1 Laboratory of Cellular and Molecular Biophysics, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda, MD
20892-1855, USA
2 Unit on Biologic Computation, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
3 Department of Physiology and Biophysics, Neuroscience Research Group, Faculty
of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
* Authors for correspondence (e-mail: jcoorsse{at}ucalgary.ca or joshz{at}helix.nih.gov)
Accepted 20 January 2003
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Summary |
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Key words: Secretory vesicles, Exocytosis, Membrane fusion, Sea urchins, Quantitative immunoblotting
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Introduction |
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To differentiate between the density of CV SNAREs, changes in the
Ca2+ sensitivity of CV fusion, and the rate and extent of CV
fusion, advantage was taken of the size, purity homogeneity and high
preparative yields that make CV useful for coupled functional-biochemical
analyses (Coorssen et al.,
1998; Tahara et al.,
1998
; Zimmerberg et al.,
2000
). Endogenously docked CVs from eggs of Strongylocentrotus
purpuratus are fully Ca2+ sensitive and release-ready, and
they fuse with the plasma membrane (PM) within milliseconds of exposure to
optimal [Ca2+]free (exocytosis in vitro)
(Shafi et al., 1994
).
Ca2+-triggered homotypic CV fusion retains the essential features
of regulated exocytosis (Coorssen et al.,
1998
; Zimmerberg et al.,
2000
); only by removing CVs from the PM is there full access to
all vesicle surface proteins. Our approach uses proteases with differing
spectra of endogenous substrates on intact CVs, to quantitatively change the
density of different sets of proteins; the
[Ca2+]free-dependent rate and extent of CV fusion is
then tested, and the density of SNARE proteins measured. This approach (1)
makes no assumptions as to which proteins are essential, (2) allows for the
involvement of low-abundance proteins, (3) uses native vesicles of endogenous
size and composition, and (4) is not subject to issues of compensatory
proteins as are genetic knockout studies. This approach does require
quantitative assays of absolute protein amount that are antibody dependent
(Coorssen et al., 2002
).
On average, isolated CV have 5500,
700 and
330 copies of
VAMP, SNAP-25 and syntaxin, respectively
(Coorssen et al., 2002
). We
combined (1) a sensitive, quantitative immunoblotting assay and polyclonal
antibodies (to minimize underestimation due to epitope loss) to measure SNARE
proteins (Coorssen et al.,
2002
); (2) fusion assays to assess the effects of SNARE removal;
and (3) a method for determining the average number of active fusion complexes
per vesicle, n , that combines the relationship between the
extent of fusion and n , with the exponential decrease in
n after protease treatment
(Vogel et al., 1996
;
Coorssen et al., 1998
). This
analysis was important because there is redundancy in the number of fusion
complexes on CV; for any one vesicle, fusion will not be inhibited until all
functional fusion complexes on that vesicle are inactivated, although the rate
of fusion will be progressively inhibited by reductions in the density of
critical components (Vogel et al.,
1996
; Blank et al.,
2001
).
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Materials and Methods |
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The determination of proteolytic fragmentation patterns for syntaxin and SNAP-25 (Fig. 1) provided a guide for cutting the blotting membrane into three pieces that could be probed independently for SNAREs. In most experiments, using the molecular weight markers as a guide, the membrane was cut between syntaxin and SNAP-25 and just below the smallest SNAP-25 fragment. Myoglobin, recognized by the SNAP-25 antibody, was added only when blotting the entire membrane for syntaxin and SNAP-25 and was not included when processing blotting membranes for the presence of VAMP.
|
For kinetic assays, CVs were centrifuged onto coverslips and transferred to
a perfusion chamber, permitting constant monitoring both by time-resolved
light scattering and by direct visual inspection
(Blank et al., 1998a;
Blank et al., 1998b
;
Coorssen et al., 1998
). A flow
rate of
1 ml/s was used to wash CV lawns and exchange buffers. All
experiments used B-IM containing [Ca2+]free of
100
nM, 150 µM or 1 mM. Light-scattering signals were normalized so that
[Ca2+]free of
100 nM and 1 mM gave 0% and 100%
fusion, respectively. In all cases, aliquots of the same CV suspensions were
used to determine and verify Ca2+ activity.
