Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK
* Author for correspondence (e-mail: o.dolly{at}ic.ac.uk)
Accepted 9 November 2001
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Summary |
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Key words: Secretion, Membrane fusion, SNAP-25, Syntaxin, Synaptobrevin, Clostridial neurotoxins
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Introduction |
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Data in this study support the hypothesis that Ca2+ stimulates
the formation of SNARE complexes but, through the use of an improved detection
method, shows that Ca2+-induced creation of a
Mr63 K species is minimal in comparison to larger
entities that are formed mainly from SNAP-25 and syntaxin. The latter were
found to cover a wide size-range and to be differentially sensitive to
proteolysis, suggesting that they may represent folding intermediates. BoNT/A
did not reduce the abundance, or inhibit Ca2+-induced formation, of
complexes in situ despite its destabilising effect on ternary SNARE complexes
in vitro (Hayashi et al.,
1994
); instead it altered the stoichiometry of SNAP-25 to syntaxin
in binary association.
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Materials and Methods |
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Chromaffin cell preparation and culture
Bovine adrenal chromaffin cells were isolated as described previously
(Lawrence et al., 1994), and
maintained as monolayer cultures in Dulbecco's modified Eagle medium
supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 2 mM sodium
pyruvate, 50 µg/ml gentamycin, 10 µM cytosine arabinofuranoside, 8 µM
fluorodeoxyuridine, 2.5 µg/ml fungizone, 25 IU penicillin, 25 µg/ml
streptomycin, 25 mM N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]
(HEPES) pH 7.4. The cells were used for experiments between 3 and 10 days
after isolation and where stated pre-intoxicated with BoNT/A, using a protocol
that facilitates its uptake (Lawrence et
al., 1996
). Briefly, the cells were exposed for 24 hours at
37°C to BoNT/A in 5 mM NaCl, 4.8 mM KCl, 2.2 mM CaCl2, 1.2 mM
MgSO4, 1.2 mM NaH2PO4, 20 mM Hepes pH 7.4,
5.6 mM glucose, 220 mM sucrose and 0.5% [w/v] bovine serum albumin. After
washing, the cells were returned to the standard culture medium and maintained
for 24-72 hours before further manipulations.
Stimulation and assay of catecholamine release
Immediately prior to experiments, which were all performed at
22°C, cells were rinsed with a Hepes-buffered saline solution (145 mM
NaCl, 4.8 mM KCl, 1.2 mM NaH2PO4, 20 mM Hepes, pH 7.4)
before permeabilisation by exposure to 20 µM digitonin in KGEP (139 mM
potassium glutamate, 5 mM EGTA, 20 mM PIPES, pH 6.5). Aliquots of
CaCl2 were added to the KGEP to produce the desired concentrations
of buffered-free Ca2+ and, where indicated in the figures, 2 mM ATP
and 4 mM MgCl2 were also included. In all experiments,
Ca2+ and MgATP (if included) were co-applied to the cells with
digitonin to avoid deterioration of the exocytotic response (known as
`run-down'), which occurs due to loss of soluble cytosolic proteins and
metabolites through the detergent-induced pores. After the stimulation period,
an aliquot was removed and the amount of catecholamines released from the
cells assayed, as described (Lawrence et
al., 1994
). Where used, trypsin (see figure legends for details)
was added directly to the digitonin-containing KGEP from a 10 mg/ml stock in
the same buffer.
Enrichment, SDS-PAGE and western blotting of membrane-bound
proteins
At the end of the experiments, a membrane-enriched fraction was prepared by
scraping the cells from the plates with a rubber policeman, followed by
trituration through a 26 G needle. Large debris was removed from the lysed
cells by centrifugation at 1000 g for 5 minutes and the
membranes in the supernatant were pelleted at 360,000 g for 20
minutes. The sediment was dissolved in 50 mM Tris.HCl, pH 5.8 containing 1%
SDS and subjected to PAGE using the NuPAGE system (Novex, San Diego),
according to the manufacturer's instructions. Relative molecular mass values
were calculated by reference to the migration of protein standards (Multimark,
Novex). Proteins were transferred from the gels onto PVDF membrane at 50 mV
for 12-16 hours while fully immersed in 25 mM Tris, 192 mM glycine containing
20% (v/v) methanol. Western blotting was performed using standard protocols
(Lawrence et al., 1996),
binding of primary antibodies being detected using horseradish
peroxidase-conjugated secondaries and visualised by the ECL system
(Amersham-Pharmacia, UK). Digital images were captured using a flat-bed
scanner and signal intensities were determined using NIH Image. The data
presented in each of the figures are representative of results obtained
consistently and on at least three separate occasions.
