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The Journal of Neuroscience, December 15, 1998, 18(24):10250-10256
NSF Function in Neurotransmitter Release Involves Rearrangement
of the SNARE Complex Downstream of Synaptic Vesicle Docking
Leigh Anna
Tolar and
Leo
Pallanck
Department of Genetics, University of Washington, Seattle,
Washington 98195
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ABSTRACT |
The SNARE hypothesis has been proposed to explain both
constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this
model, a vesicle targeting/docking complex consisting primarily of
vesicle- and target-membrane proteins, known as SNAREs, serves as a
receptor for the cytosolic N-ethylmaleimide-sensitive
fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While
features of this model remain tenable, recent studies have challenged
fundamental aspects of the SNARE hypothesis, indicating that further
analysis of these components is needed to fully understand their roles
in neurotransmitter release. We have addressed this issue by using the
temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of
NSF in neurotransmitter release in vivo. Synaptic
electrophysiology and ultrastructure in comt mutants
have recently defined a role for NSF after docking in the priming of
synaptic vesicles for fast calcium-triggered fusion. Here we report
that an SDS-resistant neural SNARE complex, composed of the SNARE
polypeptides syntaxin, n-synaptobrevin, and SNAP-25, accumulates in
comt mutants at restrictive temperature. Subcellular
fractionation experiments indicate that these SNARE complexes are
distributed predominantly in fractions containing plasma membrane and
docked synaptic vesicles. Together with the electrophysiological and
ultrastructural analyses of comt mutants, these results
indicate that NSF functions to disassemble or otherwise rearrange a
SNARE complex after vesicle docking and that this rearrangement is
required to maintain the readily releasable pool of synaptic vesicles.
Key words:
N-ethylmaleimide-sensitive fusion protein; NSF; comatose; neurotransmitter exocytosis; Drosophila; SNARE complex; synaptic vesicle
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INTRODUCTION |
Neurons signal to their target cells
primarily through the regulated exocytosis of chemical
neurotransmitters. Neurotransmitter release involves a discrete series
of steps, including formation of neurotransmitter-filled synaptic
vesicles, targeting of synaptic vesicles to presynaptic release sites,
and calcium-triggered fusion of synaptic vesicles with the nerve
terminal plasma membrane. Recent studies of constitutive and regulated
secretion have led to the identification of polypeptides that may
function in this process, and a model, known as the SNARE hypothesis,
proposing functions for these polypeptides (Rothman, 1994 ; Scheller,
1995; Südhof, 1995). According to this model, synaptic vesicle
targeting to and docking at release sites involves the assembly of
vesicle- and target-membrane proteins, referred to as v-SNAREs (e.g.,
synaptobrevin) and t-SNAREs (e.g., SNAP-25 and
syntaxin), respectively. This protein complex recruits the soluble
NSF attachment proteins (SNAPs) and
N-ethylmaleimide-sensitive fusion protein (NSF) to form a pre-fusion complex. NSF-dependent hydrolysis of ATP disassembles the
SNARE complex in a step postulated to initiate membrane fusion (Söllner et al., 1993).
Because the SNARE hypothesis was derived largely from analyses of
constitutive secretion and in vitro protein binding
experiments, recent efforts have been directed at examining the
validity of this model in synaptic function. This work has clearly
established an important role for SNAREs (Hunt et al., 1994; Broadie et
al., 1995; Schulze et al., 1995; Sweeney et al., 1995; O'Connor et al., 1997; Nonet et al., 1998; Deitcher et al., 1998) and SNAPs (DeBello et al., 1995) in neurotransmitter release. However, these studies have also challenged fundamental tenets of the SNARE hypothesis (Hunt et al., 1994; Broadie et al., 1995; O'Connor et al., 1997) and
have led to new models by which the SNARE components may function. Despite these advances, the precise functional roles of several key
components of the neurosecretory apparatus, including NSF, remain
relatively unexplored.
