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J. Biol. Chem., Vol. 277, Issue 51, 50030-50035, December 20, 2002
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From the Department of Molecular & Cell Biology, University of
California, Berkeley, California 94720 and
Received for publication, July 30, 2002, and in revised form, September 25, 2002
The soluble
N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and
fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the
gastric parietal cell. Here we examined the cellular localization and
functional importance of SNAP-25 in parietal cell cultures. Adenoviral
constructs were used to express SNAP-25 tagged with cyan fluorescent
protein, VAMP-2 tagged with yellow fluorescent protein, and
SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25
The secretion of HCl by the gastric parietal cell is
driven by a heterodimeric protein known as the H,K-ATPase or proton
pump. The regulation of the acid secretory process is thought to be accomplished by secretagogue-dependent trafficking of the
proton pump to and from the apical membrane (1, 2). Upon stimulation, the H,K-ATPase-containing cytoplasmic tubulovesicles fuse with the
apical (canalicular) membrane and the process is reversed when the
stimulus is removed. Evidence to support this recycling model of acid
secretion is extensive, although the regulatory mechanisms underlying
the trafficking and fusion events remain elusive.
Originally from the study of synaptic transmission, we have come to
recognize that membrane fusion is mediated by a set of highly conserved
proteins called SNARE proteins (3, 4). According to the
SNARE1 hypothesis, the plasma
membrane maintains a set of closely associated proteins known as target
SNAREs such as syntaxin-1A and SNAP-25 (5). Complementary proteins
located on cytoplasmic vesicle membranes are known as v-SNAREs such as
VAMP-2. Docking of the vesicle at the plasma membrane occurs through
complementary interaction of v-SNAREs and target SNAREs, ultimately to
form a stable SNARE complex that perhaps leads to or catalyzes the
fusion of the two membranes (4). Additional factors, both
membrane-attached and cytosolic ones, ensure the necessary control of
the process.
In this study, we used a recombinant adenoviral system to express two
of these SNARE proteins, SNAP-25 and VAMP-2 fused to spectrally
distinct green fluorescent proteins, in primary cultures of gastric
parietal cells. Our goal was to localize the SNARE proteins within the
cells and to study their dynamic redistribution when the cells were
activated from the resting to stimulated state. We also used a
C-terminal deletion mutant of SNAP-25 (SNAP-25 Tissue Preparation and Membrane Fractionation--
Gastric
mucosal cell fractions were obtained from rabbit and guinea pig
stomachs as described previously (6) and in accordance with procedures
approved by the local Animal Care and Use Committee. After
tranquilization, animals were injected with 20 mg/kg cimetidine 1 h before sacrifice to put parietal cells in a resting state. Gastric
mucosa was homogenized in 25 volumes of buffer containing 5 mM PIPES/Tris, pH 6.7, 125 mM mannitol, 40 mM sucrose, 1 mM EDTA, 30 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin. After centrifugation at 80 × g for 10 min to remove whole cells and connective tissue, the resulting supernatant was used to generate three crude particulate fractions and a final supernatant by sequential centrifugation steps: P1 (4300 × g for 10 min) containing large cellular structures including
nuclei and plasma membranes; P2 (13,000 × g for 10 min) containing mainly mitochondria and large granules; and P3
(100,000 × g for 1 h) containing ER and
H,K-ATPase-rich tubulovesicles (TV). The final high speed supernatant
(S3) was saved as the cytosol. An enriched plasma membrane (PM)
fraction was prepared from P1 by centrifugation at 100,000 × g for 1 h through a density step gradient made of 12 and 18% Ficoll (type 400) with membranes collected from atop each
layer (6). A TV fraction highly enriched in H,K-ATPase was prepared
from P3 by density gradient centrifugation on a sucrose step gradient
as described previously (7).
Isolation of Gastric Glands and Parietal Cells and Parietal Cell
Culture--
Isolated gastric glands and parietal cells were prepared
from New Zealand White rabbits by a combination of high pressure perfusion and collagenase digestion in HEPES-minimal essential medium
(MEM), pH 7.4, as described previously (8). The resulting suspension
containing both glands and cells was strained through a 40-µm mesh to
remove connective tissue and large debris. The resulting supernatant
was left standing on ice for 5-10 min, time sufficient for large
gastric glands to settle leaving individual cells suspended in the
medium. Intact cells for primary culture were recovered from the
suspension by three repetitions of centrifugation (200 × g for 5 min) followed by resuspension in fresh HEPES-MEM. This low speed centrifugation favors the harvesting of the larger parietal cells, although few other epithelial cells and fibroblasts were recovered. Cells were incubated for 30 min at 37 °C in medium A
consisting of Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 20 mM HEPES, 0.2% bovine serum albumin,
10 mM glucose, 8 nM epidermal growth factor,
1× SITE medium (Sigma), 1 mM glutamine, 100 units/ml
penicillin/streptomycin, 400 µg/ml gentamicin sulfate, and 15 µg/liter Geneticin or 20 µg/ml novobiocin, pH 7.4, containing 25 µg/ml amphotericin B to destroy contaminating yeast and bacteria.
Cells were then plated onto Matrigel (Collaborative Biomedical) coated
coverslips in 12-well plates and incubated at 37 °C in culture
medium A. For binding studies where we wished to harvest relatively
large amounts of SNAP-25 and its C-terminal deletion mutant, settled
glands were collected, washed twice in HEPES-MEM, and plated onto
Matrigel in medium A as described for cell culture.
