SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics.

Abstract

Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q-->E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.

Full text

Page 1

INTRODUCTION
Intracellular membrane fusion involves a highly conserved
machinery that functions in essentially all vesicular traffic steps
and is conserved throughout evolution (Bennett and Scheller,
1993; Ferro-Novick and Jahn, 1994; Hanson et al., 1997;
Rothman, 1994). The core of this machinery is formed by a
complex of the SNARE (soluble NSF (N-ethylmaleimide-
sensitive factor) attachment protein receptor) proteins. In
regulated exocytosis in neurons, and certain other cell types,
this SNARE complex is formed from syntaxin 1, SNAP-25 and
VAMP (synaptobrevin) (Sollner et al., 1993b). These proteins
can form a highly stable complex in vitro (Hayashi et al., 1994;
Hayashi et al., 1995) due to the formation of a four-helix
bundle with one helix donated each by syntaxin 1 and by
VAMP and two helices donated by SNAP-25 (Poirier et al.,
1998; Sutton et al., 1998). So far it has been shown that a
SNARE complex of similar organisation consisting of related
members of the SNARE family also functions in constitutive
exocytosis in yeast (Katz et al., 1998; Rossi et al., 1997) and
in endosome-endosome fusion (Antonin et al., 2000).
Complexes of this type are likely, however, to have general
functions in membrane fusion as SNARE homologues appear
to be essential for all traffic steps (Hay and Scheller, 1997;
McNew et al., 2000a; Pelham, 1999; Rothman, 1994). It has
been suggested that a stable SNARE complex, similar to that
seen in the crystal structure, represents a conserved
intermediate in membrane fusion reactions (Hanson et al.,
1997; Sutton et al., 1998; Weber et al., 1998). While others
have suggested that it is not involved in fusion itself but in a
preceeding step (Tahara et al., 1998; Ungermann et al., 1998),
accumulating evidence is more consistent with the SNAREs
alone or with other proteins playing a major role in bilayer
fusion (Chen et al., 1999; Grote et al., 2000; Hanson et al.,
1997; McNew et al., 2000b; Weber et al., 2000).
A current model for SNARE action in membrane fusion
(Bock and Scheller, 1999; Brunger, 2000; Chen et al., 1999;
Xu et al., 1998; Xu et al., 1999b) suggests that the interaction
between vesicular and target SNAREs leads to the formation
of an initial ‘loose complex’ (sensitive to the action of
Clostridial neurotoxins) that brings the two opposing bilayers
together. Membrane fusion would then be triggered by the
progressive assembly (Fiebig et al., 1999) or zipping up of the
SNAREs into a neurotoxin-insensitive ‘tight’ complex believed
to be similar to the four-helix bundle visualised in the crystal
structure (Sutton et al., 1998). While this is a plausible
explanation for membrane fusion it leaves unexplained the
observed biophysical aspects of fusion detected during
exocytosis. In particular, an accumulation of data supports the
idea that the initial fusion occurs through the formation of a
fusion pore (Albillos et al., 1997; Ales et al., 1999; Alvarez de
Toledo et al., 1993; Breckenridge and Almers, 1987; Fernandez
et al., 1984; Lindau and Almers, 1995). This is a reversible
structure that can return to the prefusion state even after a phase
of pore expansion (Ales et al., 1999; Rosenboom and Lindau,
1994) that may be under physiological regulation (Burgoyne
and Alvaraz de Toledo, 2000; Fernandez-Chacon and Alvarez
de Toledo, 1995; Fisher et al., 2001; Hartmann and Lindau,
1995; Scepek et al., 1998). In synaptic vesicle exocytosis, this
reversibility could enable a kiss-and-run type fusion (Fesce et
4397
Considerable data support the idea that intracellular
membrane fusion involves a conserved machinery
containing the SNARE proteins. SNAREs assembled in
vitro form a stable 4-helix bundle and it has been suggested
that formation of this complex provides the driving force
for bilayer fusion. We have tested this possibility in assays
of exocytosis in cells expressing a botulinum neurotoxin E
(BoNT/E)-resistant mutant of SNAP-25 in which additional
disruptive mutations have been introduced. Single or
double mutations of glutamine to glutamate or to arginine
in the central zero layer residues of SNAP-25 did not
impair the extent, time course or Ca2+-dependency of
exocytosis in PC12 cells. Using adrenal chromaffin cells, we
found that exocytosis could be reconstituted in cells
transfected to express BoNT/E. A double Q→E mutation
did not prevent reconstitution and the kinetics of single
granule release events were indistinguishable from control
cells. This shows a high level of tolerance of changes in the
zero layer indicating that the conservation of these residues
is not due to an essential requirement in vesicle docking or
fusion and suggests that formation of a fully stable SNARE
complex may not be required to drive membrane fusion.