To investigate the role of NSF in neurotransmitter release, we
initiated a genetic analysis of NSF function in the model organism Drosophila melanogaster by identifying a
Drosophila NSF gene (termed dNSF1) (Ordway et al., 1994) and
a pre-existing temperature-sensitive dNSF1 mutant, known as
comatose (comt) (Pallanck et al., 1995). comt was originally identified in a classical genetic screen
for recessive mutations causing temperature-sensitive paralysis
(Siddiqi and Benzer, 1976), and recent electrophysiological and
ultrastructural studies have demonstrated an activity-dependent
reduction in neurotransmitter release and a marked accumulation of
docked synaptic vesicles at restrictive temperature in comt
mutants (Kawasaki et al., 1998). These and other recent studies
of NSF function in regulated secretion (Banerjee et al., 1996;
Schweizer et al., 1998) lead to the conclusion that NSF serves to prime
vesicles for calcium-triggered fusion after docking.
We have investigated the molecular nature of this priming event by
characterizing the composition, state of assembly, and subcellular
distribution of the SNARE complex in comt mutants. Here we
report a marked accumulation of a ternary SNARE complex composed of
syntaxin, n-synaptobrevin, and SNAP-25 in comt mutants after
exposure to restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes accumulate
predominantly in plasma membrane fractions. Examination of SNARE
complex abundance in comt and other Drosophila
neural signaling mutants indicates that SNARE complex abundance is
specifically affected in mutants with synaptic vesicle trafficking
defects. Together with other recent work in comt mutants,
these results indicate that dNSF1 functions to disassemble or otherwise
rearrange the SNARE complex after synaptic vesicle docking in the
process of neurotransmitter exocytosis.
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MATERIALS AND METHODS |
Drosophila strains
Flies were cultured in standard medium at room temperature
(22°C). The three comt strains used in this analysis,
ST17, ST53, and TP7, each contain
single amino acid substitutions in the dNSF1 coding sequence with
respect to the wild-type strain, Canton S. The ST17 and
ST53 mutations have been reported previously (Pallanck et
al., 1995), and the TP7 mutation substitutes a serine for a proline at amino acid position 398 of the dNSF1 open reading frame. Transgenic expression of wild-type dNSF1 protein was accomplished using
a P-element construct consisting of a dNSF1 cDNA under the control of
an hsp70 heat shock promotor (Pallanck et al., 1995). Expression was
induced by heat-shocking flies at 38°C once per day for 3 consecutive
d (15 min, 30 min, and 30 min). Heat-shocking comt
flies that lacked the transgene did not alter the comt
phenotypes. With the exception of the membrane fractionation
experiments, all of the studies described in this manuscript involved
flies aged 3-6 d.
Preparation and analysis of SNARE complexes
Sample preparation, electrophoresis, and Western
blotting. After exposure to a given temperature for a given period
of time, flies were rapidly frozen in liquid nitrogen. Heads were
removed from frozen flies by vortexing and homogenized in SDS sample
buffer (Laemmli, 1970). After a brief centrifugation to pellet cuticle, an aliquot of the supernatant was loaded onto a discontinuous SDS-polyacrylamide gel, with the upper three-fourths and lower one-fourth of the separating gel being adjusted to 9 and 15%
acrylamide, respectively. An additional aliquot was boiled for 5 min to
disrupt SNARE complexes before gel loading. After electrophoresis, gels were electroblotted onto nitrocellulose filters according to
manufacturer's (Bio-Rad, Hercules, CA) directions. Blots were
incubated with a monoclonal anti-syntaxin antibody (MAb 8C3) (Wu et
al., 1998), with a monoclonal anti-SNAP-25 antibody (MAb CI.71.1) (Otto
et al., 1997), or with polyclonal antisera that recognize sequences in
the cytoplasmic (NSYB1) or intravesicular domains (NSYB2) of neuronal
synaptobrevin (n-synaptobrevin) that are highly divergent in the
ubiquitously expressed synaptobrevin isoform (DiAntonio et al., 1993;
van de Goor et al., 1995). After blots were stained with antisera,
bands were detected by enhanced chemiluminescence (Amersham, Arlington
Heights, IL). Quantitation of SNAREs and SNARE complexes was performed
using a GS-700 imaging densitometer (Bio-Rad). Standard curves for
densitometry were prepared by diluting samples prepared from normal
(Canton-S) flies. Each quantitation experiment was performed in
triplicate, and only signals falling within the linear range of the
standard curves were used. Results of quantitation experiments (see
Table 1) are expressed as ratios of SNARE complex to SNARE monomer to
exclude gel loading artifacts.