Western Blots--
Membrane fractions were solubilized in
SDS-PAGE sample buffer (1% SDS, 0.4 M urea, 5% 0.7 M 2-mercaptoethanol, 0.25 mM EDTA, 10%
glycerol, 0.0025% bromphenol blue, and 30 mM Tris-HCl, pH 6.8). Samples were run on 12% gels and transferred to nitrocellulose membranes and then probed with a rabbit polyclonal antibody directed against SNAP-25 (provided by H. P. Moore, University of
California, Berkeley, CA) followed by secondary probing with
horseradish peroxidase-tagged goat anti-rabbit IgG (Jackson
Laboratories). For the detection of VAMP, we used a polyclonal antibody
made in rabbit using GST-VAMP-2 fusion protein as antigen (provided by
Dr. A. W. Lowe, Stanford University, Palo Alto, CA). This was
followed by the goat anti-rabbit IgG tagged with horseradish
peroxidase. H,K-ATPase was detected by monoclonal antibody 2G11, a
mouse monoclonal antibody against its [14C]Aminopyrine (AP) Uptake
Assay--
Stimulation of parietal cells was quantified using the AP
uptake assay as described for cultured parietal cells (9). Culture medium was removed from the wells and replaced with 0.5 ml of HEPES-MEM
containing 11 nCi/ml [14C]AP. Cells were either
held in a resting state by the H2-receptor blocker cimetidine (100 µM) or stimulated by the addition of histamine and IBMX
(100 and 30 µM, respectively). Cultures were gently
shaken for 30 min at 37 °C. The coverslips were removed from the
medium, quickly dipped in phosphate-buffered saline to remove external radioactivity, and incubated in solubilization buffer (125 mM Tris-Cl, 2% SDS, and 10% 2-mercaptoethanol, pH 6.8)
for 1 h at room temperature. Cells were then scraped from the
coverslip, and the protein content of the cell scrapings was assayed
using the filter paper blot method (10). Aliquots of both the reaction medium and cell scrapings were assayed for [14C]AP
by liquid scintillation counting. These data were used to calculate the
AP accumulation ratio (ratio of
[AP]i:[AP]e). AP uptake values were
normalized among the various preparations by expressing data as a
fraction of the stimulated control.
Adenoviral Infection of Gastric Parietal Cells and Gastric
Glands--
Adenoviruses were obtained as previously described with
the following mouse cDNA constructs (11, 12): SNAP-25 fused to cyan
fluorescent protein (CFP); VAMP-2 fused to yellow fluorescent protein
(YFP); SNAP-25 wild type and GFP unfused; and C-terminal deletion
mutant SNAP-25
Infections with rAd were executed by the addition of ~2 × 106 viral particles/ml to the culture medium immediately
after isolation of gastric glands or 6-h post-plating for cultured
parietal cells. Various concentrations of viruses were tested with our
culture system, and we chose our experimental conditions based on the level of fluorescent protein expression. After 36-h infection, AP
uptake experiments were performed on cultured cells. Wild type and
mutant SNAP-25 were expressed by rAd infection for 18 h in gastric
glands, which were then solubilized with Triton X-100 and used for
binding assay.
Immunofluorescence Microscopy--
After rAd infection, cultured
parietal cells were either held in a resting state or stimulated with
histamine plus IBMX. In some cases, cells were treated with a proton
pump inhibitor (5 µM SCH-28080) as described previously
(13). Infected cells were observed in the presence of Dulbecco's
modified Eagle's medium (Invitrogen) for the duration of live cell
imaging. Images were collected using conventional epifluorescence
microscopy (excitation/emission: GFP, 488/509 nm; YFP, 500/20 nm; CFP,
436/10 nm) with a Nikon Microphot FX-2 using Isee Imaging software. In
the case of cells double-labeled with CFP-SNAP-25 and YFP-VAMP-2,
images are presented for each of the fluorescent proteins along with
images recorded by differential interference microscopy (DIC). To
compare and distinguish the distribution of two fluorophores, we also
performed a subtraction of the YFP image from the CFP image. This was
done using Isee Imaging software by first adjusting the total intensity of the two images to approximately the same level and then performing a
pixel by pixel subtraction.
In Vitro SNARE Core Complex Formation--
Recombinant rat
GST-syntaxin-1 was expressed in bacteria as fusion protein as described
previously (12). Wild type and mutant SNAP-25 were expressed in gastric
glands for 18 h by rAd infection, which were then solubilized with
Triton X-100 and used for binding assay. For binding assays, aliquots
of total soluble protein were added to 20 µl of glutathione-agarose
beads pre-adsorbed with GST-syntaxin-1 and incubated overnight at
4 °C in 300 µl of binding buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EGTA, and 1% Triton X-100).
Beads were washed three times with 0.5 ml of binding buffer at 4 °C.
Samples were separated into supernatant and pellet, solubilized in
sample buffer, and analyzed by Western blot. Blots were first probed
with antibody against SNAP-25, stripped, and then probed with antibody
against syntaxin-1 (HPC-1, Sigma).
Identification of SNAP-25 in Gastric Parietal Cells--
The
relative abundance of SNAP-25 in crude subcellular fractions and
partially purified membrane fractions from rabbit and guinea pig
gastric mucosa was assayed by SDS-PAGE and immunoblotting using a
SNAP-25 antibody. The same blots were stripped and reprobed for
relative abundance of H,K-ATPase using an antibody against the
60-80-kDa
Earlier experiments from our laboratory (14) and that of Calhoun and
Goldenring (15) demonstrated that another SNARE protein, VAMP-2 (also
known as synaptobrevin), was associated with the H,K-ATPase-rich
tubulovesicle fraction from parietal cells. To distinguish the
distribution of SNAP-25 and VAMP-2, another set of cell fractions was
prepared from rabbit gastric mucosal homogenate and Western blots were
probed for these two proteins along with H,K-ATPase as a control. The
results shown in Fig. 2 demonstrate that
VAMP-2 was most highly enriched along with tubulovesicles and
H,K-ATPase-rich membranes, whereas SNAP-25 was more highly enriched in
the fractions containing plasma membranes.