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Results |
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The amount of SNAP-25 in SDS-resistant complexes is reduced by
MgATP
Exposure of permeabilised chromaffin cells to MgATP had minimal effect on
basal level of exocytosis, but significantly enhanced
Ca2+-triggered release (2.2-times;
Fig. 1A). By contrast, cells
exposed to the nucleotide in the absence of Ca2+ showed a reduced
amount of SDS-resistant complexes compared with the level in MgATP-free
controls (Fig. 1B,C; in C, the
signal intensity of SNAP-25 in complexes in cells treated with MgATP is 80% of
the control level). The amount of complexes in cells exposed to both the
nucleotide and Ca2+ (Fig.
1B,C) was lower than in cells treated with the cation alone
(again, 80%); this is most likely due to SNARE complex disassembly mediated by
the ATPase, NSF (Hayashi et al.,
1995;
Söllner et
al., 1993
). Thus, the amount of complexes present within the cells
is not proportional to the extent of exocytosis elicited: secretion is
greatest from cells exposed to Ca2+ and MgATP together, but SNARE
association is most abundant in cells stimulated with the cation alone.
Syntaxin and synaptobrevin are also found in the SDS-resistant
complexes in situ
Second dimension gels of the samples used in
Fig. 1C were probed for
syntaxin (Fig. 2A) and
synaptobrevin (Fig. 2B). As
expected, both proteins were also found in SDS-resistant complexes and the
amounts recovered were increased (1.6-times for Sbr and 1.5-times for
syntaxin) by Ca2+, but decreased (to 20% [Sbr] and 70% [syntaxin]
of control amounts) by MgATP, as found for SNAP-25. The
Ca2+-induced increase in the level of synaptobrevin within
complexes was blocked by MgATP, which had minimal effect upon the increased
incorporation of syntaxin. However, synaptobrevin was only just detectable in
the complexes, despite extensive development of the blot such that the signal
for monomer (at Mr=18 K) was over-exposed. This suggests
that a large fraction of the SDS-resistant complexes contain SNAP-25 and
syntaxin but lack synaptobrevin.
|
BoNT/A alters the composition of SNARE complexes in situ
Pre-treatment of chromaffin cells with BoNT/A potently blocks
agonist-evoked release of catecholamines
(Foran et al., 1996;
Lawrence et al., 1996
). This
toxin cleaves SNAP-25 between Gln197-Arg198, thereby, removing nine amino
acids from its C-terminus (Niemann et al.,
1994
; Schiavo et al.,
2000
). This region is important for stability of the ternary SNARE
complex in vitro (Hayashi et al.,
1994
); complexes formed with BoNT/A-truncated SNAP-25 are 50% less
likely to become SDS-resistant than are those incorporating fulllength SNARE.
Thus, it was suspected that cell poisoning with BoNT/A would reduce the
amounts of SDS-resistant SNARE complexes in both resting and
Ca2+-stimulated chromaffin cells. Exposure of cells to the toxin,
using a protocol that results in cleavage of >95% of their SNAP-25, is
accompanied by a similar reduction in depolarisation-induced exocytosis
(Lawrence et al., 1996
).
Control and BoNT/A-poisoned chromaffin cells were permeabilised and exposed
for 45 minutes with or without Ca2+, before isolating a
membrane-enriched fraction for analysis by 2D SDS-PAGE and western blotting.
Unexpectedly, in the absence of Ca2+, the level of syntaxin
associated with SDS-resistant complexes was increased (5.7-times) following
BoNT/A (Fig. 3A). By contrast,
the amount of SNAP-25 associated with complexes was reduced (to 70% of the
control amount). As noted for toxin-free cells
(Fig. 2; Fig. 3A), there was virtually
no detectable synaptobrevin associated with these complexes. The same pattern
was observed for cells exposed to Ca2+; in the presence of the
cation, the amounts of SNAP-25 and syntaxin in complexes were increased in
BoNT/A-treated cells, as noted previously for control cells. Synaptobrevin
remained below the limit of detection.