Isolation and analysis of the 73 kDa SNARE complex. A
fly head lysate, prepared as described above, was subjected to
SDS-PAGE. After electrophoresis, the region corresponding to the
73 kDa band (containing polypeptides in the 55-85 kDa size range), and for control purposes the region below the 73 kDa band (40-55 kDa), were excised, boiled 10 min, and each placed separately on top of a
second 12% SDS gel. Polypeptides in the gel slices were then electrophoretically separated in these second SDS gels and subjected to
Western blot analysis as above.
Subcellular membrane fractionation
Separation of unbound synaptic vesicles from plasma membrane was
performed using glycerol velocity sedimentation, essentially as
described (van de Goor et al., 1995). Modifications to this published
membrane fractionation procedure included use of a 10-30% glycerol
gradient for velocity sedimentation experiments, 6 gm of flies per
gradient, and an SW 41 Ti ultracentrifuge rotor (Beckman, Fullerton,
CA). These modifications were introduced to increase the separation
between peak synaptic vesicle- and plasma membrane-containing glycerol
gradient fractions. Synaptic vesicle- and plasma membrane-containing gradient fractions were identified by subjecting aliquots of the gradient fractions to SDS-PAGE and Western blotting using antisera to
the synaptic vesicle antigens synapsin (SYNORF1) (Klagges et al.,
1996), n-synaptobrevin (NSYB1 and NSYB2; see above), and synaptotagmin
(DSYT2) (Littleton et al., 1993), and to the 42 kDa plasma membrane
antigen [the putative  -subunit of the
Na+/K+ ATPase (van de Goor et
al., 1995)] recognized by a rabbit anti-horseradish peroxidase
antiserum (ICN Pharmaceuticals, Costa Mesa, CA), as described above.
Synaptic vesicle- and plasma membrane-containing fractions were
combined into separate pools, mixed with an equal volume of 2×
SDS-PAGE loading buffer (Laemmli, 1970), aliquoted, and stored frozen
at  20°C. Quantitation of SNARE complexes was performed using
densitometry as described above.
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RESULTS |
A Drosophila SNARE complex, composed of syntaxin,
n-synaptobrevin, and SNAP-25, is resistant to denaturation by SDS
If NSF is normally required to disassemble a ternary SNARE complex
after synaptic vesicle docking, as suggested by the SNARE hypothesis,
loss of comt function should lead to increased levels of the
ternary complex. To investigate this issue, we sought to develop a
method that would allow us to determine the abundance of the ternary
SNARE complex in comt and wild-type flies. Previous work in
mammals has demonstrated that neural SNARE complexes are resistant to
denaturation by SDS, except when boiled, and that de novo
assembly of these complexes does not occur in SDS-containing buffer
(Hayashi et al., 1994). Thus we reasoned that if Drosophila SNARE complexes are similarly resistant to SDS, the quantity of SDS-resistant neural SNARE complex recovered from a fly head homogenate should reflect the quantity of SNARE complex present in
vivo.
To assay the presence of an SDS-resistant ternary SNARE complex in
Drosophila, Western blot analysis was performed on an SDS lysate prepared from wild-type fly heads using monoclonal antibodies to
the Drosophila t-SNAREs syntaxin and SNAP-25, and a rabbit polyclonal antiserum to the synaptic vesicle v-SNARE, n-synaptobrevin. In experiments conducted using antisera to syntaxin and
n-synaptobrevin, prominent bands corresponding in size to the monomeric
forms of these proteins (35 kDa and 23 kDa, respectively) (Schulze et
al., 1995; van de Goor et al., 1995) as well as a less abundant,
heat-sensitive species of 73 kDa were detected (Fig.
1A). Overloading of SDS gels, or prolonged exposures of the immunoblots, resulted in the appearance of additional higher molecular weight bands that might represent higher order SNARE complexes (data not shown). In contrast, the SNAP-25 monoclonal antibody appeared to detect only the monomeric form of SNAP-25 [30 kDa (M. N. Wu, personal
communication)], perhaps because the epitope recognized by this
antibody was masked in the 73 kDa complex (Fig.
1A).
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Figure 1.
Identification of an SDS-resistant neural SNARE
complex in Drosophila. A, Western blot
analysis of an SDS lysate from wild-type Drosophila
heads, using monoclonal antibodies to syntaxin (syx) and
SNAP-25 (snp25) and rabbit polyclonal antisera to the
neuronal isoform of synaptobrevin, n-synaptobrevin
[(n-syb); results using the NSYB2 antiserum are shown;
however, similar results were obtained with the NSYB1 antiserum].
