©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vesicle-associated Membrane Protein (VAMP)/Synaptobrevin-2 Is Associated with Dense Core Secretory Granules in PC12 Neuroendocrine Cells (*)

(Received for publication, August 1, 1994; and in revised form, October 20, 1994)

Emanuele Papini (1) Ornella Rossetto (2) Daniel F. Cutler (1)(§)

From the  (1)MRC Laboratory for Molecular Cell Biology and the Department of Biology, University College London, WC1E 6BT London, United Kingdom and the (2)Dipartimento di Scienze Biomediche, University of Padova, Via Trieste 75, 35100 Padova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 alpha/beta- 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.


MATERIALS AND METHODS

Antibodies

Peptides comprising residues 1-33 of rat VAMP-1 and VAMP-2 were synthesized by Dr. O. Marin of Centro di Ricerco Interdipartimentale per le Biotecnologie Innovative, Padua, using Applied Biosystems FastMoc chemistry. The two peptides were purified by HPLC using a reverse phase Ultra-Sphere preparative column (Beckman), conjugated to keyhole limpet hemocyanin (Pierce), and used to immunize rabbits as described(23) . Antibodies were affinity purified using the peptides bound to an AH-Sepharose 4B matrix (Pharmacia). Rabbit polyclonal antibodies against rat secretogranin II (SgII) and against synaptophysin have been prepared as described(24) . Mouse monoclonal antibodies against secretogranin II were obtained from a panel of monoclonals raised against PC12 granules. Antibodies against the TfR (H68.4) were a kind gift from from Dr. I. Trowbridge. Mouse monoclonal antibodies against Synaptophysin were from Boehringher (Mannheim, Germany).

Toxins

The L-chain of tetanus neurotoxin was purified from C. tetani acellular filtrate as described(25) . Botulinum neurotoxin F was a kind gift from Dr. Clifford C. Shone and purified by HPLC using IMAC chromatography as described(26) . Botulinum toxin G was a kind gift of Dr. Eric A. Johnson and was purified as described(27) .

Cell Growth and Labeling

PC12 cells were grown, and DCG were labeled by incubating the cells for 2 h with 0.5 µCi/ml [^3H]dopamine (Amersham International, Amersham, United Kingdom) in Dulbecco's modified Eagle's medium as described(24) .

Immunofluorescence

Immunofluorescence was carried out essentially as described(28) . Results were analyzed with a Bio-Rad MRC-1000 laser scanning confocal unit in conjugation with a Nikon Optophot microscope.

Subcellular Fractionation

All operations were carried out at 4 °C. Cells were washed three times with phosphate-buffered saline, once with 0.32 M sucrose, 10 mM NaHepes, pH 7.4 (HB), scraped into HB supplemented with a mixture of protease inhibitors (leupeptin, 1 µg/ml; pepstatin A, 1 µg/ml; antipain, 1 µg/ml; aprotinin, 1 µg/ml; TPCK, 50 µM; TLCK, 50 µM; phenylmethylsulfonyl fluoride, 100 µM) and homogenized by 15 passages through a ball-bearing homogenizer, with a 0.012-mm clearance (EMBL workshop). The cell homogenate was spun at 3500 rpm for 15 min, and 3 ml of the supernatant (PNS), equivalent to three 9-cm cell dishes, were layered on 10 ml of a 1-16% Ficoll linear gradient made in HB plus protease inhibitors. The gradients were centrifuged for 45 min at 35,000 rpm in a SW 40 Ti rotor (Beckman), and fractionated in 1-ml fractions from the top. The pellet present in the bottom of the tube was resuspended in 1 ml of HB plus protease inhibitors. Samples of each fraction were kept for immunoblot analysis, and the granule distribution was determined by measuring [^3H]dopamine radioactivity with a liquid scintillation counter (Packard). Four ml from the DCG peak were diluted with 4 ml of HB and layered on 30 ml of an 0.5-2 M sucrose gradient in 10 mM KHepes, pH 7.4, plus protease inhibitors. Gradients were centrifuged to equilibrium for 5 h at 25,000 rpm with a SW 25 Ti rotor (Beckman). Two-ml fractions were collected from the top of the tube and analyzed or frozen by liquid nitrogen and stored at -70 °C. The yelds of [^3H]dopamine and of SgII in DCGs fractions were corrected for leakage, estimated by measuring the percentage of these markers found in the loading regions of the two gradients. Protein concentration was determined by BCA (Pierce) using bovine serum albumin as standard. SSVs from rat brain cortex were prepared according to (29) . Chromaffin granules from the bovine adrenal medulla were prepared as described in (37) .