Key words: Exocytosis, SNARES, Membrane fusion, Chromaffin
cells, PC12 cells
SUMMARY
SNAP-25 with mutations in the zero layer supports
normal membrane fusion kinetics
Margaret E. Graham1, Philip Washbourne2,*, Michael C. Wilson2and Robert D. Burgoyne1,‡
1The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK
2Department of Neurosciences, University of New Mexico, Albuquerque, New Mexico, USA
*Present address: Center for Neuroscience, University of California at Davis, Davis, California, USA
‡Author for correspondence (e-mail: burgoyne@liverpool.ac.uk)
Accepted 13 September 2001
Journal of Cell Science 114, 4397-4405 (2001) © The Company of Biologists Ltd
RESEARCH ARTICLE

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4398
al., 1994; Stevens and Williams, 2000) to occur with release of
vesicle content through the pore on a sub-millisecond time-
scale. The high stability of the SNARE structure formed in
vitro from recombinant proteins or present in detergent extracts
and the fact that it can only be disassembled by the action of
the chaperones α-SNAP and NSF (Sollner et al., 1993a), is
difficult to reconcile with a rapidly reversible fusion process
(Burgoyne and Alvaraz de Toledo, 2000). It is not clear,
therefore, if the stable structure seen in vitro is the structure
that drives fusion or if it is a post-fusion ground state of the
complex. In the case of influenza haemaglutinin-mediated
fusion it appears that the stable low-pH structure originally
thought to represent the fusogenic state of the protein may
instead be an inactive conformation existing after the
completion of fusion (Lentz et al., 2000).
The neuronal SNARE complex is stabilised by hydrophobic
interactions between the four helices in a series of layers (–1
to –7 and +1 to +8) (Sutton et al., 1998). Mutations in these
layers disrupt SNARE-SNARE interactions, compromise
SNARE complex stability and are functionally disruptive
(Chen et al., 1999; Fasshauer et al., 1998; Washbourne et al.,
1999). At the 0 layer are ionic interactions between three
conserved glutamines, two from SNAP-25 and one from
syntaxin, and a conserved arginine from VAMP. These residues
are absolutely conserved thoughout evolution leading to a
suggested classification of the SNAREs as the ‘Q’ and ‘R’
SNAREs (Fasshauer et al., 1998). The conserved Q and R
residues were suggested to be important either in stabilising
the SNARE complex or for its disassembly by α-SNAP and
NSF (Sutton et al., 1998). Mutagenesis of these residues in
yeast in the exocytotic SNAREs has shown that they are
biologically relevant and confirmed their crucial importance
for growth and secretion (Katz and Brennwald, 2000; Ossig et
al., 2000). These studies in yeast do not, however, provide
information on when and where these residues are of
importance. Mutation of conserved Q residues in SNAP-25 or
the yeast homologue Sec9 reduces the affinity of SNARE-
SNARE interactions and reduces the thermal stability of the
SNARE complex assembled in vitro (Chen et al., 1999; Katz
and Brennwald, 2000; Ossig et al., 2000; Wei et al., 2000).
Surprisingly mutation of one or other of the conserved
glutamines of SNAP-25 did not modify the ability of individual
helices to reconstitute exocytosis in populations of PC12 cells
in which endogenous SNAP-25 was cleaved with botulinum
neurotoxin E (BoNT/E) (Chen et al., 1999; Scales et al., 2000).
This toxin cleaves SNAP-25 within the C-terminal helix to
release a 22-residue fragment and inactivate the endogenous
protein (Binz et al., 1994; Schiavo et al., 1993). It has been
pointed out, however, that the assay used with PC12 cells
would not reveal subtle effects of these mutations such as on
the kinetics of membrane fusion (Katz and Brennwald, 2000;
Ossig et al., 2000). In another study, a SNAP-25 (Q174L)
mutant was expressed in adrenal chromaffin cells (Wei et al.,
2000). This mutant reduced the extent but not the initial overall
kinetics of exocytosis from the releasable pool. The
interpretation of the data from this study is complicated as the
cells still retained their endogenous wild-type SNAP-25 intact
and so this could contribute to the apparently normal
exocytosis kinetics.
In this study we have examined the importance of the
conserved Q residues in SNAP-25 in cells that express
BoNT/E-resistant constructs, in which endogenous SNAP-25
has been inactivated by BoNT/E treatment. This has allowed
us to examine, in the absence of a wild-type background, the
consequences of mutations in the Q residues not only on the
overall level of exocytosis but also on the kinetics of single
granule release events measured
amperometry (Wightman et al., 1991). We show that disruptive
mutations in the 0 layer that impair SNARE-SNARE
interactions and SNARE complex stability do not prevent
exocytosis, and do not affect the kinetics of single release
events. These data show that SNARE complex function in
membrane fusion is tolerant of significant modifications in the
0 layer.