Syntaxin and n-synaptobrevin are detected as monomers of 35 and 23 kDa,
respectively, and as part of a higher molecular weight complex (73 kDa)
in unboiled samples (). The 73 kDa complex is disrupted in lysates
that were boiled (+) before loading. B, Detection of
SNAREs released from the 73 kDa complex by boiling. A head SDS lysate
prepared from wild-type flies was electrophoretically separated on an
SDS gel, and the 73 kDa region was excised, boiled, and layered onto a
second SDS gel. Polypeptides in this gel strip were then subjected to
SDS-PAGE and Western blot analysis using the antibodies to syntaxin
(syx), n-synaptobrevin (n-syb), and
SNAP-25 (snp25) described above. Lanes marked + are
samples boiled before loading of the first gel, and those marked are
unboiled samples loaded onto the first gel.
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To determine whether the SDS-resistant 73 kDa species does indeed
represent a complex composed of syntaxin, n-synaptobrevin, and SNAP-25,
the 73 kDa region (containing polypeptides in the 55-85 kDa size
range) and for control purposes the region below the 73 kDa band
(40-55 kDa) were excised from a gel and boiled to disrupt
protein-protein interactions in the putative complex. These gel strips
were then layered on top of second SDS gels and subjected to SDS-PAGE
and Western blot analysis with antisera to the three
Drosophila SNAREs. Bands corresponding to the expected sizes
for monomers of syntaxin, n-synaptobrevin, and SNAP-25 were detected
only from gel strips containing the 73 kDa material (Fig. 1B; and data not shown), thus establishing the
identity of the 73 kDa band.
Excess accumulation of the ternary SNARE complex is observed in
comt mutants at restrictive temperature
The abundance of the SDS-resistant 73 kDa ternary SNARE complex
was analyzed in three different comt mutants at permissive (22°C) and restrictive (38°C) temperatures (Fig.
2, Table 1; and data not shown). For all three comt alleles, SNARE
complex abundance relative to monomeric syntaxin and n-synaptobrevin
was similar to that seen in wild-type flies at permissive temperature. However, in contrast to wild-type flies, exposure of all three comt mutants to restrictive temperature resulted in a
striking elevation in abundance of the SNARE complex. The increased
level of the SNARE complex is paralleled by corresponding decreases of
monomeric syntaxin and n-synaptobrevin, indicating that these polypeptides accumulate in a SNARE complex when NSF activity is disrupted in comt mutants. The ratio of 73 kDa SNARE complex
to monomeric syntaxin is increased 6- to 11-fold in comt
mutants compared with wild-type after a 20 min exposure to restrictive temperature, reflecting both increases in the SNARE complex and decreases in syntaxin monomer. Although this ratio was less severely affected for n-synaptobrevin, similar increases in SNARE complex abundance were detected using antisera to this protein (data not shown).
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Figure 2.
SDS-resistant SNARE complexes accumulate in
comt mutants at elevated temperatures. A,
Western blot analysis of the 73 kDa SNARE complex from
comt mutants and wild-type controls (CS)
using a syntaxin monoclonal antibody. Flies were maintained at 22°C
or exposed to 38°C for 20 min before SNARE complexes were extracted.
Samples marked + were boiled before gel loading. Similar results were
obtained using antisera to Drosophila n-synaptobrevin.
B, SDS lysates prepared from the heads of wild-type
flies and comtST17 mutants after
exposure to 38°C for 20 min were electrophoretically separated on an
SDS gel. After electrophoresis, the 73 kDa region was excised, boiled,
and layered onto a second SDS gel, and polypeptides in this gel strip
were then subjected to SDS-PAGE and Western blot analysis using
antisera to syntaxin (syx), n-synaptobrevin
(n-syb) (results using the NSYB1 antiserum are shown),
and SNAP-25 (snp25). C, Expression of a
dNSF1 transgene rescues SNARE complex accumulation in
comt mutants. Western blot analysis of the 73 kDa SNARE
complex from three different comt mutant alleles
(ST17, TP7, and ST53) and
wild-type controls (CS) using a syntaxin monoclonal
antibody. Flies with (+) or without () the wild-type dNSF1 transgene
were exposed to 38°C for 20 min before sample preparation. Similar
results were obtained using the n-synaptobrevin antisera.