The analysis of fusion is based on the number of active fusion
complexes/CV, a Poisson distributed random variable in which the average
number of committed fusion complexes,
, varies between 0 and
9
depending on [Ca2+]free
(Vogel et al., 1996
;
Coorssen et al., 1998
;
Blank et al., 2001
). Although 1
mM [Ca2+]free is routinely used to saturate
Max and establish the sigmoid
plateau,
100% fusion occurs at much lower
[Ca2+]free; cumulative log-normal sigmoid curves can be
characterized by the distribution midpoint and width and/or the midpoints and
corners. Thus, for comparing activity curves, both the width and the
[Ca2+]free associated with 5% and 50% fusion are used.
The relationships between extent of fusion (%F),
, and trypsin treatment are
%F=100x(1exp(
=
Maxx
exp([trypsin]/
), respectively, where
is the concentration
decay constant (Vogel et al.,
1996
). Changes in %F as a function of the trypsin concentration
were fit using the equation %F=100x
(1exp(
Maxxexp([trypsin]/
))).
For kinetic studies, low solution flow was used to minimize disruption of CV
on coverslips. Under these conditions, the transition from active to committed
state is not resolved; the kinetic data were fit using the original two-state
model and a temporal offset (Vogel et al.,
1996
; Blank et al.,
2001
). The initial rate of fusion can be approximated by
xp
(Vogel et al.,
1996
;Blank et al.,
2001
).With
p=
x
(Blank et al., 2001
), where
represents a characteristic efficiency constant, the initial rate can
be expressed as
x
2. This
function suggests that
is proportional to a rate constant, whereas
is analogous to a concentration
term. With
proportional to a fusion rate constant, the change in
energy resulting from papain treatment can be calculated using the
relationship
control/
papain=exp((EcontrolEpapain)/kT).
The exponential correlation between SNARE density and the midpoint of the
calcium activity curve, Midpoint=Ax exp(Density/B)+C, was
evaluated with weighting using 1/(s.e.)2 of the midpoint estimates.
The error in SNARE density was ignored in the fitting after determining that
the error range in the parameter estimates (95% confidence) was greater than
changes in the estimates arising from fitting the midpoint vs. the density
values shifted by ± the error in the density. Overall, the error in the
density divided by the density was 19%, 16% and 5% for VAMP, SNAP-25 and
syntaxin, respectively. We could determine syntaxin density to better than 1
molecule/CV.
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Results |
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|
|
Trypsin had a systematic and progressive affect on Ca2+ activity
(sensitivity and extent) (Fig.
2A; Table 1). 700
units/ml trypsin (1 hour) shifted Ca2+ activity rightward, but with
no loss in maximal extent of fusion elicited by a saturating
[Ca2+]free; the 5%/50% fusion level occurs at 3/17
µM. 3500 units/ml trypsin (1 hour) caused a further rightward shift in
Ca2+ activity and 30% loss of fusion. Prolonged incubations or
higher trypsin doses further digested certain CV proteins, correlating with
losses in Ca2+ activity and progressively greater inhibition of
fusion; 700 units/ml (3 hours) resulted in
70% inhibition (data not
shown). In comparison, 35,000 units/ml trypsin (1 hour) rendered CV fusion
incompetent (99±1% inhibition at saturating
[Ca2+]free). Like N-ethylmaleimide (NEM), trypsin
inhibited fusion in a manner consistent with a single, crucial site of action
and the number of these sites, n
Max=8.4±1.0, agrees with previous determinations
(Vogel et al., 1996
;
Coorssen et al., 1998
). By
contrast, papain (2000 units/ml, 1 and 4 hours) shifted and broadened
Ca2+ activity without altering the maximal extent of fusion
(Fig. 2A,B;
Table 1); 5%/50% fusion occurs
at 3/45 and 68/197 µM [Ca2+]free, respectively,
following 1 and 4 hour treatments. Clostripain (100 units/ml, 1 and 4 hours)
had minimal effect on Ca2+ activity
(Fig. 2A,B;
Table 1, 5%/50% fusion occurs
at 2/11 and 40/69 µM [Ca2+]free, respectively,
following 1 and 4 hour treatments), but distinct effects on the CV protein
profile (Fig. 2C). When added
hourly, fresh papain (but not clostripain) gradually inhibited fusion
(
20-30% fusion at saturating [Ca2+]free after 3
hours treatment, data not shown). In all cases, comparable results were
obtained after simply settling protease-treated CV into contact, confirming
that the measured fusion is dependent on proteins rather than on
centrifugation (Coorssen et al.,
1998
). Similar results were also obtained for exocytosis in vitro
after 1 hour treatment of CV-PM preparations
(Fig. 2D;
Table 2). Trypsin does not
appear to affect docking per se, as CV undocking was not observed. The effects
of papain (3000 units/ml) were less dramatic, suggesting limited access to the
CV-PM contact site. Thus, each of these proteases had a different effect on CV
fusion, enabling us to distinguish between changes in the Ca2+
sensitivity or extent of fusion.