To further evaluate the cellular localization of SNAP-25, we used
fluorescence microscopy in cultured parietal cells infected with
adenovirus containing the CFP-SNAP-25 cDNA construct. By 12 h
post-infection, parietal cells clearly expressed CFP, and high levels
of expression were observed by 36 h post-infection. Cyan
fluorescence was detected in ~90% of the parietal cells. Fig.
3 shows the intracellular localization of
CFP-SNAP-25 in resting (non-stimulated) rabbit parietal cells 24 h
post-infection. At this low magnification, some diffuse fluorescence
was found in the cytoplasm of many parietal cells, but a prominence of
CFP fluorescence is clearly seen outlining the apical membrane
vacuoles. Because these membrane vacuoles in cultured parietal cells
are derived from the apical plasma membrane of parietal cells in
situ (13), we call them apical membrane vacuoles. No fluorescence was detected on the "basolateral" membrane surrounding the cells. Thus, the results indicate that SNAP-25 is clearly targeted to the
apical plasma membrane.
Effect of C-terminal Mutant SNAP-25
To further explore the molecular involvement of SNAP-25 in the gastric
acid secretion process, an in vitro binding assay was performed. SNAP-25 wild type and C-terminal deletion mutant were expressed in cultured rabbit gastric glands by adenovirus infection similar to that used for cell cultures. Membrane proteins were solubilized with Triton X-100 and incubated overnight with
GST-syntaxin-1A immobilized on glutathione beads. After separating
pellet and supernatant by centrifugation, the fractions were separated
by SDS-PAGE and successively probed for SNAP-25 and syntaxin-1 by immunoblotting. As shown in Fig. 5,
wild-type SNAP-25 was effective in facilitating the assembly of a
complex with syntaxin-1, whereas the C-terminal deletion mutant was
much less effective. Wild type SNAP-25 was detected entirely in the
binding fraction, but the majority of the C-terminal deletion mutant of
SNAP-25 was detected in the non-binding fraction. The mutant, which
runs at a lower molecular weight, allows native SNAP-25 to be seen
recovered only in the binding fraction (also seen in the non-infected
gland lysates).
Intracellular Localization and Distribution of CFP-SNAP-25 and
YFP-VAMP-2 in Gastric Parietal Cells--
In previous studies, we have
shown that expressed GFP-VAMP-2 was localized to cytoplasmic membranes
along with H,K-ATPase in resting parietal cell cultures and that the
two proteins were translocated to apical membrane commensurate with
stimulation of acid secretion (16). Here we sought to monitor the
dynamic changes in response to stimulation of live parietal cells that were co-infected with adenovirus containing cDNA constructs for CFP-SNAP-25 and YFP-VAMP-2. Cyan fluorescence was detected in ~90%
of the parietal cells. Yellow fluorescence was detected in ~75% of
the parietal cells, most of which also displayed cyan. The distribution
of CFP-SNAP-25 and YFP-VAMP-2 in parietal cells during different
functional states is shown in Figs. 6 and
7 along with a comparable image using DIC
optics. In addition, for each cell there is a "coincident fused
image" that is a mathematical reconstruction showing only pixels that
were positive for both probes. In a series of resting cells shown in
Fig. 6, CFP-SNAP-25 was clearly localized to the apical membrane
vacuoles, although there was usually some diffuse cytoplasmic labeling.
In these same resting cells, YFP-VAMP-2 was observed almost exclusively throughout the cytoplasm surrounding the apical membrane vacuoles but
with very limited yellow fluorescence directly on the apical membrane
vacuoles. In no case was either CFP-SNAP-25 or YFP-VAMP-2 associated
with the basolateral plasma membrane or the visible lamellipodial
extensions. Because there was considerable overlap in the CFP and YFP
signals, especially in the cytoplasmic region surrounding the apical
vacuoles, we performed a mathematical subtraction of YFP signal from
that of CFP. The resulting difference is shown in Fig. 6 as the last
column of images. In every case, there is a ring of remaining CFP
fluorescence denoting that CFP-SNAP-25 is more preferentially localized
to the apical membrane vacuoles in the resting parietal cells. Note
that the nuclear membrane, which is denoted on the DIC image by an
asterisk, is approximately the same size as the apical vacuoles, but it
is not labeled by CFP fluorescence and does not appear in the
subtractive image. Thus, separate compartments can be identified for
the SNAP-25 and VAMP-2 in resting cells, although there is a subset of
overlapping coincidence in the cytoplasm.