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We also sought to determine whether increased levels of SNAP-25 were
recruited into SNARE complexes in comt mutants at
restrictive temperature. However, because of the apparent inability of
the anti-SNAP-25 antibody to detect SNAP-25 protein in the SNARE
complex, we performed Western blot analyses on boiled gel slices
containing the 73 kDa SNARE complex, as described above, to compare the
levels of incorporation of this protein into SNARE complexes in
comt mutants and wild-type flies. To allow direct comparison
of the level of recruitment of SNAP-25, syntaxin, and n-synaptobrevin into the SNARE complex in comt mutants with wild-type flies,
this analysis was also performed with antisera to syntaxin and
n-synaptobrevin. Results from this analysis confirm the increased
incorporation of syntaxin and n-synaptobrevin into the SNARE complex in
comt mutants at restrictive temperature and demonstrate that
SNAP-25 also accumulates in this complex in parallel with these
polypeptides (Fig. 2B). Results from this analysis
also indicate similar increases in the levels of incorporation of these
three polypeptides into the 73 kDa complex in comt mutants
at restrictive temperature with respect to wild-type flies. Expression
of wild-type dNSF1 protein from a transgene rescues the comt
defect in SNARE complex accumulation but does not alter SNARE complex
abundance in normal flies, demonstrating that this phenotype
specifically reflects the loss of dNSF1 activity (Fig.
2C).
SNARE complex abundance is reduced in a Drosophila
synaptic vesicle recycling mutant and unaffected in
Drosophila mutants with altered membrane excitability
To examine whether the increased abundance of the SDS-resistant
ternary SNARE complex observed in comt mutants is a specific feature of the comt phenotype and not a general feature of
nervous system dysfunction, we compared SNARE complex abundance in
various Drosophila mutants with defects in different aspects
of nervous system signaling with SNARE complex abundance in wild-type
flies. Specifically, SNARE complex abundance was analyzed in the
Drosophila neural signaling mutants Shaker
(Sh), Hyperkinetic (Hk), and
ether-a-go-go (eag) (Wu and Ganetzky, 1992). In
addition, SNARE complex abundance was analyzed in the
Drosophila temperature-sensitive paralytic mutants no
action potential (nap), paralyzed
(para), seizure (sei), shibire (shi), and slowpoke
(slo), at permissive and restrictive temperatures (Kosaka
and Ikeda, 1983; Koenig and Ikeda, 1989; Wu and Ganetzky, 1992; van de
Goor et al., 1995). Within this large collection of neural signaling
mutants, only shi exhibited an appreciable alteration in the
abundance of the ternary SNARE complex (Fig.
3; and data not shown). However, in
contrast to results observed in analyses of comt mutants,
the abundance of the ternary SNARE complex isolated from shi
mutants is significantly reduced after the shift to restrictive
temperature (Fig. 3, Table 1). As was observed for the comt
alleles, the ratio of SNARE complex abundance relative to monomeric
syntaxin was similar in shi and wild-type flies at
permissive temperature.
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Figure 3.
SNARE complex abundance is reduced in
shi mutants at restrictive temperature. Western blot
analysis of the 73 kDa SNARE complex from
shiTS1 mutants using a syntaxin
monoclonal antibody. Flies were maintained at 22°C or exposed to
38°C for 20 min before extracting SNARE complexes. Samples marked + were boiled before gel loading. Similar results were observed using
antisera to n-synaptobrevin.
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Previous analyses of eag, Hk, nap,
para, sei, Sh, and slo
indicated that these mutations affect features of membrane excitability (Wu and Ganetzky, 1992), whereas the shi mutation is the
only one in this group that specifically affects synaptic vesicle
trafficking (Kosaka and Ikeda, 1983; Koenig and Ikeda, 1989; van de
Goor et al., 1995). At elevated temperatures, nerve terminals in
shi mutants become depleted of synaptic vesicles within
several minutes because of a block in the endocytic retrieval of
synaptic vesicle membrane (Kosaka and Ikeda, 1983; Koenig and Ikeda,
1989; van de Goor et al., 1995). Thus, the reduction in SNARE complex
abundance observed in shi mutants at restrictive temperature
is consistent with models for SNARE complex formation occurring during
or after synaptic vesicle docking. These results also demonstrate that
alterations in SNARE complex abundance are not general features of
nervous system dysfunction, but rather are the specific consequences of mutations affecting components that function in synaptic vesicle trafficking.
