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INTRODUCTION |
The mixing of specific membrane compartments by the process of
fusion is a highly regulated event in eukaryotic cells. The fusion
process is exquisitely controlled in a manner that does not disturb the
structural and functional identity of organelles (1, 2). Several
proteins that are involved at different steps in the transport process,
from the formation of a transport vesicle until its fusion with the
target membrane, have been identified (3-7). The
SNARE1 proteins play a
crucial role in the process of intracellular membrane fusion. For
fusion to occur, transport vesicles with distinct v-SNAREs have to pair
with cognate t-SNAREs at the appropriate target membrane. Syntaxin and
SNAP-23/25 are members of the t-SNARE family of proteins, whereas
synaptobrevin/vesicle-associated membrane protein are v-SNARE
proteins (3-7).
Syntaxin, SNAP-25, and vesicle-associated membrane proteins mediate
fusion through the formation of helical bundles that span opposing
membranes. Hydrophobic C-terminal domains anchor syntaxin and
vesicle-associated membrane proteins to the lipid bilayer of the plasma
and vesicle membranes, respectively. The crystal structure of the SNARE
complex has identified the regions of SNAP-25 that are involved in
binary and ternary interactions (8, 9). SNAP-25 and its non-neuronal
homolog, syndet/SNAP-23 (10, 11) possess cysteine residues clustered in
a relatively unstructured segment between two helical domains. The
cysteine residues in this segment are palmitoylated (12-14). Mutants
of syndet and SNAP-25 lacking cysteines, and therefore not
palmitoylated, are localized predominantly in the cytoplasm of
transfected cells (15, 16). Based on mutagenesis of SNAP-25, a minimal
plasma membrane-targeting domain composed of residues 85-120 has been
identified. This segment has the ability to target green fluorescent
protein to plasma membranes in transfected cells (17). There
have been reports which suggest that cysteines and consequently
palmitoylation, are not essential for targeting of SNAP-25 to
membranes, as membrane association is facilitated by syntaxin 1A (18).
However, recent reports suggest that membrane targeting of SNAP-25 is
independent of interaction with syntaxin 1A and the cysteine-rich
domain is necessary for function in intact cells (19).
To understand the role of the proposed membrane targeting segment of
the SNAP family of proteins in membrane assembly of the parent protein
and its possible role in the fusion process, we have studied the
interaction of synthetic peptides spanning the cysteine-rich region of
SNAP-23, with and without covalently linked palmitic acids, with model
and natural membranes. A schematic sketch showing the various domains
of SNAP-23, the primary structure of the segment with the cysteine
cluster in SNAP proteins from different species, and the synthetic
peptides investigated in the present study are presented in Fig. 1,
A and B. We observed that palmitoylation is
essential for the peptides to cause membrane fusion. Our results also
provide insights into dependence of membrane fusion on peptide length,
position of cysteines, and extent of palmitoylation.
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EXPERIMENTAL PROCEDURES |
Materials--
Fmoc amino acids used in peptide syntheses were
purchased from Advanced Chemtech (Louisville, KY). All phospholipids
including those labeled with fluorescent tags were purchased from
Avanti Polar Lipids (Birmingham, AL). Calcein was from Sigma. All other chemicals were of the highest grade commercially available.
Peptide Synthesis and On-resin Acylation--
Peptides were
synthesized manually using solid phase procedures and Fmoc chemistry.
Cysteine sulfhydryl groups were blocked with acetamidomethyl group. The
N termini of peptides were acetylated in a mixture of acetic
anhydride/triethylamine/dimethyl formamide (0.1:0:0.02:1, v/v).
Palmitoylation was carried out as described before (20). Briefly,
cysteine sulfhydryl groups of resin-bound peptides were deprotected
using 5 eq of mercuric acetate (per cysteine) in dimethyl formamide for
1 h, followed by treatment with 50 eq of
-mercaptoethanol for
another hour. The procedure was repeated three times to ensure complete
removal of acetamidomethyl. On-resin palmitoylation of free -SH groups
was carried out using palmitic acid activated with
N-hydroxybenzoatriazole and
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Peptides were cleaved from the resin purified by
reverse phase high performance liquid chromatography/reverse phase fast
performance liquid chromatography and characterized by matrix-assisted
laser desorption ionization-time of flight mass spectrometry. Expected
mass was observed for the nonpalmitoylated and palmitoylated peptides.