using carbon-fibre
MATERIALS AND METHODS
Plasmids
The plasmid pEGFP-C1 was obtained from Clontech (Basingstoke,
UK). The plasmid encoding BoNT/E light chain as an EGFP fusion
construct (pEGFP-BoNT/E) was described previously (Graham et al.,
2000). The BoNT/E-resistant construct of SNAP-25 (Em2) was
described previously (Washbourne et al., 1999). Specific mutations
were introduced into Em2 using site directed mutagenesis with a
Quickchange site-directed mutagenesis kit (Stratagene Europe,
Amsterdam, The Netherlands). The primers used were as follows with
the changed codons underlined:
5′-GTTATGTTGGATGAGGAAGGCGAACAACTG-3′
sense); 5′-CAGTTGTTCGCCTTCCTCATCCAACATAAC-3′ (Q53E
antisense); 5′-GAGATTGACACCGAGAATCGCCAGATTGAC-3′
(Q174E sense); 5′-GTCAATCTGGCGATTCTCGGTGTCAATCTC-3′
(Q174E antisense); 5′-TTATGTTGGATGAGCGAGGCGAACAAC-
TG-3′ (Q53R sense); 5′-CAGTTGTTCGCCTCGCTCATCCAACA-
TAAC-3′
(Q53R antisense);
CGCCAGATTGAC-3′ (Q174R sense); 5′-GTCAATCTGGCGAT-
TCCGGGTGTCAATCTC-3′ (Q174R antisense). The SNAP-25
delta35-44 mutant was created by the megaprimer PCR method using
the primer 5′-CGTCGCATGCTGCTGCAAAGGACTTTGGTTATG-
3′, which loops out bases 113-132 of the SNAP-25 coding sequence
and introduces a silent point mutation (G112A), eliminating the PstI
site for screening purposes. This primer was used in conjunction with
the primers SBf and SBr described previously (Washbourne et al.,
1999) and the product inserted into pCDNA/HA1. All constructs were
checked by automated sequencing.
(Q53E
5′-GAGATTGACACCCGGAAT-
PC12 cell transfection and assay of growth hormone
release
The assay of release from transfected PC12 cells used a modification
of the growth hormone (GH) release assay (Wick et al., 1993). PC12
cells were maintained in culture in collagen-coated 24 well trays and
transiently co-transfected with 4 µg growth hormone plasmid pXGH5,
along with 2 µg of each test plasmid (control vector, Em2 or Em2
containing additional mutations) as previously described (Graham et
al., 1997) using lipofectamine (Gibco BRL). GH secretion was used
as a marker for transfected cells. Cells were permeabilized 72 hours
after transfection with 20 µM digitonin, incubated with or without
bacterially expressed 14 nM BoNT/E-His6 light chain (Glenn and
Burgoyne, 1996) for 40 minutes and then challenged by the addition
of buffer containing no Ca2+or with 10 µM Ca2+. Buffer samples and
the cells were processed and assayed for GH levels using an enzyme-
linked immunosorbent kit according to the manufacturer’s instructions
(Boehringer-Mannheim, Indianapolis, IN). In all experiments, the
amount of GH release is expressed as a percentage of total cellular
content of GH. To verify expression levels of the transfected Em2
constructs, cells were solubilised, separated by SDS polyacrylamide
JOURNAL OF CELL SCIENCE 114 (24)

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4399SNARE complex and exocytosis
gel electrophoresis and samples probed by immunoblotting with
mouse monoclonal anti-HA at 1:1000 dilution.
Chromaffin cell culture and transfection
Freshly isolated bovine adrenal chromaffin cells (Burgoyne, 1992)
were plated on non-tissue culture-treated 10 cm Petri dishes at a
density of 1×106/ml. The following day non-attached cells were
pelleted by centrifugation and re-suspended in growth medium at a
density of 1.5×107/ml. The cells (1 ml) were mixed with 22.5 µg of
pEGFP, 7.5 µg of pEGFP-BoNT/E and 30 µg of the Em2 construct
to be tested. 1 ml of cells and plasmids were electroporated at 250 V
and 975 µF for one pulse, using a Bio-Rad Gene Pulser II (Bio-Rad,
CA) and 4 mm cuvettes. Cells were then diluted as rapidly as possible
to 1×106/ml with fresh growth media and 1×106cells grown on 35
mm Petri dishes in a final volume of 3 ml of growth medium for a
further 3-5 days.