|
To determine whether the differential protease effects on Ca2+
activity and fusion extent were due to differential proteolysis of SNARE
proteins, we used the fact that known SNAREs have substantial numbers of
potential trypsin, papain and clostripain cleavage sites throughout their
cytoplasmic domains (Table 3).
The initial number of accessible proteolytic cleavage sites is expected to be
lower due to structural constraints
(Hubbard et al., 1998;
Hubbard, 1998). Using the program Nickpred
(Hubbard et al., 1998
),
examination of SNAREs in the Brookhaven Protein Data Base predicted multiple
limited proteolytic sites. Subsequent loss of structure is expected to expose
additional sites to further proteolysis. Indeed, ladders of multiple
proteolytic fragments migrating below the main VAMP and SNAP-25 protein bands
were detected with lower protease concentrations or shorter treatment time (1
hour). Following 4 hour protease treatments, syntaxin appears to be either
intact or fully proteolyzed, as no fragments were detected
(Fig. 1). This observation is
consistent with the complete loss of syntaxin complex immunoreactivity
observed following trypsin proteolysis
(Lawrence and Dolly, 2002
).
SNAP-25 immunoreactivity, which may correspond to fragments, was detected in
both control and treated samples (Fig.
1); VAMP immunoreactivity often appeared as a doublet (data not
shown). The amounts of these SNARE fragments decreased with increased protease
concentration or treatment time and were included in the quantitative
determinations of SNARE density. Thus, unlike Clostridial toxins,
these proteases could destroy all known SNAREs (including toxin-insensitive
homologs).
|
Trypsin (700 units/ml, 1 hour) digested substantial amounts of VAMP,
SNAP-25 and syntaxin but had no effect on the extent of fusion
(Fig. 3). Increased trypsin
(3500 units/ml) removed >90% of VAMP and syntaxin; 60% SNAP-25
remained (Fig. 4). Higher
trypsin doses (Fig. 4) or
longer incubation times (700 units/ml, 3 hours) did not reduce SNAREs to
levels lower than detected after 1 hour with 3500 units/ml, but did block
fusion. Papain (2000 units/ml) and clostripain (100 units/ml) removed most
SNAREs after 1 hour; there was little added affect of 4 hour incubations.
Papain reduced VAMP, SNAP-25 and syntaxin by 94%, 91% and >99%,
respectively, and clostripain by 92%, 87% and 94%; CV remained fully
Ca2+ sensitive and 100% fusion competent
(Fig. 3). A post-protease wash
with chaotropic buffer resulted in some further loss of SNARE fragments
nonspecifically bound to CV, but there was no change in fusion at saturating
[Ca2+]free, indicating that free SNARE fragments were
not promoting fusion; although ineffective, these fragments were still
included in our density assessments to ensure exhaustive SNARE quantification.
Intact SNAREs are not removed by the chaotropic buffer wash (data not shown).
Post-protease fusion was not due to nonphysiological Ca2+-induced
membrane destabilization, or fusion would remain high after extensive
trypsinization; this was not the case (Figs
2,
3 and
Fig 4A; 35,000 units/ml
trypsin). Thus, despite SNARE removal, fusion activity remained. Furthermore,
when added hourly, fresh papain, but not clostripain, gradually inhibited
fusion despite comparable reductions in SNARE densities. Fusion appears to
require proteins other than the SNAREs.
|
|
Reducing syntaxin to <1 copy/CV (`biochemical knockout'; 0.1 or 0.4
copy/CV at the 95% confidence level for each of two samples with a range from
0-0.8) by the most potently SNARE-destructive treatment
(papain; 2000 units/ml replaced every hour for 3 hours)
(Fig. 5) decreased but did not
abolish the extent of fusion elicited by saturating
[Ca2+]free (two trials, final extent 20-30%). Eight more
trials with 2000 units/ml papain gave 100±1% fusion with only 1-<3
syntaxin copies/CV in 4 trials and >3 copies/CV in 4 trials; 50% fusion
occurred at 45 and
200 µM [Ca2+]free after 1 and
4 hour treatments, respectively. Overall, the reduced syntaxin density as a
function of papain treatment ranged from <1-80 copies/CV depending upon the
papain concentration (3005000 units/ml) and treatment time (1-4 hours).