The SNARE complex colocalizes with plasma membrane markers
To determine where SNARE complexes accumulate in comt
mutants at nonpermissive temperature, we compared the subcellular
distribution of SNARE complexes in comt mutants and
wild-type flies at permissive and restrictive temperatures. To perform
this analysis, postnuclear membrane fractions prepared from head
homogenates of comtST17 mutants or
wild-type flies were sedimented on a glycerol gradient to separate
plasma membrane from synaptic vesicle membrane (van de Goor et al.,
1995). After centrifugation, fractions were collected from the bottom
of the gradient and subjected to Western blot analysis using an
anti-horseradish peroxidase antiserum that recognizes a 42 kDa plasma
membrane marker [the putative -subunit of the Na+/K+ ATPase (van de Goor et
al., 1995)], as well as antisera to the synaptic vesicle proteins
synapsin, synaptotagmin, and n-synaptobrevin, to identify peak plasma
membrane- and synaptic vesicle-containing fractions (Fig.
4). In addition, a monoclonal
anti-syntaxin antibody was used to identify peak syntaxin- and SNARE
complex-containing gradient fractions (Fig. 4).
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Figure 4.
The SNARE complex resides in the plasma membrane.
A postnuclear head homogenate from wild-type flies
(A) or comtST17
mutants (B) was sedimented on a glycerol
gradient, and aliquots of the gradient fractions (numbered 1-13 from
the bottom to the top of the gradient) were subjected to Western blot
analysis to identify peak plasma membrane fractions, peak synaptic
vesicle fractions, and SNARE complexes. Western blot analyses were
performed using antisera to a 42 kDa plasma membrane marker [the
putative -subunit of the
Na+/K+ ATPase, which is
recognized by rabbit antisera to horseradish peroxidase (van de Goor et
al., 1995)] and to the synaptic vesicle proteins n-synaptobrevin (a
mixture of the NSYB1 and NSYB2 antisera), synaptotagmin (DSYT2)
(Littleton et al., 1993), and synapsin (data not shown), as well as a
monoclonal anti-syntaxin antibody to detect SNARE complexes and monomer
syntaxin. Analyses to identify syntaxin- and n-synaptobrevin-containing
gradient fractions were performed using samples boiled 5 min to disrupt
SNARE complexes. SNARE complex-containing gradient fractions were
detected using unboiled samples. No comparison of SNARE complex
abundance can be made between A and B
because immunoblot exposure times varied in these experiments. Results
obtained with the anti-synapsin antiserum were consistent with those
obtained with antisera to the other synaptic vesicle antigens analyzed.
Results using flies exposed to restrictive temperature (38°C) for 20 min before preparation of head homogenates are shown. Similar results
were obtained using flies maintained at permissive temperature
(22°C).
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In accordance with previous work in Drosophila (van de Goor
et al., 1995), peak plasma membrane-containing fractions were found to
be distributed at the bottom of the glycerol gradient (heavy
membranes), and two peaks of synaptic vesicle antigenicity were
observed: a rapidly sedimenting peak colocalizing with heavy membranes
and a slowly sedimenting peak (light membranes) (Fig. 4). Previous work
(van de Goor et al., 1995) indicates that the light membrane fractions
contain unbound synaptic vesicles (attributable to the identical
sedimentation rate of material in these fractions with rat brain
synaptic vesicles), whereas the rapidly sedimenting peak of synaptic
vesicle antigenicity likely represents synaptic vesicle proteins
distributed in the plasma membrane as well as synaptic vesicles bound
(docked) to plasma membrane. The subcellular distributions of syntaxin
and the SNARE complex were also analyzed and found to exhibit
sedimentation profiles indistinguishable from the plasma membrane
marker used in this analysis (Fig. 4). Quantitative Western blotting of
the pooled light membrane fractions (fractions 7-11) and the preceding
heavy membrane fractions (fractions 1-6) was used to further document
the subcellular distribution of SNARE complexes in comt
mutants and wild-type flies at permissive and restrictive temperatures.
Results from this analysis indicate that >97% of SNARE complexes
sediment with plasma membrane markers in both comt mutants
and wild-type flies at permissive and restrictive temperatures.