Carbon-fibre amperometry
The cells were washed three times with Krebs-Ringer buffer (145 mM
NaCl, 5 mM KCl, 1.3 mM MgCl2,1.2 mM NaH2PO4, 10 mM glucose
and 20 mM Hepes, pH 7.4), incubated in bath buffer (139 mM
potassium glutamate, 20 mM Pipes, 0.2 mM EGTA, 2 mM ATP, 2
mM MgCl2, pH 6.5) and viewed using a Nikon TE300 inverted
microscope. Transfected cells were identified as those expressing
EGFP. A pre-cut 5 µm diameter carbon fibre electrode (NPI,
Germany), was placed in direct contact with the surface of a cell. For
stimulation of the cells, a digitonin-permeabilisation protocol
(Jankowski et al., 1992) was used. A micropipette was filled with cell
permeabilisation buffer (139 mM potassium glutamate, 20 mM Pipes,
5 mM EGTA, 2 mM ATP, 2 mM MgCl2, 20 µM digitonin and 10 µM
free Ca2+, pH 6.5) and positioned on the opposite side of the cell from
the carbon fibre and buffer pressure ejected on to the cell for 20
seconds. A holding voltage of +700 mV was applied between the
carbon fibre tip and the Ag/AgCl reference electrode in the bath.
Amperometric responses were monitored with a VA-10 amplifier (NPI
Electronic, Tamm, Germany), collected at 4 kHz, digitised with a
Digidata 1200B acquisition system and monitored online with the
AxoScope 7.0 program (Axon Instruments, CA). Data were
subsequently analysed using an automated peak detection and analysis
protocol within the technical graphics program Origin (Microcal,
MA). Spikes were only analysed in detail if they had a base width
greater than 6 milliseconds and an amplitude greater than 40 pA. This
amplitude was chosen so that analyses were confined to spikes arising
immediately beneath the carbon fibre and to limit effects on the data
of diffusion times from exocytotic sites distant from the carbon fibre.
RESULTS
Reconstitution of exocytosis in PC12 cells by
BoNT/E-resistant SNAP-25 and effect of 0 layer
mutations
We have previously developed a mutated form of SNAP-25b
(Em2) that is resistant to cleavage by BoNT/E and can
reconstitute exocytosis following transfection in BoNT/E-
treated PC12 cells (Washbourne et al., 1999; Washbourne et
al., 2001). This construct allowed the testing, in the present
study, of the effect of additional mutations in SNAP-25 on
exocytosis. The Em2 mutant (Washbourne et al., 1999)
contains three mutations including a change of the conserved
isoleucine in the +2 layer to glutamate (Fig. 1). The mutations
in Em2 do not affect its ability to become incorporated into a
SNARE complex but its interactions with other SNAREs are
compromised as it cannot form a binary complex with VAMP
suggesting that this mutation could already impair SNARE
complex formation and stability in vivo (Washbourne et al.,
Fig. 1. The organisation of SNARE
helices and mutated residues in
SNAP-25B. (A) Domain structure
of SNAP-25B. (B) Alignment of the
SNAP-25B helices that participate
in the core complex. Residues are
indicated that are mutated in the
BoNT/E-resistant construct, Em,
and in the Q/E mutants. (C) The
structure of the core SNARE
complex showing the helices from
VAMP (red), syntaxin 1 (blue) and
SNAP-25 (green).

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4400
1999). The intention in this study was to introduce further
disruptive mutations in the 0 layer residues and to examine the
consequences for the extent and kinetics of exocytotic fusion
events supported by the constructs. A major advantage of this
assay is that it is not confounded by potentially disruptive
effects of introduced inhibitory constructs. In addition,
reconstitution will only occur if constructs are correctly post-
translationally modified and targeted.
As shown in Fig. 2, treatment of control-transfected PC12
cells with BoNT/E light chain after permeabilisation
essentially abolished Ca2+-induced exocytosis (in cells
transfected with pcDNA3) measured as growth hormone (GH)
release from co-transfected cells. Prior transfection with the
Em2 construct completely reconstituted exocytosis as
described previously (Washbourne et al., 1999). It has been
shown that the C-terminal helix of SNAP-25 alone can, at least
partially, restore exocytosis in BoNT/E-treated PC12 cells
(Chen et al., 1999), although co-addition with the N-terminal
helix is more effective (Scales et al., 2000). To determine
whether the reconstitution by full-length SNAP-25 in our
studies required a functional N-terminal helix or if the C-
terminal helix of the protein was sufficient, a disruptive
deletion (∆35-44) was introduced within the N-terminal helix
of Em2 removing residues from layers –3 to –5. The ∆35-44
construct was expressed to similar levels as Em2 (Fig. 2A) but
gave almost no reconstitution (Fig. 2B). This shows that the
reconstitution of exocytosis in our assay involved contributions
from both the N- and C-terminal helices of the expressed full-
length protein and would, therefore, allow testing of the effect
of mutations in either helix. The result with the ∆35-44
construct also rules out a trivial explanation for the
maintenance of exocytosis by the toxin-resistant mutant due to
binding and sequestration of the BoNT/E toxin.
To disrupt 0 layer interactions, the conserved glutamines
Gln53 and Gln174 were mutated individually or together to
glutamate to introduce additional charges into the 0 layer. Fig.