In the case of cells stimulated by histamine and IBMX, the apical
membrane vacuoles become highly enlarged filling with HCl and water
because of the action of newly incorporated H,K-ATPase pump. The images
of maximally stimulated cells shown in Fig. 7A demonstrate
an almost coincident distribution of CFP-SNAP-25 and YFP-VAMP-2 on the
enlarged apical vacuoles, consistent with the notion that YFP-VAMP-2
translocated to the apical membrane after stimulation. However, the
relative diminution of free cytoplasmic space in these maximally
stimulated cells makes it difficult to exclude the possibility that
some spatial artifact might contribute to apparent co-localization of
the two signals. We previously showed that treatment of the cells with
a proton pump inhibitor eliminates the swelling artifact by inhibiting
the delivery of HCl and water while permitting other events in the
signaling cascade including the recruitment of H,K-ATPase to the apical
membrane vacuoles (13). Thus, to avoid the swelling artifact parietal cells were stimulated and treated with the proton pump inhibitor SCH-28080. The corresponding images in Fig. 7B show that
CFP-SNAP-25 and YFP-VAMP-2 are primarily associated with the vacuolar
membranes, even while the cytoplasmic area was preserved by the
treatment of stimulated cells with SCH-28080. Thus, in live parietal
cells, there is a dynamic stimulation-dependent
co-localization of VAMP-2 and SNAP-25 in the apical plasma membrane vacuoles.
In this study, we examined the distribution dynamics and
functional importance of SNAP-25 in gastric parietal cells. In the cell
fractionation studies, SNAP-25 immunoreactivity was mostly associated
with the enriched plasma membrane fraction. SNAP-25 was also detected
in the P3 (microsomal) fraction, but because it did not purify with
H,K-ATPase-rich tubulovesicles, we suspect that this was may be a
contamination of the rough ER. On the other hand, VAMP-2 clearly
associates with the P3 fraction and is further enriched when H,K-ATPase
is purified by density gradient purification. These data are consistent
with earlier work suggesting that VAMP-2 is associated with
H,K-ATPase-rich membranes typical of a v-SNARE, whereas SNAP-25 may be
associated with the apical plasma membrane more typical of a target
SNAREs (14, 17). However, the present SNAP-25 data are in disagreement
with the findings of Jöns et al. (17) who
report that SNAP-25, syntaxin-1, and synaptobrevin (VAMP-2) all
co-localize with H,K-ATPase in tubulovesicles of resting parietal cells
and redistribute to the apical membrane after stimulation. In their
study, Jöns et al. (17) did not establish
co-localization by double labeling but rather used comparative staining
patterns observed in sections of resting and secreting gastric mucosa
and in isolated parietal cells. Therefore, it is not possible to
conclude that none of the three proteins was associated with the apical
membrane in resting cells. Furthermore, the isolated parietal cell
preparations used by these authors were highly rounded and appeared to
lack apical membrane vacuoles, indicating that they may have been
functionally and morphologically compromised. To localize SNAP-25 more
specifically and because we found that existing commercial antibodies
against SNAP-25 were not suitable for immunocytochemistry, we used the
recombinant adenovirus technique as an ideal vector for introducing
specific gene expression into primary cultures of parietal cells. This
allowed us to localize targeted protein expression, observe the dynamic
changes associated with functional activity, and introduce mutations to
alter that activity.
Gastric acid secretion by the parietal cell involves
secretagogue-regulated recycling of the H,K-ATPase to and from the
apical membrane (1, 2). These regulated trafficking events, which deliver the pump to the apical cell surface, result in massive morphological transformations of the parietal cell. The trafficking of
the H,K-ATPase and the remodeling of the apical membrane during this
process probably involve the coordinated function of vesicular trafficking machinery (1, 13). The dramatic nature of the morphological
transformation facilitates superior visualization of changes in the
distribution of proteins such as SNAP-25 and VAMP-2, affecting fusion.
By introducing both CFP-SNAP-25 and YFP-VAMP-2 adenoviral constructs
into the same gastric parietal cells, we have successfully studied the
subcellular localization and dynamic distribution of these proteins. In
resting cells, YFP-VAMP-2 was distributed throughout the cytoplasm in a
pattern similar to that of H,K-ATPase. YFP-VAMP-2 was largely
translocated to the apical membrane vacuoles upon stimulation. These
data are consistent with previous work where GFP-VAMP-2 was expressed
in parietal cells (16). In the case of parietal cells expressing CFP-SNAP-25, the majority of signal was localized to the apical membrane vacuoles in both resting and stimulated cells; however, there
was always a component of CFP-SNAP-25 detected in cytoplasm, especially
in the resting state. An artifactual localization is always a
possibility when one is inducing overexpression, and the specific
location of the expressed protein is frequently related to the
integrity of the product such as correct folding, glycosylation, and so
forth (18, 19). In cases of overexpression, we would expect to find
abundant signal associated with the ER or recycling endosomal
compartment as well as in the final targeted cytolocation.
The formation and stability of the SNARE complex including VAMP-2,
syntaxin, and SNAP-25 are believed to play a central role in the
molecular mechanism underlying exocytosis (3, 4, 20). The cleavage of
SNAREs by Clostridial neurotoxins has been established as an essential
tool to investigate the role of these proteins in neurotransmission
(21, 22). The C terminus of SNAP-25 that contributes one helix to the
four-helix cytoplasmic bundle is believed to be critical for SNARE
complex formation and has been implicated in the final step of
calcium-triggered exocytosis (4, 23-27). More specifically, botulinum
neurotoxins A and E cleave the C-terminal domain of SNAP-25, causing
complete or partial inhibition of secretion in neuronal and endocrine
systems as well as pancreatic acinar cells (22, 28). Moreover, mutation studies have shown important details regarding the participation of
SNAP-25 in the exocytic process (12, 29-32). These results suggest
that SNAP-25 may promote membrane fusion by contributing two
cytoplasmic-helices including the C-terminal helix to stabilize the
four-helix core complex. Yang et al. (12) recently report that the overexpression of the C-terminal deletion mutant A major function of the gastric parietal cell is to produce HCl
secretion. Because of the massive membrane transformations required for
the regulation of its functional activity, the parietal cell may be a
model system to characterize the protein-protein interactions that
regulate membrane trafficking in other cell types (2, 33). Previous
studies demonstrate the presence of v-SNARE and target SNARE proteins
on tubulovesicle membranes including the association of VAMP-2 and
syntaxin-3 with H,K-ATPase-rich tubulovesicles (14, 15, 34). SNAP-25
and syntaxin isoforms 1, 2, and 4 were also identified in parietal cell
membrane fractions (14, 17). Earlier functional studies (16, 35)
demonstrate that VAMP-2 (16) and syntaxin-3 (35) are essential for the parietal cell activation. The present results now implicate a functional role for SNAP-25 in the regulated trafficking and recycling of H,K-ATPase and add further evidence for the general importance of
SNARE proteins for the secretory activation of parietal cells.
