(Received for publication, August 1, 1994; and in revised form, October 20, 1994)
From the
The presence and intracellular distribution of vesicle-associated membrane protein-1 (VAMP-1) and VAMP-2 were investigated in the PC12 neuroendocrine cell line using isotype-specific polyclonal antibodies. VAMP-2 was detected in the total membrane fraction, while VAMP-1 was undetectable. Subcellular fractionation demonstrates that a substantial amount of the VAMP-2 (24-36%) is associated with dense core, catecholamine-containing granules (DCGs). This was confirmed by immunofluorescence microscopy. The L chain of tetanus neurotoxin, known to inhibit granule mediated secretion in permeabilized PC12 cells, as well as botulinum neurotoxins F and G, effectively cleaved DCG-associated VAMP-2. These data demonstrate that VAMP-2 is present on the secretory granules of PC12 cells.
Regulated exocytosis, a mechanism allowing cells to modulate
their response to the environment, is carried out by the fusion of a
variety of intracellular storage compartments with the plasma membrane
(reviewed in (1) ). Small synaptic vesicles (SSVs) ()accumulate and release neurotransmitters in presynaptic
nerve terminals. Similar organelles called synaptic-like microvesicles
(SLMVs) have also been identified in neuroendocrine cells. Dense core
granules (DCGs) represent the major vehicle for regulated secretion of
proteolytic enzymes, active peptides, small mediators, and hormones in
a variety of exocrine, endocrine, and neuronal cells. Other
intracellular membrane compartments found in various tissues accumulate
specific membrane proteins such as glucose or ion transporters, whose
level on the cell surface can be rapidly up-regulated following
suitable hormonal stimulation.
An essential role for VAMPs (also called synaptobrevins), a family of 18-20-kDa membrane proteins enriched in synaptic vesicles (2, 3) , in the fusion of storage organelles with the plasma membrane is strongly suggested by the action of some clostridial neurotoxins in ablating neurotransmitter release. Tetanus neurotoxin (TeNT) from Clostridium tetani, a powerful block to neurosecretion in the inhibitory interneurons in the spinal cord, and serotypes B, D, F, and G of botulinum neurotoxins (BoNT) from Clostridium botulinum, which inhibit secretion at the neuromuscular junction, are Zn-endopeptidases able to selectively cleave VAMPs (reviewed in (4) ). Because intoxication with TeNT does not prevent the docking of SSVs in squid giant axons(5) , VAMPs may be acting at a late step of neurotransmitter release involving their interaction(s) with syntaxins, and SNAP 25, proteins present in the presynaptic plasma membrane(6) .
VAMP function is not
restricted to SSV fusion. VAMPs bind in vitro to NSF
(NEM-sensitive factor) and to /
- and
-SNAPs (soluble NSF
attachment proteins), cytosolic factors thought to participate in many
intracellular membrane fusion steps along both the secretory and the
endocytic pathway(7, 8) . The concept of a general
role for VAMP-like proteins is also backed by their detection in
non-neural tissues (3, 9, 10) and in
intracellular compartments other than synaptic vesicles, such as
Glut4-positive vesicles in adipocytes (11) , endocytic
compartments(12) , and in zymogen granules in the exocrine
pancreas(13, 14) , as well as by the presence of yeast
homologues involved at various steps of the secretory pathway (reviewed
in (15) ). These data have been included in a hypothesis (the
SNARE hypothesis) which suggests that VAMP-like proteins are key
components of all intracellular membrane fusion events and may be
critical in specifying which vesicles can fuse with which
organelles(8) .
In this context we have investigated the distribution of VAMP-1 and VAMP-2 in a system where we can monitor the proteins present on the membrane of more than one regulated secretory organelle. At present, it is not known whether the machinery responsible for the regulated fusion of DCGs with the plasma membrane is the same as that responsible for SSV exocytosis. Although TeNT and BoNT can prevent evoked secretion from granules in permeabilized PC12(16) , and bovine chromaffin cells (17) , the failure of synthetic peptides resembling the cleavage site of VAMP-2 and VAMP-1 to block the activity of TeNT (18) and the lack of VAMP immunoreactivity on DCGs (3) have led to suggestions of an alternative mechanism and a different target for TeNT(19) . Thus it has been suggested that VAMP may neither play a general role in membrane fusion nor be a unique target for TeNT and BoNT. One possible reason for these apparently contradictory findings may be that the presence of two isoforms of the protein is complicating the interpretation of experimental results. In this paper, the presence and distribution of VAMP-1 and VAMP-2, the two known isoforms found in nervous tissue(20) , are re-examined in PC12 cells, a neuroendocrine cell line derived from a rat pheochromocytoma endowed with both SLMV and DCG-mediated regulated secretion(21, 22) . We show that VAMP-2 molecules, readily cleaved in vitro by clostridial neurotoxins are present in substantial amounts on the membranes of the DCGs.