2B shows that these additional mutations did not affect the
overall reconstituting effect of transfected toxin-resistant
SNAP-25 in PC12 cells. Since this assay would not reveal
subtle effects of the mutations, exocytosis from the transfected
cells was looked at in more detail by comparing Em2 with the
double Q/E mutant. The time course of evoked release from
transfected BoNT/E-treated cells was followed but no
differences in the time course of release was seen between the
two constructs (Fig. 3A). Cleavage of SNAP-25 by botulinum
neurotoxins has been shown to modify the Ca2+-sensitivity of
exocytosis implying that SNAP-25 may interact with the Ca2+-
sensor (Gansel et al., 1987) such as synaptotagmin (Schiavo
et al., 1997). We therefore, examined the effect of the Q/E
mutations on the Ca2+-dependency of exocytosis in PC12 cells.
No difference in Ca2+-dependency was observed, however,
between exocytosis supported by Em2 and the double Q/E
mutant in BoNT/E-treated cells (Fig. 3B).
Effect of 0 layer mutations on the extent and kinetics
of exocytosis assessed using amperometry in
adrenal chromaffin cells
In contrast to the apparent lack of effect of Q mutations in
SNAP-25 in PC12 cells seen here and elsewhere, a partial
reduction in exocytosis was observed in chromaffin cells
overexpressing a SNAP-25 (Q174L) construct. Therefore, we
examined the effect of the double Q/E mutant in adrenal
chromaffin cells. These experiments gave us the opportunity to
analyse in detail the kinetics of single granule release events
by the use of carbon-fibre amperometry (Schroeder et al., 1996;
Wightman et al., 1991) and thereby analyse the importance of
the SNAP-25 0 layer residues for the kinetics of membrane
fusion. In these experiments the cells were stimulated by local
application of digitonin and Ca2+to permeabilise the cells and
directly activate exocytosis.
To inactivate endogenous SNAP-25 in chromaffin cells, they
were transfected with a GFP-BoNT/E light chain construct for
3-5 days to allow time for effective cleavage of endogenous
SNAP-25 and so avoid complications due to the functioning of
residual wild-type protein. Only three of nine GFP-BoNT/E
transfected cells still responded to stimulation and there was
an overall 90% reduction in the mean number of evoked
amperometric spikes (Fig. 4A) similar to previous findings
(Graham et al., 2000). Co-transfection of the SNAP-25 Em2
construct along with the GFP-BoNT/E light chain effectively
JOURNAL OF CELL SCIENCE 114 (24)
Fig. 2. Effect of expression of SNAP-25 constructs on exocytosis in
BoNT/E treated PC12 cells. The cells were co-transfected with a
plasmid encoding growth hormone (GH) along with control vector
(pcDNA3), plasmid encoding toxin-resistant mutant (Em2) or Em2
containing additional mutations as indicated. (A) Expression of
SNAP-25 constructs demonstrated by blotting with anti-HA three
days after transfection. (B) Transfected cells were permeabilised with
20 µM digitonin for 6 minutes and treated with recombinant BoNT/E
light chain for 40 minutes. The cells were then challenged with 0 or
10 µM Ca2+as indicated and GH release over a 20 minute period
assayed. Total GH content for each well was determined and GH
release is shown as a percentage of total GH expressed.

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4401SNARE complex and exocytosis
reconstituted exocytosis in chromaffin cells so that the evoked
amperometric spikes due to the function of expressed SNAP-
25 mutants could be examined. From the examination of a large
A
number of cells it was apparent that Em2 expressing cells
produced, on average, as many release events as seen in control
non-transfected cells. While the double Q/E mutant did
C
AB
Double Q/E
Em2
GH release (% of total)
Time (min)
Em2
Double Q/E
GH release (% of total)
[Ca ] ( M)
2+
µ
40
0
10
20
30
40 01020
30
25
0
10
15
20
5
150102030525
Fig. 3. Effect of Q/E mutations in SNAP-25 on the time
course and Ca2+-dependency of exocytosis in PC12 cells.
The cells were transfected with the GH plasmid along
with the Em2 plasmid or Em2 harbouring the double Q/E
mutations. After 3 days, the cells were permeabilised with
digitonin and treated with BoNT/E light chain followed
by incubation with Ca2+to stimulate exocytosis. (A) Time
course of GH release in response to 10 µM Ca2+from
cells co-transfected with either Em2 or the double mutant.
(B) Ca2+-dependency of GH release from cells transfected
with Em2 or the double Q/E mutant.