Although previous work indicates that ternary SNARE complexes can
reside within the synaptic vesicle membrane (Otto et al., 1997), we
sought to determine whether the low level of SNARE complexes detected
in the light membrane fractions from our gradients reside in the
synaptic vesicle membrane or, alternatively, represent contamination of
the light membrane fractions with plasma membrane-derived SNARE
complexes. To facilitate detection of SNARE complexes in the unbound
synaptic vesicle pool, large aliquots of glycerol gradient fractions
4-13 were subjected to Western blot analysis using antisera to
syntaxin, n-synaptobrevin, and the 42 kDa plasma membrane marker.
Results of this analysis reveal a decreasing gradient of SNARE complex,
syntaxin, and the 42 kDa plasma membrane marker extending into the
unbound synaptic vesicle peak fractions (Fig.
5). No second SNARE complex peak,
colocalizing with the peak of unbound synaptic vesicles, was detected.
Quantitative Western blotting of the light membrane fractions
(fractions 7-11) and the preceding heavy membrane fractions (fractions
1-6) using the anti-horseradish peroxidase antiserum indicated that
the distribution of the plasma membrane marker recognized by this
antiserum closely approximates the distribution of SNARE complexes in
these fractions (~3% resides in the unbound synaptic vesicle pool).
Further evidence that SNARE complexes in the unbound synaptic vesicle
fractions derive from plasma membrane contamination is provided by the
uniform distribution of SNARE complex to monomer syntaxin ratios across the gradient, indicating a single common pool of syntaxin and SNARE
complexes (data not shown). In contrast, the ratio of SNARE complex to
monomer n-synaptobrevin is increased >25-fold in plasma membrane
fractions with respect to the unbound synaptic vesicle fractions in
both comt mutants and wild-type flies, indicating that there
are two distinct pools of n-synaptobrevin: a plasma membrane pool that
is largely in complex with t-SNAREs and an uncomplexed pool associated
with unbound synaptic vesicles. Together, these results indicate that
most, if not all, of the SNARE complexes present in light membrane
fractions arise from contamination with plasma membrane SNARE
complexes.
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Figure 5.
Plasma membrane contamination provides an
explanation for the SNARE complexes detected in synaptic vesicle
fractions from comt mutants. Aliquots of glycerol
gradient fractions 4-13 from
comtST17 mutants were subject to
Western blot analysis using antisera to (A)
n-synaptobrevin (a mixture of the NSYB1 and NSYB2 antisera),
(B) syntaxin, and (C)
horseradish peroxidase (anti-HRP), which recognizes a 42 kDa plasma membrane marker. Results using flies exposed to restrictive
temperature (38°C) for 20 min before preparation of head homogenates
are shown.
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DISCUSSION |
Work presented here describes characterization of an SDS-resistant
Drosophila neural SNARE complex composed of syntaxin,
n-synaptobrevin, and SNAP-25. In wild-type flies the SNARE complex
appears to represent a small fraction of the total SNARE protein, and
its abundance is not substantially altered as a result of temperature
changes or mutations affecting membrane excitability. In contrast,
SNARE complex abundance is greatly altered from wild-type levels in the
temperature-sensitive paralytic mutants comt and
shi during exposure to restrictive temperature.
Interestingly, SNARE complex abundance in comt and
shi strongly correlates with the state of vesicle docking in
these mutants, suggesting that SNARE complexes form during or after
synaptic vesicle docking. Furthermore, subcellular fractionation
experiments indicate that most, if not all, of the SNARE complex in
both comt mutants and wild-type flies colocalizes with
fractions containing plasma membrane and docked synaptic vesicles.
Although these results are consistent with general features of the
SNARE hypothesis, including a role for SNAREs in vesicle docking and an
NSF-mediated disassembly of the SNARE complex after docking, they are
also compatible with other models.