|
Several SDS-resistant SNARE complexes exist
The observed separation of multiple SNARE species may indicate their
multi-merisation or, possibly, occurrence in several folded states. These
possibilities were evaluated by examining their susceptibility in situ to
degradation by trypsin. This was performed in tandem with the experiment
documented in Fig. 3A. The
cells were treated identically, except that trypsin was added 15 minutes after
permeabilisation and maintained for the remaining 30 minutes before preparing
membranes for biochemical analysis. In the absence of Ca2+,
virtually all the syntaxin was proteolysed but a significant amount of SNAP-25
remained (Fig. 3B). In
BoNT/A-treated cells, a lower level of SNAP-25 was recovered
(Fig. 3B), which is not
surprising as less SNAP-25 is associated with the complexes following
intoxication (Fig. 3A). The
notable appearance of trypsin-resistant synaptobrevin was unexpected
(Fig. 3B) in view of it being
absent from complexes in cells not exposed to trypsin
(Fig. 3A); perhaps the protease
blocked turnover of ternary complexes. Less synaptobrevin was found in
complexes from BoNT/A-treated cells (Fig.
3A). After Ca2+ stimulation, the amount of
trypsin-resistant SNAREs recovered increased in all cases
(Fig. 3B); this accords with
the fact that the cation stimulates complex formation (Figs
1,
2,
Fig. 3A). Likewise, the
induction by Ca2+ of protease-resistant SNARE complexes was not
blocked by BoNT/A (Fig. 3B;
SNAP-25 and synaptobrevin increased 2.4- and 7.8-times, respectively, compared
with 2.1- and 1.1-times in control. The fold-induction of trypsin-resistant
syntaxin is not quantifiable, but clearly it was not inhibited), as expected,
because this toxin does not inhibit complex formation in trypsin-free cells
(Fig. 3A). Notably, different
sized complexes offered varying degrees of protection to each of the SNAREs.
It was the highest mobility complexes that best protected synaptobrevin
(Mr=100 K), intermediate complexes for syntaxin
(Mr=100-155 K) and SNAP-25 was protected over a broad
range (Mr=55-155 K). This suggests that the relative
accessibility of each SNARE to trypsin is not the same for complexes of
different molecular mass which, in turn, may imply that these represent
distinct folded states rather than multimers of SNARE complexes (see
Discussion).
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Discussion |
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Binary SNAP-25:syntaxin complexes predominate over ternary SNARE
forms
Another notable finding from our investigation is that the majority of
SDS-resistant complexes in chromaffin cells lacks synaptobrevin. There have
been prior reports of association of SNAP-25 with the cytosolic domain of
syntaxin, but this was deemed not SDS-resistant
(Fasshauer et al., 1997;
Hayashi et al., 1994
),
although they could gain added stability from the presence of the
transmembrane anchor of syntaxin (Poirier
et al., 1998
). Thus, the composition of these complexes might be
important for fast secretory responses, as BoNT/A slows down the rate of the
exocytotic burst and the C-terminus of SNAP-25 has been implicated in a late
step of exocytosis (Xu et al.,
1999
). Curiously, trypsin increased the amount of synaptobrevin
recovered in the SDS-resistant complexes, possibly due to a dynamic
association of this protein that is upset in trypsin-treated cells.
Different sized complexes could be distinct folded states that may
facilitate fast exocytosis
The spread of SDS-resistant complexes over a wide size-range suggests that
they occur in a variety of states (Hayashi
et al., 1994; Otto et al.,
1997
; Brunger,
2001
). This would explain the non-overlapping trypsin-sensitivity
profiles for the individual components. It is tempting to speculate that
complexes of varied mobility on SDS-PAGE represent distinct folded
intermediates. Fully-folded complexes would be more compact and, hence, likely
to migrate faster on SDS-PAGE and be more resistant to protease attack. An
alternative hypothesis, that the less mobile bands could be oligomers of SNARE
heterotrimers (Tokumaru et al.,
2001
), is difficult to reconcile with the greater protease
resistance observed for smaller complexes. Site-directed mutagenesis of
residues in one of the four helices of the SNARE bundle creates complexes
exhibiting different degrees of thermal stability
(Chen et al., 1999
), indicating
that stable, SDS-resistant abnormally folded states can be created in vitro.
Likewise, stable folded complexes can be formed between SNAP-25, syntaxin and
C-terminally-truncated forms of synaptobrevin; these appear to be folded
completely N-terminus to the truncation site, but are unstructured at the
C-terminal end (Margittai et al.,
2001
).
Ca2+ triggered the formation of complexes across the entire size
range, which could be achieved by stimulation of an early stage of a SNARE
association and folding reactions that can then proceed in its absence.
Alternatively, there could be various intermediate steps that are also
accelerated by Ca2+; indeed, several kinetically distinct phases of
secretion can be distinguished by their Ca2+-sensitivities
(Bittner and Holz, 1992).