050100 150200
Time (s)
0
100
200
300
Current (pA)
Em2
050100 150200
0
100
200
300
Time (s)
Current (pA)
Non-transfected
p<0.001
0
5
10
15
20
25
Spikes per cell
Control
BoNT/E
p<0.01
0
5
10
15
20
25
Spikes per cell
Double Q/E
Non-transfected
Em2
Double Q/E
0
100
200
300
Current (pA)
050100 150200
Time (s)
D
B
E
Fig. 4. Effect of Q/E mutations in SNAP-25 on the extent of exocytosis in adrenal chromaffin cells measured using amperometry. Adrenal
chromaffin cells were transfected with a plasmid-encoding GFP-BoNT/E light chain along with the Em2 plasmid or Em2 containing the double
Q/E mutations. After 3-5 days the cells were stimulated by local application of 20 µM digitonin and 10 µM Ca2+and responses measured using
carbon-fibre amperometry. Non-transfected cells were assayed as controls in the same dishes as GFP-expressing transfected cells and using the
same carbon-fibres. (A) Mean numbers of amperometric spikes elicited in BoNT/E light-chain expressing cells (n=9 cells) compared to controls
(n=10 cells) in the same dishes. (B) Mean numbers of spikes in control cells (n=39) or in cells co-transfected with Em2 (n=31 cells) or Em2
containing the double Q/E mutations (n=40 cells). Typical traces are shown from a non-transfected cell (C) or from cells co-transfected with
GFP-BoNT/E light chain and Em2 (D) or the double Q/E mutant (E).

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4402
reconstitute exocytosis, the number of amperometric
spikes elicited in cells expressing this mutant was
reduced by around 50% compared with non-transfected
cells (Fig. 4B). Fig. 4C-E shows typical overall responses
from control non-transfected cells (in the same dishes as
transfected cells) and cells transfected to express GFP-
BoNT/E along with Em2 or the double Q/E mutant. Apart
from the reduction in number of spike events in cells
expressing the double Q/E mutant, the overall responses
looked similar. To determine whether the double Q/E
mutations affected the dynamics of the individual release
events, the kinetics of individual amperometric spikes
were analysed. We concentrated on analysis of the rise
times of the spikes as this reflects the time course of
initial fusion pore expansion (Schroeder et al., 1996). We
have previously shown this parameter to be modified
under certain specific
overexpression of cysteine string protein (Graham and
Burgoyne, 2000; Graham et al., 2000) or a munc-18
mutant (Fisher et al., 2001), although it is unaffected by
changes in vesicle catecholamine content. This parameter
can therefore report modifications in the fusion
machinery. Comparison of the rise-times of spikes from
control non-transfected, Em2-expressing and double Q/E
mutant expressing cells showed no significant differences in
the mean values (Fig. 5A) or in the frequency distribution of
the individual spike values for the rise-times (Fig. 5B-D).
conditions including
Effect of Q→R mutations in the 0 layer of SNAP-25
on the reconstitution of exocytosis in PC12 cells
The above data suggest that the 0 layer residues of SNAP-25
do not have a crucial role in determining the kinetics of
SNARE complex assembly during exocytotic membrane
fusion. To confirm this idea we also examined the effect of
Q→R mutations recently shown to significantly impair the
affinity of SNARE interactions and to be functionally
disruptive in yeast. Single substitutions of R for Q in either
helix of Sec9 is sufficient to impair growth in yeast (Katz and
Brennwald, 2000). Responses were determined in cells co-
transfected with GH and with Q/R mutants in SNAP-25. The
Q174R mutant (data not shown) and a double Q/R mutant were
able to functionally replace the BoNT/E-cleaved endogenous
SNAP-25 in exocytosis in PC12 cells (Fig. 6A), even though
the double Q/R mutant was less well expressed than Em2 (Fig.
6B).
DISCUSSION
In this study we have exploited the use of a BoNT/E-resistant
mutant of SNAP-25 to allow expression of engineered
constructs in the absence of functional endogenous SNAP-25.
As shown previously (Washbourne et al., 1999), the toxin-
resistant form of SNAP-25 (Em2) fully supported exocytosis
in permeabilised PC12 cells treated with BoNT/E light chain.
In addition, the Em2 construct supported exocytosis in
chromaffin cells co-transfected to express GFP-BoNT/E. In
these cells the single granule release events supported by the
BoNT/E-resistant mutant had identical kinetic properties to
those in control non-transfected cells when assayed by
amperometry. We examined the effect of additional mutations
in the 0 layer residues by introducing them into the Em2
construct. In these assays support of exocytosis required a
functional N-terminal helix as well as the C-terminal helix
needed to replace that of the endogenous protein cleaved by
BoNT/E. This differs from the situation in a previous study
using permeabilised PC12 cells where addition of the C-
terminal helix alone was stimulatory, although this was
enhanced by co-addition of the N-terminal helix (Chen et al.,
1999; Scales et al., 2000). The difference is likely to be due to
our study being based on the use of a full length SNAP-25
protein rather than free helices. In our study, mutation of either
or both conserved glutamines to glutamate had no effect on
the reconstitution in PC12 cells in terms of the extent, time
course or Ca2+-dependency of release. We have previously
demonstrated that the rise-time of amperometric spikes, a
JOURNAL OF CELL SCIENCE 114 (24)
0
Mean rise time (ms)
Double Q/E
Non-transfected
Em2
1
2
4
3
6
5
0
50
100
150
Non-transfected
5
101520
Rise-time (ms)
Frequency
0
20
40
60
80
Em2
5
101520
Rise-time (ms)
Frequency
0
20
40
60
80
Double Q/E
5
101520
Rise-time (ms)
Frequency
AB
C
D
Fig. 5. Effect of Q/E mutations of the kinetics of single granule
release events. Data was taken from chromaffin cells following
transfection. Non-transfected cells in the same dishes were
used as controls for comparison with cells expressing Em2 or
the double Q/E mutations. Identified spikes were analysed
using Origin and the rise time to peak determined for each
spike. (A) Values of rise time shown as mean±s.e.m. (B-
D) Frequency distribution of rise-times for control (n=727
spikes), Em2 expressing (n=440 spikes) and double-mutant
expressing (n=343 spikes) cells.