We thank J. G. Duman, A. Sawaguchi, R. Zhou, R. Jain, and M. Turkoz for technical assistance, C. Chan and Dr.
H.-P. Moore (University of California, Berkeley, CA) for SNAP-25
antibody, and Dr. A. W. Lowe (Stanford University, Palo Alto, CA)
for VAMP-2 antibody.
*
This work was supported in part by National Institutes of
Health Grants DK10141 and DK38972 (to J. G. F.), DK56292 (to X. Y.),
and NS35167 and NSF, National Institutes of Health Grant IBN0110980 (to
Y. L.).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: Dept. of Molecular & Cell Biology, University of California at Berkeley, 145 Life Sciences
Addition, Number 3200, Berkeley, CA 94720-3200. Tel.: 510-642-1544;
Fax: 510-643-6791; E-mail: jforte@uclink4.berkeley.edu.
Published, JBC Papers in Press, October 16, 2002, DOI 10.1074/jbc.M207694200
The abbreviations used are:
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
SNAP-25, soluble N-ethylmaleimide-sensitive factor
attachment protein-25, VAMP-2, vesicle-associated membrane protein-2;
CFP, cyan fluorescent protein;
YFP, yellow fluorescent protein;
GFP, green fluorescent protein;
ER, endoplasmic reticulum;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
PM, plasma membrane;
DIC, differential interference microscopy;
IBMX, isobutylmethylxanthine;
MEM, minimum Eagle's medium;
AP, aminopyrine;
TV, tubulovesicle;
rAd, adenoviruses with the incorporated cDNA;
GST, glutathione S-transferase.
Localization and Function of Soluble
N-Ethylmaleimide-sensitive Factor Attachment Protein-25 and
Vesicle-associated Membrane Protein-2 in Functioning Gastric Parietal
Cells*
, and Department
of Pathology, University of Oklahoma, Health Sciences Center, Oklahoma
City, Oklahoma 73190
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
181-206). Membrane fractionation experiments and fluorescent
imaging showed that SNAP-25 is localized to the apical plasma membrane.
The expression of the mutant SNAP-25 181-226 inhibited the acid
secretory response of parietal cells. Also, SNAP 181-226 bound
poorly in vitro with recombinant syntaxin-1 compared with
wild type SNAP-25, indicating that pairing between syntaxin-1 and
SNAP-25 is required for parietal cell activation. Dual expression of
SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with
yellow fluorescent protein revealed a dynamic change in distribution
associated with acid secretion. In resting cells, SNAP-25 is at the
apical plasma membrane and VAMP-2 is associated with cytoplasmic
H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins
co-localize on the apical plasma membrane. These data demonstrate the
functional significance of SNAP-25 as a SNARE protein in the parietal
cell and show the dynamic stimulation-associated redistribution of
VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25
on the apical plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
181-206) to examine
the role of SNAP-25 in the activation of parietal cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (Affinity
Bioreagents) followed by secondary antibody with horseradish
peroxidase-tagged goat anti-mouse IgG. Bands were visualized by
chemiluminescence with the Renaissance kit (PerkinElmer Life Sciences).
181-206 and GFP unfused. Adenoviruses with the
incorporated cDNAs (rAd) were used to infect cells and glands with
the respective cDNAs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit. As shown in Fig.
1A for crude cell fractions from rabbit stomach, a visible immunoreactive band was detected in the
25-kDa SNAP-25 region of the low speed fraction (P1) and in the
microsomal fraction (P3). There was little or no detectable SNAP-25
reactivity in either the mitochondrial-rich fraction (P2) or the
cytosolic supernatant (S3). To better characterize the localization of
SNAP-25 in parietal cells, P1 and P3 were further fractionated by
density gradient centrifugation. SNAP-25 was highly enriched in the PM
fraction purified from P3. In contrast, there was virtually no
enrichment of SNAP-25 in the H,K-ATPase-enriched TV fraction purified
from P3. The cross-reactivity of the anti-SNAP-25 antibody prepared
from immunized rabbit serum resulted in the appearance of several minor
immunoreactive bands in the rabbit cell fractions, some more prominent
than others (e.g. P1). To circumvent this problem,
comparable cell fractions were prepared from guinea pig gastric mucosa.
The results shown in Fig. 1B were similar to those for the
rabbit; that is SNAP-25 was most highly enriched in the plasma
membrane-rich fractions (P1 and PM), whereas H,K-ATPase was most
enriched in the membrane fraction derived from TV P3.
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Fig. 1.