Stock solutions were made in trifluoroethanol or dimethyl sulfoxide.
Peptide concentration was determined by monitoring absorbance at 280 nm.
Preparation of Lipid Vesicles--
Lipid vesicles with
compositions PC, PC:PE (1:1), PC:PE:SM:CL (1:1:1:1.5), and PC with 30 mol % cholesterol were prepared by the extrusion method or by
sonication. Lipid stocks were diluted in chloroform to appropriate
molar concentrations and dried to form a thin film. The lipid films
were left under vacuum overnight to remove residual solvent and
subsequently hydrated in 5 mM HEPES, pH 7.4, containing 150 mM NaCl. SUV were obtained by sonication of the lipid
suspension. LUV were obtained by extrusion through polycarbonate
filters of 100 nm pore size (21). For circular dichroism studies,
peptide was added to lipid solution in chloroform and dried to form a
mixed film. The film was hydrated and SUV were prepared by sonication.
Fusion Assay in the Presence of Peptides--
Resonance energy
transfer between NBD-PE (energy donor) and Rho-PE (energy acceptor) was
used to monitor membrane fusion (22). Two populations of liposomes were
prepared, one labeled with 2 mol % each of NBD-PE and Rho-PE and the
other unlabeled. The labeled and unlabeled vesicles were mixed at a
ratio of 1:9 to a final concentration of 200 µM in 1.2 ml
in 5 mM HEPES containing 150 mM NaCl. Peptides
were added and after 5 min, fluorescence was recorded with excitation
at 465 nm and emission set at 530 nm. The slit widths of the excitation
and emission monochromators were 5 nm. Percent fusion was calculated
using the equation (F
Fo)/(Ft
Fo) × 100, where Fo = fluorescence intensity before adding peptide, F = fluorescence intensity after adding peptide, and Ft = fluorescence intensity after complete dilution by detergent (Triton
X-100). Fusion assays were done in the presence of 10 mM
dithionite or lysophosphatidylcholine (LPC) (23) as follows. The
labeled vesicles were incubated on ice for 1 h in the presence of
10 mM dithionite. Excess dithionite was removed by passing
through Sephadex G-50 spin columns. To check the effect of LPC, 10 µg/ml LPC was added to the vesicles before recording the RET changes
induced by the peptides. Three independent experiments were carried out.
Binding of Peptides to Lipid Vesicles--
Peptides (5 µM) were titrated with increasing amounts of lipid
vesicles. The tryptophan fluorescence was monitored with the excitation
monochromator set at 280 nm, and the emission spectrum was recorded
from 320 to 400 nm with 5-nm slit widths. All fluorescence measurements
reported in this study were done in a F-4500 Hitachi fluorescence
spectrometer. The accessibility of tryptophan to the aqueous quencher
iodide in the presence of lipid vesicles was determined as follows. KI
was added in increasing amounts from a 4 M stock containing
1 mM Na2S2O3. The data
were analyzed according to the Stern-Volmer equation,
Fo/F = 1 + KSV[Q], where Fo
and F represent the fluorescence intensities in the absence
and presence of quencher [Q], respectively and KSV is the Stern-Volmer quenching constant.
Normalized accessibility factor was calculated from the ratios of
KSV at a lipid:peptide ratio of 50:1 and
KSV for peptide in the absence of lipid.
Circular Dichroism (CD) Spectroscopy--
Spectra were recorded
in a JASCO J-715 spectropolarimeter in water and also in SUV composed
of PC:PE:SM:CL (1:1:1:1.5). Scanning was done in the wavelength range
of 195 to 250 nm. Path length was 1 mm. Data are represented as mean
residue ellipticity.