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4403SNARE complex and exocytosis
correlate of fusion pore expansion, can be modified by
overexpression of Csp (Graham and Burgoyne, 2000) or a
Munc18 mutant (Fisher et al., 2001). Expression in chromaffin
cells of the double Q/E mutant not only supported exocytosis
but the single release events were not modified compared with
control cells. Disruptive mutations within the 0 layer of one or
both glutamines to arginine in the double Q/R mutant did not
prevent the reconstitution of exocytosis in PC12 cells. Some
differences are apparent in this and in previous studies (Chen
et al., 1999; Scales et al., 2000; Wei et al., 2000) between PC12
and chromaffin cells. Mutations in Q residues reduced the
overall extent of exocytosis in chromaffin but not in PC12 cells
in this study and a Q174L mutation was shown to reduce
sustained secretion in chromaffin cells (Wei et al., 2000). One
possible explanation for these differences between cell types is
that much of the exocytosis triggered in PC12 cells comes from
docked granules. while sustained secretion in chromaffin cells
requires recruitment of granules from a non-docked pool. Since
α-SNAP and NSF appear to be important for recruitment
(priming) and sustained secretion in chromaffin cells
(Chamberlain et al., 1995; Xu et al., 1999a) these effects of
mutations in SNAP-25 may be consistent with the Q residues
of SNAP-25 being important in the disassembly of cis-SNARE
complexes during priming.
The lack of effect of the Q→E and Q→R mutations and
those previously reported (Chen et al., 1999; Scales et al.,
2000; Wei et al., 2000) suggest a surprising level of tolerance
to mutation in the 0 layer considering the evolutionary
invariance of these residues and their conservation in all
SNAREs acting in distinct vesicle fusion steps. Previous
mutations examined in the 0 layer in assays for exocytosis have
involved substitution of glutamine with uncharged residues
(alanine, isoleucine or leucine) that may not have markedly
altered the packing of the SNARE helices. We chose to
substitute each glutamine with a glutamate so that the
introduction of two negative charges in the 0 layer of the
double Q/E mutant would cause electrostatic repulsion and
disruption in this layer and potential weakening of the
interactions between helices. Surprisingly even these mutations
were tolerated, although the overall level of exocytosis was
partially reduced in the chromaffin cells but not PC12 cells. In
the case of the double Q/R mutant the presence of two bulky
positively charged arginines would result in both electrostatic
and physical distortion within the 0 layer, modifying the
packing of the SNARE helices (such changes have been
predicted for only a single arginine substitution within the 0
layer) (Ossig et al., 2000). The double Q/R mutant was,
nevertheless, still functional in exocytosis. Overall, the results
suggest that conservation of the 0 layer residues is not required
for membrane fusion to proceed with normal kinetics.
Examination of mutations in the SNAREs involved in
constitutive exocytosis in yeast have demonstrated the
importance of the conserved 0 layer residues for growth and
secretion (Katz and Brennwald, 2000; Ossig et al., 2000).
However, these assays would be dependent not only on
effective SNARE function in exocytosis but also on SNARE
recycling. It has been suggested that the 0 layer may be more
important for SNARE disassembly and recycling by α-SNAP
Fig. 6. Effect of Q/R mutations in SNAP-25 on the extent of
exocytosis in PC12 cells. PC12 cells were transfected with a
plasmid-encoding growth hormone along with control plasmid,
plasmid encoding Em2 or plasmid encoding Em2 containing the
double Q/R mutations. After 3 days the cells were permeabilised,
treated with BoNT/E for 40 minutes and responses to 0 or 10 µM
Ca2+determined. (A) GH release shown as a percentage of total GH
expressed. (B) Immunoblot with anti-HA showing the expression of
Em2 and the double Q/R mutant in the same cells used for the GH
assay.