Localization of SNAP-25 in subcellular
fractions of gastric parietal cells. Cell fractions isolated from
rabbit (A) and guinea pig (B) gastric mucosa were
developed by SDS-PAGE (25 µg protein for each of the primary
fractions, P1-S3; 20 µg for PM; 12 µg for TV), blotted to
nitrocellulose membrane, and immunoprobed for SNAP-25 (SN25)
as shown in the lower half of each panel. The blots were then stripped
and re-probed with an antibody against the -subunit of H,K-ATPase
(HK) as indicated in the upper
portion of each panel. Among the crude cell fractions,
SNAP-25 was identified in P1 and P3. SNAP-25 was then highly enriched
in the PM fraction purified from P1. HK, which runs as a broad band
between 60 and 80 kDa, was most highly enriched in the microsomal
fraction (P3) and in the TV membranes purified from P3. The
extra band at 52 kDa in P3 is the high mannose form of HK from the
ER (pre). Small amounts of SNAP-25 were seen in the P3
and L2 fractions. Adenoviral infection of COS-7 cells was used as a
positive control for SNAP-25 expression (ctrl).
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Fig. 2.
Triple probe for SNAP-25, VAMP-2, and
H,K-ATPase in rabbit gastric mucosal cell fractions. Crude
cell fractions (P1, P2, P3, and
S3) and purified membrane fractions including PM fraction
and an H,K-ATPase-rich TV fraction were isolated from rabbit gastric
mucosa as described under "Experimental Procedures." The fractions
were solubilized, developed by SDS-PAGE, blotted to nitrocellulose
membrane, and probed for H,K-ATPase (HK), SNAP-25
(SN25), and VAMP-2 as indicated. In the blot for HK and
SN25, all lanes had 20 µg of protein with the exception of TV, which
had 10 µg of protein. The blot for VAMP-2 had 37.5 µg of protein
for each lane. As usual, H,K-ATPase was most highly enriched in P3 and
TV. SNAP-25 was detected in P1 and enriched in PM. VAMP-2 was most
enriched in TV.
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Fig. 3.
Localization of CFP-SNAP-25 in gastric
parietal cells. Primary cultures of gastric parietal cells were
infected with recombinant CFP-SNAP-25 adenovirus. After 36 h,
cells were examined in presence of 100 µM cimetidine for
general morphology and CFP fluorescence. Two separate fields including
several parietal cells are shown with corresponding images of
epifluorescence (CFP) and DIC optics (DIC).
Parietal cells are easily distinguished as large cells with obvious
internalized vacuoles of apical membrane. Although there is some cell
to cell variation in the extent of CFP-SNAP-25 expression, CFP
fluorescence was found associated with most of the vacuolar membranes
in the parietal cells. Bar marker is 20 µm.
181-206 on Gastric Acid
Secretion--
The C terminus of SNAP-25 has been shown to be
important in forming a SNARE complex; therefore, we used a C-terminal
deletion mutant of SNAP-25 181-206 to probe for a direct functional
role of SNAP-25 in parietal cell activation. After 36 h, cultures
infected with adenovirus including either wild type or mutant SNAP-25
or a control adenovirus containing the -galactosidase gene were compared with uninfected cultures for their acid secretory response to
stimulants. Parietal cell cultures from each condition were either
maintained in a resting state (cimetidine) or stimulated to maximum
secretion with histamine plus IBMX. The [14C]AP uptake
was measured as an index of acid secretion (Fig.
4). To account for variations among four
separate preparations, the [14C]AP uptake ratio was
normalized to the control non-infected, stimulated parietal cells that
was set at 100% for each experiment. Compared with uninfected
controls, [14C]AP uptakes were not significantly lower in
cells infected with wild type SNAP-25 adenovirus (85.7 ± 19.2%
of stimulated control; p = 0.9) or control adenovirus
containing -galactosidase gene but no SNAP-25 (70.4 ± 14.9%
of stimulated control; p = 0.24). However, we detected
almost 70% inhibition of acid secretion by cells infected with the
C-terminal mutant SNAP-25 (AP ratio was 31.0 ± 5.9% of the
stimulated control, p < 0.003).
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Fig. 4.
C-terminal mutant SNAP-25 inhibits acid
secretion by cultured parietal cells. Cultured parietal cells were
infected for 36 h with wild type SNAP-25 (S-25 WT),
C-terminal 181-206 mutant (S-25 mutant), or adenovirus
with -galactosidase instead of SNAP-25 (Adenovirus).
Additional cultures of non-infected parietal cells served as untreated
control (Control). Cells were then incubated for 30 min with
cimetidine (REST) or stimulated in the presence of 100 µM histamine and 30 µM IBMX
(STIM), and the [14C]AP accumulation ratio was
measured. The data shown here for four experiments have been normalized
by setting the value for the stimulated control to 100% for each
experiment. Values are the mean ± S.E. of four independent
experiments, each with duplicate determinations. Acid secretion was
significantly inhibited only by the SNAP-25 mutant infection (inhibited
by 69 ± 5.9%; p = 0.003).
View larger version (46K):
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Fig. 5.
Ineffective binding of mutant SNAP-25 to
syntaxin-1. Gastric gland cultures infected with wild type SNAP-25
(SN25 wt) or C-terminal 181-206 deletion mutant
(SN25 mut) were solubilized with Triton X-100 and incubated
overnight with GST-syntaxin-1 attached to glutathione beads as
described under "Experimental Procedures." The beads were
centrifuged and washed with phosphate-buffered saline containing 0.1%
Triton X-100 three times. The pellet (P) and supernatant
(S) from each sample were first analyzed by Western blot
using anti-SNAP-25 antibody (lower panel). Blots were then
stripped and re-probed with an antibody against syntaxin-1 (upper
panel). The wild type SNAP-25 was exclusively retained in the
syntaxin-1 pellet. Endogenous SNAP-25 of non-infected control cells
(non-inf) was also retained in the syntaxin-1 pellet.