Rabbit polyclonal antibodies, raised against sequences of VAMP-1 and VAMP-2 (methods) chosen because they are different from each other and from cellubrevin(12) , were tested on rat brain SSVs and on PC12 membranes by immunoblotting. In SSVs, anti-VAMP-2 recognizes a single polypeptide chain of 20 kDa, whereas anti-VAMP-1 detects a protein with a slightly higher molecular weight of 21.5 kDa (Fig. 1A). The different mobilities of VAMP-1 and VAMP-2 in SDS-PAGE has been already reported(31) . TeNT L-chain, known to cleave rat VAMP-2 at a much faster rate than rat VAMP-1(31) , completely abolishes the 20-kDa band identified by anti-VAMP-2 antibody but scarcely affects the 21.5 kDa detected by anti VAMP-1 (Fig. 1B). Together, these data strongly suggest that the two antibodies employed in this study are isotype-specific, although we cannot rule out the possibility that they may also recognize previously unidentified VAMPs of similar apparent molecular weight and toxin sensitivity to VAMP-1 and -2. When a total membrane fraction from PC12 cells was analyzed, a protein with the electrophoretic mobility of VAMP-2 was observed, whereas VAMP-1 was undetectable (Fig. 1A). Although immunoreactivity from uncharacterized VAMPs has already been reported in PC12 cells(22) , these data reveal that of the VAMP isoforms so far identified in nervous tissue, only VAMP-2 is present in detectable amounts in these cells using our antibodies.
Figure 1:
Expression of VAMP-2 in PC12 cells. A, rat brain SSVs (2.3 µg) or a total membrane fraction
from PC12 cells (100 µg) were separated on a 15% SDS-PAGE,
transferred to nitrocellulose, and tested with anti-VAMP-1 and
anti-VAMP-2. Immunoreactivity was detected by enhanced
chemiluminescence (ECL), followed by exposure to an autoradiographic
film. Migration of molecular weight standards is shown on the left of the figure. The antibodies used and the samples being tested
are indicated at the top of the panel. B, SSVs from
rat brain were treated with 200 nM TeNT L-chain at 37 °C
and the residual amounts of the 21.5 kDa protein identified by antibody
anti VAMP-1 () and of the 20-kDa protein identified by antibody
anti VAMP-2 (
) were determined by immunoblot, followed by
quantitation. Data are expressed as a percentage of the mock-treated
sample.
Previously, it has been reported that VAMPs in PC12s are present in the SLMVs(22) . To determine the distribution of VAMP-2 between the regulated secretory organelles of PC12 cells, double label indirect immunofluorescence microscopy was performed (Fig. 2). When compared with the distribution of synaptophysin (Fig. 2a), a well characterized marker for SLMVs, the distribution of VAMP-2 (Fig. 2b) is similar but not identical, being markedly more punctate. We therefore double-labeled with antibodies to SgII, a marker of the DCGs Fig. 2, c and d). Again while similar, the two markers do not completely overlap. In addition, we have compared the distribution of the transferrin receptor (Fig. 2e) with VAMP-2 (Fig. 2f), since cellubrevin, a VAMP-like molecule, has been reported to be in the recycling endosome. VAMP-2 and the transferrin receptor are largely separate by this assay. These experiments suggested that VAMP-2 may be present on the membranes of the DCGs as well as the SLMVs.
Figure 2: Immunofluorescent co-localization of VAMP-2 with SLMVs, DCGs, and TfR in PC12 cells. PC12 cells were fixed and processed for inmmunofluorescence as described (``Materials and Methods''). The primary antibodies used were as follows: synaptophysin(a) and VAMP-2 (b), SgII (c) and VAMP-2(d), TfR (e) and VAMP-2 (f).