Studies of the neural SNAREs in Drosophila (Broadie et al.,
1995) and at the squid giant synapse (Hunt et al., 1994; O'Connor et
al., 1997) indicate that disruption of individual SNARE proteins, either n-synaptobrevin or syntaxin, or disruption of SNARE protein interactions, fails to prevent synaptic vesicle targeting or docking. Furthermore, recent in vitro studies of homotypic vacuole
fusion in yeast have provided evidence that the yeast NSF homolog
SEC18p is required for docking (Mayer et al., 1996; Mayer and Wickner, 1997). These experiments also indicate that SEC18p is required only
before the mixing of donor and acceptor membranes, and thus a role for
SEC18p downstream of docking appears unlikely. How can we resolve these
results with those obtained in biochemical, ultrastructural, and
electrophysiological studies of comt mutants? Although
parallel accumulation of docked vesicles and SNARE complexes in
comt mutants is consistent with a role for SNAREs in vesicle docking, these results are also compatible with a model in which SNARE
complex formation follows morphological vesicle docking. Thus, SNAREs
may be dispensable for vesicle targeting and docking (defined by
ultrastructural criteria) and instead may function in downstream
events, such as bilayer fusion.
Although the conflicting results of molecular studies of yeast vacuole
fusion and regulated secretion are currently unresolved, it is possible
that these conflicts reflect fundamental differences between homotypic
and heterotypic fusion. In the former case, the donor and target
membranes presumably contain the same complement of vesicle and target
SNAREs. Under these conditions, NSF may be required to disrupt SNARE
interactions on the same membrane as a prerequisite to docking
(Ungermann et al., 1998). At the synapse, v-SNAREs and t-SNAREs are
largely restricted to synaptic vesicles and plasma membrane,
respectively, and thus no such requirement for NSF would be expected.
An alternative possibility, equally consistent with studies of NSF
function in vacuole fusion and the present study of NSF function in
neurotransmitter release, is that NSF function is required after
synaptic vesicle fusion to disassemble the SNARE complex, thereby
reactivating SNAREs for another round of membrane fusion. This model
differs in a fundamental way from the SNARE hypothesis in that it
proposes a post-fusion role for NSF in the recycling of SNAREs. Recent work showing that SNAREs alone are capable of promoting bilayer fusion
in vitro provides further support for such a model (Weber et
al., 1998).
A feature that distinguishes these two models of NSF function is the
subcellular distribution of SNARE complexes that accumulate under
conditions of reduced NSF activity. The SNARE hypothesis predicts that
SNARE complexes would accumulate at sites of synaptic vesicle docking
under conditions of impaired NSF function. In contrast, a post-fusion
role for NSF in SNARE complex disassembly does not place any such
constraints on SNARE complex localization because the SNARE complex
would be composed of SNAREs residing as neighbors in the same membrane.
One possibility raised by the post-fusion model for NSF function is
that SNARE complexes accumulating under conditions of reduced NSF
activity may become incorporated into recycling synaptic vesicles.
However, the subcellular membrane fractionation experiments reported
here clearly argue against this possibility. In comt mutants
(and wild-type flies), essentially all of the cellular SNARE complexes
were found to be distributed with membrane fractions consisting of
plasma membrane and docked synaptic vesicles. Although these results do
not allow us to distinguish between models of NSF function, they
strongly argue against unbound synaptic vesicles as sites of NSF
function and provide novel information on SNARE complex distribution in
neural tissue.
In summary, data presented here, together with the results of recent
physiological and ultrastructural studies of the comt mutant
(Kawasaki et al., 1998), clearly establish a role for NSF in the
regulated exocytosis of neurotransmitter. The finding that defects in
NSF function result in accumulation of docked synaptic vesicles and
ternary SNARE complexes in the plasma membrane support a model in which
NSF functions after vesicle docking in the disassembly or rearrangement
of a neural SNARE complex to maintain the readily releasable pool of
synaptic vesicles. Ongoing ultrastructural and biochemical analyses of
comt mutants should further refine the function of
NSF in neurotransmitter release.
| |
FOOTNOTES |
Received Aug. 11, 1998; revised Oct. 5, 1998; accepted Oct. 5, 1998.
This work was supported in part by a grant from the Whitehall
Foundation (A98-29) to L.P. We thank Hugo Bellen, Erich Buchner, Ed
Giniger, Reinhard Jahn, Regis Kelley, Tom Schwarz, and Konrad Zinsmaier
for providing antisera, and Rick Ordway for communication and
discussion of unpublished data. Special thanks to Jesse Goldmark for
technical assistance in analyses of SNARE complexes.
Correspondence should be addressed Leo Pallanck, Department of
Genetics, Box 357360, University of Washington, Health Sciences, J113,
1959 N.E. Pacific Street, Seattle, WA 98195.
| |
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Top
Abstract
Introduction
Gene