However, most of the C-terminal deletion mutant was recovered in the
supernatant. A small amount of the mutant SNAP-25 along with endogenous
glandular SNAP-25 was associated with the syntaxin-1 pellet. Positive
control samples were also run and probed for wild type SNAP-25 and
C-terminal 181-206 deletion mutant SNAP-25 expressed in COS-7 cells
and for the GST-syntaxin-1 beads without solubilized cell
samples.
View larger version (52K):
[in a new window]
Fig. 6.
Intracellular distribution of
CFP-SNAP-25 and YFP-VAMP-2 in live resting parietal cells.
Primary cultures of gastric parietal cells were co-infected with
recombinant CFP-SNAP-25 and YFP-VAMP-2 adenoviruses. Cells were
maintained in culture in the presence of the H2-receptor blocker
cimetidine (100 µM) to maintain a resting, non-secreting
state. After 36 h, cells were examined for general morphology
(DIC) and for CFP-SNAP-25 fluorescence (CFP.SN25)
and YFP-VAMP-2 fluorescence (YFP.VMP2). To compare and
discriminate the fluorescent signals, the YFP image was subtracted from
the CFP image (CFP-YFP) as described under "Experimental
Procedures." Apical membrane vacuoles are clearly seen in all images
surrounded by both CFP and YFP fluorescence extending into the
cytoplasm. In almost all cases, there appears to be a concentration of
CFP intensity specifically associated with the apical membrane
vacuoles. This is emphasized by the subtractive image of CFP-YFP where
the vacuoles always have some degree of intensity over surrounding
structures. Note that nuclei (indicated by asterisk in DIC
image), which are about the same size as vacuoles, are not ringed by
CFP or YFP fluorescence. Bar marker is 10 µm.
View larger version (58K):
[in a new window]
Fig. 7.
Dynamic changes in the intracellular
distribution of CFP-SNAP-25 and YFP-VAMP-2 in live parietal cells and
following stimulation. Primary cultures of gastric parietal cells
were co-infected with recombinant CFP-SNAP-25 and YFP-VAMP-2
adenoviruses and maintained in culture in the presence of 100 µM cimetidine as described in Fig. 6. After 36 h,
cimetidine was removed and cells were stimulated with 100 µM histamine plus 30 µM IBMX, either in the
absence (A) or presence (B) of the proton pump
inhibitor SCH-28080 (5 µM) as indicated. After 25 min of
stimulation, live cells were examined for general morphology
(DIC) and for CFP-SNAP-25 fluorescence
(CFP.SN25) and YFP-VAMP-2 fluorescence
(YFP.VMP2). Apical membrane vacuoles are much enlarged in
the stimulated state because of the accumulation of HCl and water,
whereas the vacuoles are not enlarged when the
H+/K+ pump is inhibited by SCH-28080. For both
conditions of stimulation, there is a high degree of co-localization of
the CFP-SNAP-25 and YFP-VAMP-2 fluorescence on the apical membrane
vacuoles and a relative diminution of signal in the cytoplasm. The
co-localization is substantiated by the subtractive image
(CFP-YFP) in which the vacuolar signal all but disappears.
The asterisk in DIC image indicates nucleus. Bar marker is
10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
180-206 significantly inhibited glutamate release from neuronal cells. Because
of the functional effects in neuronal cells, we used the C-terminal
deletion mutant to study the function of SNAP-25 in parietal cells. In
gastric parietal cells, we observed that cells expressing C-terminal
deletion mutant SNAP-25 180-206 showed consistent and significant
diminution of stimulated acid secretion as compared with wild type
expression. In addition, our binding data demonstrate that the
C-terminal deletion mutant unlike wild type SNAP-25 was relatively
ineffective in associating with syntaxin-1. These secretion and binding
data agree with a previous study involving cerebellar neurons, which
showed that overexpressed SNAP-25 180-206 mutant altered the
vesicle fusion kinetics, reaffirming the importance of SNAP-25 and
specifically this C-terminal segment in exocytosis (12). The results
also add further evidence for the importance of SNARE proteins for the
activation of parietal cells.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Forte, J. G.,
and Yao, X.
(1996)
Trends Cell Biol.
6,
45-48[CrossRef][Medline]
[Order article via Infotrieve]
2.
Okamoto, C. T.,
and Forte, J. G.
(2001)
J. Physiol. (Lond.)
532,
287-296 3.
Jahn, R.,
and Sudhof, T. C.
(1999)
Annu. Rev. Biochem.
68,
863-911[CrossRef][Medline]
[Order article via Infotrieve]
4.
Chen, Y. A.,
and Scheller, R. H.
(2001)
Nat. Rev. Mol. Cell. Biol.
2,
98-106[CrossRef][Medline]
[Order article via Infotrieve]
5.
Rothman, J. E.,
and Warren, G.
(1994)
Curr. Biol.
4,
220-233[CrossRef][Medline]
[Order article via Infotrieve]
6.
Reenstra, W. W.,
and Forte, J. G.
(1990)
Methods Enzymol.
192,
151-165[CrossRef][Medline]
[Order article via Infotrieve]
7.
Tyagarajan, K.,
Chow, D. C.,
Smolka, A.,
and Forte, J. G.
(1995)
Biochim. Biophys. Acta
1236,
105-113[Medline]
[Order article via Infotrieve]
8.
Chew, C. S.
(1994)
Annu. Rev. Physiol.