For comparison, the reduced syntaxin density as a function of clostripain
treatment ranged from 5 to 50 copies/CV. If an individual SNARE complex
catalyzes fusion, and proteolysis is random, then Poisson analysis
(Vogel et al., 1996
;
Coorssen et al., 1998
;
Blank et al., 2001
) can be used
to predict the extent of fusion at saturating
[Ca2+]free. The extents of fusion predicted by the
measured syntaxin densities following papain treatment are not consistent with
the observed fusion (
2, P<0.05); neither syntaxin
nor syntaxin-containing complexes have the properties of a Poisson-distributed
fusogen. Three SNARE complexes per vesicle fusion event have been suggested
(Hua and Scheller, 2001
), and
it is thus significant that the maximum extent of fusion (100%) elicited by
saturating [Ca2+]free is independent of syntaxin density
from 1 to 330 copies/CV (papain treated relative to control).
|
Shifts in Ca2+ activity preserving fusion competence are
observed in both control (4 hours) and papain-treated samples, suggesting
disruption of a Ca2+ regulatory system mediating fusion. As
clostripain-treated samples did not have large shifts in Ca2+
activity relative to control and the Ca2+ activity midpoint was not
correlated with SNARE density (slope not significantly different from zero),
it is somewhat surprising that, for papain-treated samples, the midpoints of
the Ca2+ activity curve were correlated with SNARE density
(Fig. 6). For all three SNAREs,
the correlation between [Ca2+]free (EC50) and
SNARE density was described by the exponential relationship,
midpoint=Axexp(density/B)+C, where A+C is the midpoint
(EC50, µM) of the Ca2+ activity curve in the absence
of SNAREs and B is the density decay constant. The extrapolated midpoint at
high SNARE density (parameter C) varied from 13 to 20 µM, depending on the
SNARE, and is consistent with control midpoints (620 µM for 0, 1 and
2 hour incubations). When low-density values are normalized to the density at
1 hour and the high-density midpoint constrained to the value of the 1 hour
control (7.1 µM), all three SNAREs were described by a common exponential
decay with a low-density midpoint of 250 µM
[Ca2+]free. Thus, papain treatment reveals an intrinsic
Ca2+-dependent process with an activity midpoint that can be
modulated by proteolysis between
6 and
250 µM
[Ca2+]free. If this reflects the inherent
Ca2+ sensitivity of the native regulated fusion complex, then the
EC50 of this endogenous fusion complex is
250 µM
[Ca2+]free.
|
Decreases in the density of components crucial to triggered fusion are
predicted to inhibit the rate of fusion
(Vogel et al., 1996;
Blank et al., 2001
). As
clostripain (100 units/ml, 4 hours) failed to affect Ca2+ activity
(Fig. 2B) and had no effect on
fusion kinetics (Fig. 7A), but
did digest SNARES, it is unlikely that the identified SNARES in this system
are crucial to fusion. Papain (2000 units/ml, 4 hours) reduced the kinetics
and extent triggered by 150 µM Ca2+ but not the final extent of
fusion triggered by saturating Ca2+, indicating that both the
probability per unit time (p) and n decreased
(Fig. 7A). However, a limited
papain treatment (2000 units/ml, 3 hours) altered the kinetics triggered by
150 µM Ca2+ with minimal changes in the extent
(Fig. 7B). Analysis (see
Materials and Methods) ruled out a direct reduction in n ,
indicating that papain reduced P, the probability that any given
vesicle will fuse (Vogel et al.,
1996
; Blank et al.,
2001
). After 3 hours, p/n (a measure of
efficiency) in papain-treated CV is only
17% of control, but is
unaffected by clostripain (4 hours) (Fig.