Moreover, in an attempt to reconcile biochemical and electrophysiological
data, it has been proposed that the fastest phase of exocytosis may involve
maturation of a partially folded SNARE complex intermediate. Accordingly, an
antibody that binds to SNAP-25 in partially, but not fully, folded complexes
delays the exocytotic burst, whereas another that recognises only free (i.e.
non-complexed) SNAP-25 retarded only the slower phases of secretion
(Xu et al., 1999
). Low
[Ca2+]I (<1 µM) may stimulate the early stages of
SNARE association and higher levels (>3 µM) could trigger exocytosis by
inducing maturation of partially folded complexes
(Voets, 2000
). Therefore, it
is notable that the putative Ca2+-sensor, synaptotagmin, exhibits a
dual Ca2+ affinity for promotion of its binding to syntaxin, with
EC50=0.7 µM and 180 µM, respectively
(Chapman et al., 1995
). In
permeabilised neuroendocrine cells, secretion is triggered by >1 µM
[Ca2+]i and is optimal at
20 µM [although higher
levels accelerate the fastest phase
(Bittner and Holz, 1992
);
therefore, high [Ca2+]i is not essential for complex
maturation, but only for acceleration of the final steps
(Xu et al., 1998
). This is
almost exactly equivalent to the Ca2+-dependence for stimulation of
conformational changes in synaptotagmin
(Davletov and Sudhof, 1994
) and
for inducing it to bind acidic phospholipids
(Davletov and Sudhof, 1993
).
Thus, the latter may be critical interactions that, by bringing complexes in
the process of maturation into close proximity with the cell membrane
(Davis et al., 1999
), drive the
fusion reaction. Accumulation of partially folded SNARE complex intermediates
could be an important mechanism for synaptic plasticity; indeed, enhancement
of the content of SNAP-25-syntaxin complexes in synaptosomes caused an
increase in the ready releasable pool of neurotransmitter
(Lonart and Sudhof, 2000
).
BoNT/A inhibits secretion by perturbing binary complexes that may be
vital for fast exocytotic response
In chromaffin cells poisoned with BoNT/A, the composition and state
(trypsin sensitivity profile) of the SNARE complexes were perturbed. These
findings support the proposal (Xu et al.,
1999) that the partially folded complexes are important for the
fastest phase of secretion, as BoNT/A slows down the exocytotic burst
(Xu et al., 1998
). The toxin
blocks secretion in response to depolarising stimuli almost completely; this
is in accordance with the hypothesis that such responses require a pool of
partially folded SNARE complexes to drive secretion rapidly, before the
[Ca2+]i signal fades. By contrast, following
permeabilisation, a lower but persistent increase of
[Ca2+]i does elicit secretion from BoNT/A-poisoned
cells. This would suggest that BoNT/A does not prevent the slower formation of
complexes: exactly as observed in situ in the cells. Hence, BoNT/A blocks the
accelerated phase of exocytosis stimulated by high
[Ca2+]i, but not slow release mediated by lower
[Ca2+]i (Xu et al.,
1998
). This proposal does not support a prior hypothesis that
BoNT/A reduces the Ca2+ sensitivity of exocytosis due to a
reduction of the Ca2+-affinity for the promotion of synaptotagmin
binding to SNAP-25 (Gerona et al.,
2000
; Schiavo et al.,
1997
). It is possible that the latter interaction could be
involved in the most rapid exocytotic burst elicited by high
[Ca2+]i (Xu et al.,
1998
). However, the interaction is not likely to mediate the much
slower Ca2+-triggered exocytosis that ensues from permeabilised
neuroendocrine cells; the latter response requires less Ca2+ and
the EC50 is not (or only modestly, at most) altered by poisoning of
the cells with BoNT/A (Gerona et al.,
2000
; Lawrence et al.,
1996
).
In summary, high-resolution 2D SDS-PAGE has demonstrated that the relationship between Ca2+-induced SNARE complex formation and triggered exocytosis is more complicated than previously reported. Specifically, large SNAP-25:syntaxin complexes are present before secretion has occurred and more are created during the reaction; ternary complexes and smaller forms are relatively rare. However, this may be due to the rapid turnover of the latter, which can be blocked by trypsin. Our interpretation of the data is that Ca2+ stimulates an early step of syntaxin association with SNAP-25 and that the resultant components go through a number of folded states before fusion is triggered by the binding of synaptobrevin and formation of the SDS and trypsin-resistant ternary complex; it is envisaged that Ca2+ would regulate this series of reactions at several points.
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Acknowledgments |
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Footnotes |
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