56,
445-461[CrossRef][Medline]
[Order article via Infotrieve]
9.
Duman, J. G.,
Tyagarajan, K.,
Kolsi, M. S.,
Moore, H. P.,
and Forte, J. G.
(1999)
Am. J. Physiol.
277,
C361-C372[Medline]
[Order article via Infotrieve]
10.
Minamide, L. S.,
and Bamburg, J. R.
(1990)
Anal. Biochem.
190,
66-70[CrossRef][Medline]
[Order article via Infotrieve]
11.
Xia, Z.,
Zhou, Q.,
Lin, J.,
and Liu, Y.
(2001)
J. Biol. Chem.
276,
1766-1771 12.
Yang, Y.,
Xia, Z.,
and Liu, Y.
(2000)
J. Biol. Chem.
275,
29482-29487 13.
Agnew, B. J.,
Duman, J. G.,
Watson, C. L.,
Coling, D. E.,
and Forte, J. G.
(1999)
J. Cell Sci.
112,
2639-2646[Abstract]
14.
Peng, X. R.,
Yao, X.,
Chow, D. C.,
Forte, J. G.,
and Bennett, M. K.
(1997)
Mol. Biol. Cell
8,
399-407[Abstract]
15.
Calhoun, B. C.,
and Goldenring, J. R.
(1997)
Biochem. J.
325,
559-564[Medline]
[Order article via Infotrieve]
16.
Karvar, S.,
Yao, X.,
Duman, J. G.,
Hybiske, K.,
Liu, Y.,
and Forte, J. G.
(2002)
Gastroenterology
123,
281-290[CrossRef][Medline]
[Order article via Infotrieve]
17.
Jons, T.,
Lehnardt, S.,
Bigalke, H.,
Heim, H. K.,
and Ahnert-Hilger, G.
(1999)
Eur J. Cell Biol.
78,
779-786[Medline]
[Order article via Infotrieve]
18.
Okamoto, C. T.,
Chow, D. C.,
and Forte, A. J.
(2000)
Am. J. Physiol.
278,
C727-C738
19.
Ward, C. L.,
and Kopito, R. R.
(1994)
J. Biol. Chem.
269,
25710-25718 20.
Weber, T.,
Zemelman, B. V.,
McNew, J. A.,
Westermann, B.,
Gmachl, M.,
Parlati, F.,
Sollner, T. H.,
and Rothman, J. E.
(1998)
Cell
92,
759-772[CrossRef][Medline]
[Order article via Infotrieve]
21.
Lawrence, G. W.,
Weller, U.,
and Dolly, J. O.
(1994)
Eur. J. Biochem.
222,
325-333[Medline]
[Order article via Infotrieve]
22.
Lawrence, G. W.,
Foran, P.,
Mohammed, N.,
DasGupta, B. R.,
and Dolly, J. O.
(1997)
Biochemistry
36,
3061-3067[CrossRef][Medline]
[Order article via Infotrieve]
23.
Chen, Y. A.,
Scales, S. J.,
Patel, S. M.,
Doung, Y. C.,
and Scheller, R. H.
(1999)
Cell
97,
165-174[CrossRef][Medline]
[Order article via Infotrieve]
24.
Chen, Y. A.,
Scales, S. J.,
Jagath, J. R.,
and Scheller, R. H.
(2001)
J. Biol. Chem.
276,
28503-28508 25.
Sorensen, J. B.,
Matti, U.,
Wei, S. H.,
Nehring, R. B.,
Voets, T.,
Ashery, U.,
Binz, T.,
Neher, E.,
and Rettig, J.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
1627-1632 26.
Criado, M.,
Gil, A.,
Viniegra, S.,
and Gutierrez, L. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7256-7261 27.
Mehta, P. P.,
Battenberg, E.,
and Wilson, M. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10471-10476 28.
Hansen, N. J.,
Antonin, W.,
and Edwardson, J. M.
(1999)
J. Biol. Chem.
274,
22871-22876 29.
Huang, X.,
Sheu, L.,
Tamori, Y.,
Trimble, W. S.,
and Gaisano, H. Y.
(2001)
Pancreas
23,
125-133[CrossRef][Medline]
[Order article via Infotrieve]
30.
Graham, M. E.,
Washbourne, P.,
Wilson, M. C.,
and Burgoyne, R. D.
(2001)
J. Cell Sci.
114,
4397-4405[Medline]
[Order article via Infotrieve]
31.
Wei, S., Xu, T.,
Ashery, U.,
Kollewe, A.,
Matti, U.,
Antonin, W.,
Rettig, J.,
and Neher, E.
(2000)
EMBO J.
19,
1279-1289[CrossRef][Medline]
[Order article via Infotrieve]
32.
Gil, A.,
Gutierrez, L. M.,
Carrasco-Serrano, C.,
Alonso, M. T.,
Viniegra, S.,
and Criado, M.
(2002)
J. Biol. Chem.
277,
9904-9910 33.
Okamoto, C. T., Li, R.,
Zhang, Z.,
Jeng, Y. Y.,
and Chew, C. S.
(2002)
J. Controlled Release
78,
35-41[CrossRef][Medline]
[Order article via Infotrieve]
34.
Calhoun, B. C.,
Lapierre, L. A.,
Chew, C. S.,
and Goldenring, J. R.
(1998)
Am. J. Physiol.
275,
C163-C170[Medline]
[Order article via Infotrieve]
35.
Ammar, D. A.,
Zhou, R.,
Forte, J. G.,
and Yao, X.
(2002)
Am. J. Physiol.
282,
G23-G33
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