Fig. 7. Schematic model of SNARE complex states before
and during exocytosis. This model is based on previous data
from adrenal chromaffin cells (Xu et al., 1998; Xu et al.,
1999b), synapses (Hua and Charlton, 1999), liposome fusion
(Weber et al., 2000) and the data presented in this paper. In
this model, secretory vesicles initially associate with the
plasma membrane via a loose SNARE complex in which the
SNAREs are sensitive to clostridial neurotoxins. Disassembly
of cis SNARE complexes may be important prior to
exocytosis to free SNAREs for assembly into the initial trans
complex (Graham and Burgoyne, 2000; Xu et al., 1999a).
Conversion to a ‘tight’ complex resistant to tetanus toxin
precedes fusion, and fusion itself is driven by a complex that
is fully toxin-resistant but not zipped up into the stable
complex. The formation of the stable SNARE complex
occurs only after full fusion has been completed and then it be can disassembled by the action of α-SNAP and NSF.
“loose”
complex
fusion
complex
reversible
fusion
neurotoxin
sensitive
SNAP/NSF
stable
complex
“tight”
complex
tetanus
toxin-resistant
fully
toxin-resistant
toxin-resistant
NSF-sensitive
Q/R
interactions

Page 8

4404
and NSF, although mutations in the 0 layer did not apparently
affect NSF-mediated disassembly in vitro (Chen et al., 1999).
Their effects on the dynamics of disassembly in vivo remain
to be determined. It is also possible that the 0 layer residues
are crucial for other protein-protein interactions occurring
post-fusion.
The stability of the SNARE complex in vivo and the extent
to which it exists in a stable confirmation, resembling that in
the crystal structure, before or during exocytosis is not possible
to assess. The only way to analyse SNARE complex formation
is after detergent solubilisation, and it is possible that the
complex could fall into the stable complex only after
solubilisation. Discrimination between cis complexes on the
same membrane and the trans complexes that mediate fusion
is also not possible. It is unclear, therefore, whether the
structure that drives bilayer fusion is equivalent to the stable
complex that assembles in vitro, but we have attempted to
address this question using high-resolution analysis of
exocytosis. The toxin-resistant SNAP-25 (Em2) that we used
already has mutations in conserved residues including
substitution of glutamate for isoleucine in the conserved
hydrophobic layer 2 (Fig. 1). These mutations impair the
binary interaction of Em2 SNAP-25 with VAMP, although it
can still associate into a complex with VAMP and syntaxin
(Washbourne et al., 1999). Mutations within the hydrophobic
layers of either the N- or C-terminal helices of SNAP-25 that
are close to the 0 layer reduced thermal stability of the SNARE
complex (Chen et al., 1999) and loss of function in the yeast
homologue sec9 (Rossi et al., 1997). Despite a potential effect
on SNARE interactions, and probably on stability, exocytosis
supported by Em2 was indistinguishable from that due to
endogenous wild-type SNAP-25. The fact that additional
mutations in residues within the 0 layer, including potentially
disruptive substitutions such as the introduction of two charged
residues in the double Q/E mutant, can also be tolerated and
that exocytosis proceeds with normal kinetics argues that the
stable SNARE complex may not be the structure that drives
fusion.
Various experimental data have suggested the existence of
multiple states of SNARE complex assembly in addition to
distinct cis- and trans-complexes (Fiebig et al., 1999; Hua and
Charlton, 1999; Weber et al., 2000; Xu et al., 1998; Xu et al.,
1999b). A comparison of the effects of two Clostridial
neurotoxins, BoNT/D and tetanus toxin, that cleave VAMP has
suggested the existence of a SNARE complex prior to fusion
of synaptic vesicles in which the N-terminal but not the C-
terminal domain of VAMP is shielded (Hua and Charlton,
1999). This partial neurotoxin-sensitive complex would be
distinct from the neurotoxin-resistant tight complex postulated
to occur in chromaffin cells (Xu et al., 1998; Xu et al., 1999b)
leading to the possibility of the existence of a series of distinct
quasi-stable SNARE complexes. Putting this information
together with the data in the present paper leads us to suggest
a model in which fusion involves the formation of a non-fully
zipped SNARE complex that can be reversible (Fig. 7). In the
in vitro liposome fusion assay the SNARE complex formed
prior to fusion is resistant to α-SNAP and NSF (Weber et al.,
2000). As suggested, this could be due to steric hindrance of
α-SNAP and NSF binding or, alternatively, the SNARE
complex that mediates fusion may be unable to recruit α-SNAP
and NSF until functional 0 layer interactions occur to allow the
formation of the stable and now NSF-sensitive complex. Our
model shown in Fig. 7 has the merit that it would be consistent
with reversible fusion pore opening and closure as
demonstrated in biophysical studies of regulated exocytosis
and the possibility that fusion pore dynamics is subject to
physiological regulation.
This work was supported by grants from the Wellcome Trust to
R.D.B.
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