SDS-PAGE and Immunoblotting

Proteins were run on 5%-15% SDS-polyacrylamide slab gels(30) , then transferred to nitrocellulose at 1 A for 80 min at 4 °C. After saturation for 1 h with 5% milk powder, 0.2% Tween 20 in phosphate-buffered saline (blotting buffer, BB), nitrocellulose sheets were incubated with primary antibodies for 1 h, washed with BB, and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies in BB for 30 min. After washing with BB, antigens were detected by the ECL reaction (Amersham) and exposed to autoradiographic films. For quantitative immunodetection the signal was measured with a GS-250 Molecular Imager (Bio-Rad) after exposure to a chemiluminescence-sensitive screen for 30 min. Linearity of the relation between signal and antigen amount was checked by serial dilution of appropriate reference samples (purified rat brain synaptic vesicles or a total membrane fraction from PC12 cells).

Toxin Treatment

Rat brain SSVs (2.3 µg/ml) or purified DCGs (10 µg/ml) were incubated for different times at 37 °C in 0.5 M sucrose, 10 mM NaHepes, pH 7.4, containing leupeptin (1 µg/ml), pepstatin A (1 µg/ml), antipain (1 µg/ml), and phenylmethylsulfonyl fluoride (100 µM), in the presence of TeNTL chain (200 nM), BoNT F (50 nM), BoNT G (50 nM), or with an equivalent amount of bovine serum albumin. Botulinal toxins were treated with 10 mM dithiothreitol for 30 min at 37 °C before use. Treated samples were recovered by trichloroacetic acid precipitation and separated by SDS-PAGE for immunoblot quantitation.


RESULTS AND DISCUSSION

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 (bullet) and of the 20-kDa protein identified by antibody anti VAMP-2 (circle) 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 [^3H]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 [^3H]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 [^3H]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 [^3H]dopamine (circle) and VAMP-2 (bullet) (A and C) and of synaptophysin (box), TfR (), and SgII (times) (B and D) were measured by liquid scintillation ([^3H]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 (circle) or with 50 nM BoNT F (bullet) and 50 nM BoNT G (times) 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.


FOOTNOTES

*
This work was supported by a Wellcome Trust grant (to D. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: MRC Laboratory for Molecular Cell Biology, University College London, Gower St., WC1E 6BT London, UK. Tel.: 71-380-7808; Fax: 71-380-7805.

(^1)
The abbreviations used are: SSVs, small synaptic vesicles; VAMP, vesicle-associated membrane protein, also called synaptobrevin; SLMVs, synaptic-like micro vesicles; DCGs, dense core granules; TeNT, tetanus neurotoxin; BoNT, botulinum neurotoxin; NEM, N-ethylmaleimide; SgII, secretogranin II; TfR, transferrin receptor; BB, Blotting Buffer; PAGE, polyacrylamide gel electrophoresis; NSF, NEM-sensitive factor; SNAP, soluble NEM-sensitive attachment protein; PNS, post-nuclear supernatant; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

The toxins used in this work were generously provided by Giampietro Schiavo of the University of Padova. H68.4 was a kind gift of I. S. Trowbridge of the Salk Institute. We thank C. R. Hopkins and C. Montecucco for helpful comments.


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