7A). The changes in fusion rate after 3 hours papain treatment are
consistent with an increased energy barrier for fusion. Treating p/n
as a rate constant (see Materials and Methods), the energy contributed
by papain-sensitive proteins is estimated to be
2 kT (0.6 kcal/mol) per
fusion complex. This is about half that required to overcome the hydration
layer at membrane surfaces (Leiken et al., 1993) or to mediate molecular
rearrangements thought necessary for bilayer merger
(Kuzmin et al., 2001
). Thus,
papain (but not clostripain or low doses of trypsin) removes or modifies
crucial modulatory proteins that have pronounced effects on the efficiency of
triggered fusion.
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Discussion |
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Lack of a direct role for SNAREs in membrane fusion: alternate
interpretations
Our conclusion, that SNAREs are neither driving CV membranes together nor
inextricably linked to Ca2+-regulated fusion via an absolute
requirement for the presence of their cytosolic domains, is dependent on a
correct interpretation of SNARE proteolysis, antibody interactions and the
fusion process. Two classes of alternative hypothesis that preserve a direct
role for SNARE proteins can be considered. The first class is that the `wrong'
SNAREs were evaluated in this study: the necessary SNAREs were still present
on protease-treated CV at a sufficient density to support fusion. This class
can be subdivided into SNARE homologs and SNARE fragments that do not
cross-react with the antibodies used. The second class is that multiple fusion
pathways exist, and include SNARE-dependent and -independent mechanisms; the
latter can be subdivided into protein-mediated and protein-independent
mechanisms.
Do we have the right SNAREs?
The interpretation of genetic studies of SNARE activity can be influenced
by the presence of gene homologs (e.g.
Vilinsky et al., 2002). By
contrast, the biochemical knockout approach relies on the structural
conservation of putative homologs. The conservation of the different potential
protease cleavage sites in all known SNARE homologs is very high
(Table 3), particularly for the
trypsin, papain and clostripain cleavage sites closest to the PM (i.e.
cleavage at this site would result in the complete release of the cytoplasmic
domain) and in the potential coiled-coil domains postulated to be essential to
SNARE-mediated fusion. Thus, although we do not know all the epitopes
recognized by the antibodies employed, complete destruction of other SNARE
homologs should also occur under the conditions of our experiments. If this
interpretation is incorrect, then a putative protease resistant `SNARE' would
either have a sequence that is significantly different from all known SNAREs,
such that not even one functional proteolytic site exists (a feature that
would question the nature of SNARE homology), or exist in a stable
conformation such that its cleavage sites were never accessible to protease
action during the extended incubation times used in our experiments.
Antibodies raised specifically against nonconserved regions of the protein
can certainly identify different homologs of SNAREs. In this study, polyclonal
antibodies raised against the whole cytosolic region (e.g. including the
extended highly conserved domains) were used. Because SNAREs maintain highly
conserved regions it is more likely that our antibodies would cross-react with
any protein containing these conserved regions than would monoclonal
antibodies. We have used antibodies against SNAREs from other species in the
past (Coorssen et al., 1998;
Coorssen et al., 2002
;
Tahara et al., 1998
), and have
always identified only these SNAREs on the CV membrane. To accept the
alternate hypothesis, that SNAREs drive the fusion step, occult SNARE homologs
to (or proteolytic fragments of) syntaxin, VAMP and SNAP-25 need to exist in
protease-resistant conformations that are not recognized by our polyclonal
antibodies (i.e. lack all the epitopes recognized by the antibodies used here)
at all protein concentrations and dilutions assessed with an ultrasensitive
immunodetection protocol (Coorssen et al.,
2002
), and the putative proteolytic fragments would have to remain
stably bound in the presence of chaotropic buffers. Although this is an
alternative explanation, we think it simpler and thus more likely that
CVCV fusion can occur in either the absence of syntaxin or with 91-99%
overall loss of SNARE proteins. This interpretation is also most consistent
with the substantial differences in SNARE densities measured on native
secretory vesicles compared with those required for fusion in reconstituted
preparations (Weber et al.,
1998
; Coorssen et al.,
2002
). In the absence of SNARE and other modulatory influences,
the fusion process has an endogenous Ca2+-activity whose midpoint
is
250 µM [Ca2+]free.