In a
second set of experiments, a 1-16% linear Ficoll velocity
gradient (24) followed by a sucrose equilibrium gradient (32) were used to purify DCGs and the distribution of VAMP-2,
synaptophysin, the transferrin receptor, dopamine, and SgII were
determined. The velocity gradient resulted (Fig. 3A) in
a major peak of VAMP-2 in fractions 4 and 5, with a broad shoulder
sedimenting at a higher rate. The major peak of VAMP-2 co-sediments
with synaptophysin, and overlaps with the distribution of TfR,
suggesting that VAMP-2 found in the upper part of the gradient is
mostly associated with SLMVs but that some may be found in recycling
endosomes. This is consistent with earlier
findings(21, 22) . The shoulder of VAMP-2 in the lower
part of the gradient co-sediments with
[H]dopamine and SgII (Fig. 3, A and B). When DCGs from a velocity gradient were further
purified by equilibrium centrifugation, the majority of VAMP-2
co-purifies with DCG markers (Fig. 3, C and D). The enrichment of VAMP-2 in purified DCGs (Table 1),
compared with another protein (synaptophysin) which is predominantly
found in the SLMVs, suggests that VAMP-2 is actively sorted into
granules. From the ratios between the yield of VAMP-2 (9.5%) and of
[
H]dopamine (26%) and SgII (35%) in purified DCGs
we estimate that about one-third of the total intracellular VAMP-2
present in a PC12 cell post-nuclear supernatant is in secretory
granules. This is consistent with the percentage of VAMP-2 found
associated with DCGs (24-26%) in a velocity gradient (Fig. 3A).
Figure 3:
Distribution of VAMP-2 after subcellular
fractionation of PC12 cells. A post-nuclear supernatant from PC12 cells
labeled with [H]dopamine was centrifuged on a
1-16% Ficoll linear (velocity) gradient (A and B), and fractions were collected and numbered from the top (fraction 14 corresponds to a resuspended pellet). The amounts of
[
H]dopamine (
) and VAMP-2 (
) (A and C) and of synaptophysin (
), TfR (
), and
SgII (
) (B and D) were measured by liquid
scintillation ([
H]dopamine), or by quantitative
immunoblotting. The fractions (9, 10, 11, 12) corresponding to the
peak of DCGs were pooled and further centrifuged on a 0.5-2 M sucrose linear (equilibrium) gradient (C and D).
Data are expressed as percentage of the amount present in the PNS
supernatant.
DCG-associated VAMP-2 was further characterized by testing its sensitivity to TeNT L-chain, which cleaves rat VAMP-2(31) , and to BoNTs F and G, which cleave both VAMP isoforms in different positions (33, 27) . Fig. 4shows that DCG-associated VAMP-2 is completely cleaved by TeNT L-chain and by BoNTs F and G. Taken together, our data demonstrate the presence on DCGs in PC12 cells of VAMP molecules that by their electrophoretic mobility, immunological reactivity, and toxin sensitivity behave as VAMP-2. This observation provides an explanation for the inhibitory action of clostridial neurotoxins on granule secretion in PC12 and chromaffin cells(17, 18) and suggests a close similarity between the mechanism of regulated fusion with the plasma membrane of DCGs and SSVs, in spite of their differences in biogenesis and their secretory properties. Thus the association of VAMP-2 with synaptotagmin, and with syntaxin and SNAP 25 present on the plasma membrane, as well as with soluble NSF and the SNAPs, may be crucial steps in DCG secretion, as has been proposed for SSVs(8) . An involvement of NSF and SNAP 25 in DCG secretion is consistent with the inhibitory action of NEM and of BoNT A (which together with BoNT E cleaves SNAP 25(34) ), on the stimulated release of noradrenaline in chromaffin cells(35, 36) . However, with respect to the similarities between the dense granules of PC12 cells and chromaffin granules, we have found that in contrast to PC12 cells, a Western blot of chromaffin granules prepared from bovine adrenal medullae (37) reveals the presence of both VAMP-1 and VAMP-2 (not shown).
Figure 4:
Cleavage of VAMP-2 associated to DCGs by
TeNT L-chain, BoNT F and BoNT G. DCGs corresponding to fractions
14-17 from an equilibrium gradient as in Fig. 4(10
µg/ml) were incubated at 37 °C with 200 nM TeNTL-chain
() or with 50 nM BoNT F (
) and 50 nM BoNT
G (
) activated with dithiothreitol. At the indicated times,
proteins were precipitated with trichloroacetic acid, and the amount of
residual VAMP-2 was measured by quantitative immunoblotting using anti
VAMP-2-specific antibodies. Data are expressed as percentage of the
signal obtained from mock-treated DCGs.
Recent work has led to hypotheses emphasizing the possibility of VAMP-like proteins being involved in controlling the specificity of intracellular fusion(10) . The data presented here demonstrate the presence of the same VAMP isoform in different types of secretory vesicles within the same cell. This apparent contradiction may reflect the fact that both DCGs and SLMVs fuse with the same acceptor membrane, the plasma membrane.