Content Leakage Assay--
Lipid films formed after drying in
vacuum were hydrated in 70 mM calcein solution made in
HEPES buffer, pH 7.4. SUV were prepared by sonication. Untrapped
calcein was separated by gel filtration on a Sephadex G-75 column in 5 mM HEPES containing 150 mM NaCl and 5 mM EDTA, pH 7.4. Release of calcein on addition of peptides was monitored with excitation at 490 nm and emission at 520 nm. Percent
release was calculated from the equation (F
Fo)/(Ft
Fo) × 100, where Fo and
F are fluorescence intensities in the absence and presence
of peptides and Ft is the fluorescence intensity
after adding Triton X-100.
Preparation of Pancreatic Zymogen Granules and Plasma
Membranes--
Granules and membranes were isolated as described by
Nadin et al. (24). The precautions mentioned by Edwardson
(25) were taken. Granules from a single pancreas were suspended in 500 µl of 5 mM MES containing 280 mM sucrose, pH
6.0, 1 mM phenylmethylsulfonyl fluoride, 1 µg of
pepstatin, and 1 mM EDTA was also included. Protein was
assayed by the Bradford method (26). Pancreatic membranes were prepared
by method of Meldolesi et al. (27). EDTA and pepstatin were
included in the buffers during processing. Membranes were resuspended
in 5 mM MES containing 280 mM sucrose, pH 6.5, and stored in aliquots at
20 °C at a protein concentration of 1 mg/ml. KCl-treated membranes were prepared by incubating the membranes
in 0.5 M KCl for 30 min at 4 °C (25). Membranes were
recovered by centrifugation at 70,000 × g for 30 min.
The pellets were resuspended in 0.5 M KCl and the recovery
step was repeated. The membrane pellet was washed in sucrose/MES buffer and resuspended at a protein concentration of 1 mg/ml in the same buffer.
Fusion between Zymogen Granules and Pancreatic Plasma
Membranes--
Membrane fusion was assayed by the measurement of
relief of self-quench of fluorescent probe R18 (28). 1 µl of
octadecylrhodamine (R18) from a 20 mM stock solution in
ethanol was added to 300 µl of granule suspension and incubated at
37 °C for 5 min. Granules were collected by centrifugation at
900 × g for 10 min and resuspended in the original
volume. In the fusion assay, 5 µl of the granules (~10 µg of
protein) were resuspended in 600 µl of MES buffer, pH 6.5, containing
1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 1 µg/ml pepstatin, and 280 mM sucrose.
Fluorescence was recorded with excitation at 560 nm and emission at 590 nm. After equilibration for 3 min, the granules were challenged with
target membranes (~20 µg of protein). In the case of KCl-treated
membranes, ~30 µg of protein was used. Other components like
peptides, Ca2+, and EGTA were added to the cuvette in
accordance with the experimental requirement. Percent fusion was
calculated considering the fusion induced by 1 mM
Ca2+ as 100%.
Preparation of Resealed Ghosts--
Red blood cells were
isolated from rat blood and resealed ghost membranes were prepared
using the method described by Mobley et al. (29).
Phospholipid concentration of the resealed ghosts was estimated by the
Fiske-Subbarow method (30). The ghost membranes were resuspended in
incubation buffer (10 mM sodium phosphate, pH 7.4, containing 120 mM KCl, 30 mM NaCl, and 1 mM EDTA). Fresh preparations were used in all the experiments.
Lipid Mixing between Red Cell Membranes--
Lipid mixing was
analyzed by measuring dequenching of R18 (29), by a procedure similar
to the zymogen granule plasma membrane fusion assay. Resealed ghost
membranes were incubated with R18 at a final concentration of 1 mol of
R18/50 mol of red cell lipid for 30 min at room temperature. Unbound
probe was removed by repelleting the ghosts at 22,000 × g for 20 min and washed in the incubation buffer. For the
assay 50 µM phospholipid containing labeled and unlabeled
ghosts at a molar lipid ratio of 1:4 were taken in incubation buffer.
Fluorescence was recorded for 3 min before the addition of peptides.
Peptides were added and fluorescence changes were monitored as a
function of time. Fluorescence intensity of intact R18-labeled red cell
ghosts was 20-30% that of labeled ghosts treated with 0.1% Triton
X-100. Percent fusion was calculated using the formula: 100 × ((F
Fo)/(Fmax
Fo)), where F, Fo, and
Fmax are the fluorescence intensities before
addition of peptide, after addition of peptide, and after complete
dilution of the probe by addition of 1% Triton X-100.