Are there multiple protein-dependent or protein-independent fusion
mechanisms?
Our analysis of the Ca2+ activity and kinetics of fusion is
based on the hypothesis that an increase in [Ca2+]free
increases the number of participating fusion complexes and that these
complexes are randomly distributed among CVs
(Vogel et al., 1996;
Blank et al., 2001
). This
analysis has consistently explained the experimental data observed in this
system (Vogel et al., 1996
;
Blank et al., 1998a
;
Blank et al., 1998b
;
Coorssen et al., 1998
;
Tahara et al., 1998
;
Blank et al., 2001
;
Ikebuchi et al., 2001
).
However, if changes in the Ca2+ activity and the kinetics of fusion
do indicate a shift from one fusion pathway to another, then perhaps SNARE
proteins normally mediate fusion, but in their absence alternative mechanisms
become evident; redundant pathways may exist. This is an intriguing and
provocative alternative hypothesis. However, no changes occurred in kinetics
following clostripain treatment, despite substantial decreases in SNARE
density, whereas comparable decreases in SNARE density following papain
treatment did alter the kinetics of fusion. This is perhaps the most direct
evidence against an essential role for the SNAREs in fusion, as any change in
the density of an essential component of the fusion complex would be predicted
to inhibit the rate of fusion (Vogel et
al., 1996
; Blank et al.,
2001
). The differential effects of protease treatments are
difficult to reconcile with a simple switch from a SNARE-mediated to a
SNARE-independent, protein-mediated fusion pathway without postulating the
existence of multiple classes of SNAREs such that clostripain preserves the
SNARE-mediated pathway by acting only on nonfunctional SNAREs while papain and
trypsin target only the functional SNAREs. Rather, the demonstrated
relationship between SNARE density and Ca2+ activity, seen in the
papain data (Fig. 6), clearly
indicates the existence of a single fusion pathway (with an inherent
Ca2+ activity). The Ca2+ sensitivity of this fundamental
fusion pathway is modulated in the `physiological' range of
[Ca2+]free either by SNAREs coupled with other
modulatory components such as `Ca2+ sensors,' or by other
components alone that track the papain-dependent changes in SNARE density via
similar proteolytic sensitivity. Redundancy in modulatory factors may be a
conserved hallmark of triggered exocytosis; the data indicate that trypsin and
papain more broadly affect these modulatory factors than does clostripain.
A switch from a SNARE-mediated to a protein-independent mechanism (e.g.
Ca2+-mediated lipid fusion) can not explain the data because
extensive trypsinization ablates the fusion response, even to several
millimolar [Ca2+]free; if any of the fusion observed was
nonphysiological or purely lipid-mediated, then it should also have been
observed under these conditions. As the fusion response is also lost after
treatments with other broad-spectrum proteases (e.g. chymotrypsin; data not
shown), it is unlikely that purely lipid-mediated fusion occurs under our
experimental conditions. In fact, even when using pure lipid vesicles (LV) as
the target membrane (CVLV fusion; Ca2+-dependent fusion at
<60 µM [Ca2+]free), proteinaceous machinery is
still essential to the triggered fusion steps, as determined by thiol
sensitivity (Vogel et al.,
1992). The shift in the midpoint of the Ca2+ activity
curve for CVCV fusion never enters the range of
[Ca2+]free required to induce purely lipid fusion;
fusion of lipid membranes containing mixtures of neutral and negatively
charged lipids requires tens of millimolar [Ca2+]free
(Cohen et al., 1980
;
Cohen et al., 1984
;
Duzgunes et al., 1981
;
Coorssen and Rand, 1995
;
Zimmerberg and Chernomordik,
1999
). The Ca2+ activity curves for all conditions,
except for papain where the distribution of Ca2+ sensitivity is
believed to be significantly altered, indicate that >95% fusion occurs
between
10 and 160 µM [Ca2+]free, a range that
is consistent with the physiology of Ca2+-triggered exocytosis.
Because it takes time for the proteases to work, we must also consider a shift
in mechanism due to incubation time. However, the invariance of p/n
with incubation time (0-4 hours), indicating no change in the
estimated energy of fusion, makes a time-dependent shift in mechanism
unlikely.
