 cell line Ca2+ was found to enhance
SNARE complex assembly and subsequently drive membrane fusion (32).
Studies in adrenal chromaffin cells have suggested that
trans-SNARE complexes (i.e. complexes of v-SNAREs with t-SNAREs from opposing membranes) exist in two interconvertible states: a "tight" state that mediates the fast component of release and a "loose" state that mediates a sustained slow phase (33). It
is interesting to note that, in general, the kinetics of non-neuronal exocytosis are far slower than neuronal exocytosis. Because
non-neuronal exocytosis involves SNARE complexes containing SNAP-23 and
not SNAP-25, it is possible that at least one contributing factor to
the diminished kinetics of exocytosis in non-neuronal cells is the
presence of the "weak" SNARE SNAP-23 in secretory
trans-SNARE complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. The
samples were heated to 95 °C for 5 min, and the supernatant
fractions were analyzed by SDS-PAGE (27). The gels were stained with
Coomassie Brilliant Blue R-250 to visualize protein bands and
impregnated with Enlightening (PerkinElmer Life Sciences) for
fluorography. The gels were dried and exposed to PhosphorImager
screens, and the intensity of each band was quantitated by
PhosphorImager analysis using a Molecular Dynamics model HHF PhosphorImager.
-hexosaminidase activity released into the medium was measured. Mock
degranulation studies were carried out in parallel using vehicle in
medium alone. At the end of the assay, cells were lysed with 1 ml of
1% Triton X-100 in RPMI to measure residual cell-associated
-hexosaminidase. To determine
-hexosaminidase activity, aliquots
(20 µl) of the supernatants and cell lysates were incubated with 50 µl of the substrate solution (1.3 mg/ml of
p-nitrophenyl-N-acetyl-
-D-glucosaminide
(Sigma) in 0.1 M citrate buffer (pH 4.5) for 90 min at
37 °C. The reaction was terminated by the addition of 100 µl of
0.2 M NaOH/0.2 M glycine. Absorbance was read
at 405 nm in an enzyme-linked immunosorbent assay reader, and the
amount of exocytosis was expressed as the percentage of total
-hexosaminidase activity present in cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mapping of VAMP 2 and syntaxin 4 binding
interacting domains of SNAP-23 in vitro.
A, schematic of exon/intron organization of SNAP-23 gene.
The amino acid boundaries between the exons encoding human SNAP-23 are
indicated. The exon structure and amino acid residues listed are based
on a complete structure analysis of the gene as described (28). The
location of the two putative coiled-coil domains of SNAP-23 are also
indicated. B D, full-length wild-type human
SNAP-23-(1-211) or human SNAP-23 deletion mutants (numbers specify
amino acids) were radiolabeled by in vitro translation and
incubated with 10 µg of GST-VAMP 2 (B) or GST-syntaxin 4 (C) immobilized on glutathione-Sepharose beads for 2 h
at 4 °C. After washing, the bound material were analyzed by SDS-PAGE
and fluorography. D, equal amounts of radioactive SNAP-25 or
SNAP-23 were added to each condition as shown by analyzing an aliquot
of the sample input for each condition.
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Fig. 2.
Binding of wild-type SNAP-23-(1-211) and
SNAP-23-(1-188) to SNAREs in vivo. HeLa cells
were transiently transfected with empty vector (mock), with
wild-type human SNAP-23-(1-211) or human SNAP-23-(1-188) together
with FLAG-VAMP 2 (A) or with syntaxin 4 (B). Cell
lysates were prepared, and anti-SNAP-23 immunoprecipitates were probed
by immunoblotting with anti-VAMP 2 and anti-syntaxin 4 antibodies.
Aliquots of the lysates were also analyzed by immunoblotting with
specific antibodies recognizing SNAP-23, VAMP 2, and syntaxin 4.
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[in a new window]
Fig. 3.
Expression of wild-type and mutant SNAP-23 in
RBL-2H3 mast cells. A, cell lysates of RBL cell clones
expressing wild-type human SNAP-23-(1-211) or the human
SNAP-23-(1-188) truncation mutant were analyzed by immunoblotting for
expression of human SNAP-23 (upper panel) or total rat and
human SNAP-23 (middle and lower panels). A longer
exposure of the immunoblot (lower panel) clearly reveals
expression of human SNAP-23-(1-188) in the RBL cell clone (indicated
by an arrow). B, confocal immunofluorescence
microscopy of mock-transfected RBL mast cells and RBL mast cell lines
expressing wild-type human SNAP-23-(1-211) and the SNAP-23-(1-188)
carboxyl-terminal deletion mutant. The cells were stained with an
antibody that preferentially recognizes human SNAP-23. Note that both
wild-type and mutant SNAP-23 are expressed primarily on the plasma
membrane.