Although extensive trypsinization (3500 units/ml, 1 hour) does not
simply create an environment for `lipid-mediated' fusion, this proteolytic
treatment did reduce VAMP and syntaxin to <10% of control, or
30
syntaxin/CV; a maximum of
30 syntaxin-limited complexes (1:1:1 VAMP,
SNAP-25, syntaxin complexes or syntaxin with other binding partners) are
possible per trypsin-treated CV. If syntaxin and fusion complexes are
stoichiometrically proportional, then reducing n
Max by 90%
(
Max, trypsin=0.84) would
still support >50% fusion at saturating [Ca2+]free.
If an individual syntaxin complex (trimeric SNARE complex or other)
(Lawrence and Dolly, 2002
)
causes fusion (i.e. is both fusogen and Ca2+ sensor)
(Sutton et al., 1998
;
Chen et al., 2001
;
Peters et al., 2001
) then,
assuming a uniform surface distribution, any contacting CV domains >130 nm
in diameter would ensure 100% fusion at saturating
[Ca2+]free (for CVs having 30 syntaxin/CV). But
extensively trypsinized CVs do not fuse
(Fig. 3,
Fig. 4A). As trypsin effects on
Ca2+ activity did not correlate with SNARE removal
(Fig. 2A,
Fig. 3,
Fig. 4A), other
trypsin-sensitive proteins must function in defining the activity curve for
Ca2+-sensitive fusion; notably, the trypsin sensitivity of
synaptotagmin is well established (Tugal
et al., 1991
).
SNAREs and the process of exocytosis
The simplest hypothesis is that there is only one endogenous mechanism of
protein-mediated fusion in this system, and our dissection of this mechanism
results in altered responses. According to this hypothesis, although SNARES
have key roles in the pathway of exocytosis, they are not members of any
minimal protein set required for Ca2+-triggered fusion in this
regulated exocytotic system certainly not in any capacity that
requires their intact cytosolic domains; their rapid intermembrane binding
does not appear to be essential to the minimal mechanism of native membrane
merger (Coorssen et al.,
1998). Of course, SNAREs are enormously important to the process
of exocytosis; there is good evidence that SNAREs affect an early stage of
target membrane recognition, vesicle docking and/or priming
(Pelham, 2001
;
McNew et al., 2000
;
Scales et al., 2000a
;
Scales et al., 2000b
). Indeed,
our new estimates indicate that SNAREs, together with other modulatory
proteins, can contribute energy to the fusion mechanism, although not enough
to directly trigger fusion, except perhaps at densities in excess of those in
native vesicle membranes (Weber et al.,
1998
; Coorssen et al.,
2002
). SNAREs probably play a role in trafficking and the
localization of CV to PM docking sites, which occurs during the oocyte-to-egg
transition (Berg and Wessel,
1997
), because fusion can be disrupted by clostridial toxins
injected into eggs whose CVs have been de-docked
(Bi et al., 1995
); these PM
docking sites may be associated with fusion complexes
(Ikebuchi et al., 2001
).
In other systems, genetic knockouts of VAMP and SNAP-25 affect the
regulation of exocytosis but not the ultimate capacity to release vesicular
contents (Nonet et al., 1998;
Yoshihara et al., 1999
;
Schoch et al., 2001
;
Rao et al., 2001
;
Washbourne et al., 2002
),
leading to two hypotheses that SNAREs catalyze fusion reactions by
stabilizing transition states (Schoch et
al., 2001
) and that multistep, Ca2+-dependent assembly
of SNAREs forces membranes together to promote fusion
(Xu et al., 1999
;
Chen et al., 2001
;
Scales et al., 2001
).
Considering the findings presented here, neither of these hypotheses is
probable because the rate of CV fusion (rather than of multiple late steps in
the exocytotic pathway) in clostripain-treated samples was independent of
SNARE density, and the removal of SNAREs and other modulatory proteins
suggests an inherent Ca2+ activity in the triggered steps of
fusion.
In regulated fusion, SNAREs may function as structural proteins that
prepare or optimize local release sites
(Coorssen et al., 1998),
perhaps by coordinating the right vesicles
(Rothman, 1994
;
McNew et al., 2000
) to the
right Ca2+ channels (Rettig et
al., 1997
; Jarvis et al.,
2000
), for which they then act as regulatory components. This
represents another interpretation of SNARE knockout data
(Schoch et al., 2001
;
Washbourne et al., 2002
). If
VAMP loss alters presynaptic architecture such that vesicles dock less
effectively (outside domains of increased intracellular Ca2+),
leading to diminished functional docking by
10%, then spontaneous release
and measures of docked, primed vesicles (i.e. sucrose pools) would be
diminished by
10% (Parsons et al.,
1995
) and the fast evoked response to Ca2+ would be
lost (Xu et al., 1998
).
A calcium regulatory complex that is papain sensitive
Although we do not yet know whether the fusion complex itself is inherently
Ca2+ sensitive, or whether this property is resident to an
associated sensor, this sensitivity can be correlated to SNARE density
following proteolysis by papain. This suggests a native papain-sensitive
complex that includes SNAREs and associated proteins. However, this complex is
not essential per se in the triggered fusion steps of exocytosis because in
our work higher [Ca2+]free overcame the block to fusion
(seen at `physiological' [Ca2+]free) caused by
proteolysis. Such recovery has also been documented in earlier experiments
with Clostridial toxins (Dreyer
and Schmitt, 1983;
Ahnert-Hilger and Weller, 1993
;
Bittner and Holz, 1993
;
Lawrence et al., 1994
;
Glenn and Burgoyne, 1996
;
Lawrence et al., 1996
;
Capogna et al., 1997
;
Land et al., 1997
;
Fassio et al., 1999
),
suggesting that the concept of a regulatory complex may extend beyond the
present system. In general, the testing and rescue of exocytosis at only one
[Ca2+]free after complex disruption tells us a great
deal about the modulatory role of the complex. Incomplete Ca2+
activity curves provide only a partial analysis of the pathway in question,
leading to models that explain only a very circumscribed stage of exocytosis,
upstream of the fusion steps. Complexes of SNAREs and accessory proteins may
promote fusion efficiency in vivo. Our finding that proteolysis by papain
reduced the probability of fusion supports this interpretation. Although
similar influences of SNAREs on the probability of fusion have been noted in
other systems (Finley et al.,
2002
; Stewart et al.,
2002
), the mechanism of this action was not discernible; here, we
hypothesize that a complex of SNAREs and associated proteins may modulate the
inherent Ca2+ sensitivity of the native fusion complex, and that
these modulatory proteins are more sensitive to papain than to
clostripain.
In summary, the isolated CV preparation is unique in its stage specificity,
allowing the dissection of post-SNARE, Ca2+-dependent steps of
exocytosis. Proteolysis has revealed two different functional activities
involving the Ca2+ sensor(s) and the fusogen, in accordance with
other studies indicating the involvement of additional proteins acting after
SNAREs (Coorssen et al., 1998;
Tahara et al., 1998
;
Ungermann et al., 1998
;
Peters et al., 2001
). In the
current study we achieved a biochemical `knockout' of syntaxin in
stage-specific native secretory vesicles, without the loss of triggered
fusion. These studies have also provided intriguing estimates of the inherent
Ca2+ sensitivity of the endogenous fusogen-fusion complex separate
from a regulatory complex that appears to include SNAREs.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
The error in determining Copy #/CV is:
Copy #/CV=Copy
#/CVx[(
fmoles/fmoles)2+(
Cv
count/CV count)2+(
Dilution factor/dilution
factor)2+(
Extraction efficiency/Extraction
efficiency)2]0.5.
Assuming that the relative error for each of these terms is 5% (0.05), an
extremely conservative value for all terms except the extraction efficiency,
then the total error can be approximated by Copy #/CV=Copy
#/CVx0.1.
Using a weighted average of the results obtained at the two different
protein loads (30 and 15 µl of sample), the average copy numbers are
0.107±0.008 and 0.404±0.032. If the calculated error is
representative of the underlying error then the Copy #/CV is significantly
less than one copy at the 99% confidence level (3xCopy #/CV). If
the calculated error represents an estimate of the unknown underlying
distribution then the appropriate t value with one degree of freedom (two
samples were averaged, n1 degrees of freedom) must be used. In
this case, the t value for one degree of freedom at the 95% confidence
interval is 12.71 and the error would be 0.107±0.102 and
0.404±0.407; these values also indicate significantly less than one
copy at the 95% confidence level. Therefore, within >95% probability, these
two experiments show that our determination of less than one copy of syntaxin
per CV is statistically significant.
![]() |
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