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[in a new window]
Fig. 4.
Expression of human SNAP-23
augments exocytosis in rat mast cells. Mock-transfected RBL mast
cells or RBL mast cells expressing wild-type human SNAP-23-(1-211)
(panel A) or wild-type human SNAP-23-(1-211) and human
SNAP-23-(1-188) (panel B) were stimulated with phorbol
myristate acetate and ionomycin for various times, and exocytosis of
-hexosaminidase into the medium was assayed as described in the
text. Cells incubated in the absence of these agents did not release
any
-hexosaminidase into the medium over the time course shown. In
A the data represent the mean ± S.E. of triplicate
determinations and is representative of two independent clones. In
B the data represent the mean ± S.E. of five different
secretion assays in two independent experiments. Data were analyzed
using an unpaired t test at the 95% confidence interval and
revealed significant differences from mock-treated cells at the level
of p < 0.0001 in A and p = 0.0003 in B.
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[in a new window]
Fig. 5.
Binding of SNAP-23 and SNAP-25 to GST-SNARE
fusion proteins. Radiolabeled, in vitro translated
SNAP-25, human SNAP-23, rat SNAP-23, or mouse SNAP-23 were incubated
with 10 µg of GST-Syntaxin 4 or GST-VAMP 2 fusion proteins
immobilized on glutathione-Sepharose beads for 2 h at 4 °C and
washed extensively, and the amount of radiolabeled protein bound was
determined by SDS-PAGE and fluorography. Equal amounts of radioactive
SNAP-25 or SNAP-23 were added to each condition as shown by analyzing
an aliquot of the sample input for each condition. The gels were
stained with Coomassie Blue R-250 to confirm that each reaction also
contained identical amounts of GST fusion protein.
View larger version (15K):
[in a new window]
Fig. 6.
Human SNAP-23 binds to SNAREs better than
does rat SNAP-23. The binding of in vitro translated
rat SNAP-23, human SNAP-23, or SNAP-25 to GST-syntaxin 4 or GST-VAMP 2 fusion proteins was quantitated by SDS-PAGE and PhosphorImager analysis
and is expressed as a percentage of the total input radioactivity. The
data represent the average ± S.E. from samples analyzed in
triplicate from three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells possess SNAP-25, and it has been shown that
SNAP-25 is involved in insulin secretion from these cells (31). Interestingly, under conditions in which SNAP-25 is cleaved by Botulinum neurotoxin E and exocytosis is diminished, human SNAP-23 (which is not cleaved by this toxin) can rescue exocytosis only if it
is expressed at extraordinarily high levels (22). In light of our
current work, we interpret this result by proposing that replacement of
SNAP-25 (an excellent SNARE-binder) by human SNAP-23 (a moderate
SNARE-binder) would require very large amounts of human SNAP-23. On the
other hand, enhancing exocytosis from mast cells that use rat SNAP-23
(a poor SNARE-binder) by introducing human SNAP-23 (a moderate
SNARE-binder) would require only small amounts of human SNAP-23. Our
data presented here, together with the functional results of Sadoul
et al. (22) are in very good agreement with this hypothesis.
Although we had hoped to overexpress the excellent SNARE-binder SNAP-25
in rat mast cells, we were unable to isolate healthy RBL transfectants
expressing significant amounts of SNAP-25. This could be due to
toxicity of overexpressed SNAP-25 in these cells, and in agreement with
this we were also unable to isolate stable RBL transfectants expressing
very large amounts of human SNAP-23. We conclude from these studies
that the efficiency of exocytosis is regulated in part by the ability of SNARE complexes to form in living cells since compensation for a
loss of SNAP-25 requires expression of large amounts of human SNAP-23
and expression of even small amounts of human SNAP-23 can potentiate
secretory processes in cells possessing rat SNAP-23.
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ACKNOWLEDGEMENTS |
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We thank Dr. Richard Scheller for the gift of various SNARE expression vectors, Dr. Elizabeth Hiltbold for assistance with confocal microscopy, Dr. Shashi Srivastava for statistical analysis, David Winkler for oligonucleotide synthesis and automated sequence analysis, and Dr. Juan Rivera for helpful discussions regarding RBL cell culture.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: National Institutes of
Health, Bldg. 10, Rm., 4B36, Bethesda, MD 20892. Tel.: 301-594-2595;
Fax: 301-496-0887; E-mail: paul.roche@nih.gov.
Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M103536200
2 V. V. Vaidyanathan, N. Puri, and P. A. Roche, unpublished observations.
3 K. Vogel and P. A. Roche, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble NSF-attachment protein receptor; t- and v-SNARE, target and vesicle membrane-associated SNARE, respectively; VAMP, vesicle-associated membrane protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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