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RESULTS |
Syntheses of Peptides--
Peptides corresponding to 14 or 27 residues of the membrane-targeting domain of human SNAP-23 were
synthesized by solid phase methods. Four of the five cysteines present
in this domain are conserved in human SNAP-25. In 27P4, the
nonconserved cysteine was replaced with phenylalanine, as observed in
the sequence of human SNAP-25 (Fig. 1).
To generate peptides with two palmitic acids, two cysteines were
substituted with serines (27P2 and 14P2). In Sc27P4, the cysteines at
positions 6 and 8 that are present in 27P4 were exchanged with the
amino acids at positions 18 and 24, to spread out the cysteines and
examine whether their clustering was important for activity. The amino
acids after the cysteine cluster in 27P4 were also scrambled (Sp27P4)
with a view to examine the role of this segment in membrane fusion.
Palmitoylation was carried out with peptides attached to the resin
after removal of the cysteine thiol protecting group. Cleavage from the
resin yielded the palmitoylated peptides. In the 14-residue peptides, tryptophan was introduced at the N terminus to facilitate quantitation as well as to monitor interaction with lipids by fluorescence spectroscopy.

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Fig. 1.
Schematic sketch of SNAP-23, sequences of the
region containing the cysteine cluster from various mammalian species
and peptides synthesized. A, sketch showing the highly
conserved coiled-coil domains of SNAP-23 and the cysteine cluster
representing the palmitoylation sites. Sequences of human SNAP-23 and
the corresponding regions of SNAP proteins from various species within
the palmitoylation domain and the adjacent residues are indicated. The
conserved cysteine residues in the SNAP proteins are
underlined. B, sequence of the peptides
synthesized. Cysteine residues that have been palmitoylated are
underlined. Corresponding nonpalmitoylated peptides are
denoted as 27C4, 27C2, Sc27C4,
Sp27C4, 14C4, and 14C2,
respectively, and indicated in parentheses.
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Fusion of Liposomes--
The ability of peptides to fuse lipid
vesicles of varying composition was examined by a
fluorescence-dequenching assay (22) that monitors lipid mixing. In this
assay, NBD-PE and Rho-PE are present in "donor" liposomes and
fluorescence of NBD is quenched as a result of energy transfer. When
donor liposomes fuse with unlabeled "acceptor" liposomes,
the average distance between the fluorophores increases, which results
in an increase of NBD fluorescence. The ability of 27P4 to fuse SUV and
LUV of varying lipid composition is shown in Fig.
2A. Considerable fusion is
observed with vesicles composed of PC:PE:SM:CL. LUV fused more
effectively than SUV. Because maximum fusion was observed with
PC:PE:SM:CL vesicles, the fusogenic ability of other peptides related
to 27P4 was examined on SUV and LUV with this composition. The data
shown in Fig. 2B indicate that 27P4 causes maximum fusion.
27P2 and 14P4 show similar extents of fusion, which is less than 27P4
but comparable with Sc27P4. The shorter peptide with two palmitic acids
causes fusion to a considerably lower extent. The peptide Sp27P4 is
marginally less effective in causing fusion at high peptide
concentrations as compared with 27P4. However, the extent of fusion
caused by Sp27P4 is more than the other peptides. The four palmitic
acids clustered at the N terminus appear to significantly influence the
fusogenic properties of the peptides. A peptide corresponding to a
segment of the Sendai virus F1 protein (IKLTQHYFGLLTAFGSNGTIG) that has
been demonstrated to induce fusion of lipid vesicles (31) is less
efficient than 27P4 and Sp27P4 in causing fusion.

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Fig. 2.
Fusogenic activity of the palmitoylated
peptides. A, fusion of SUV and LUV by 27P4 at a
peptide/lipid ratio 0.01. Lipid composition of vesicles were:
1, PC:PE:SM:CL (1:1:1:1.5); 2, PC:PE (1:1),
3, PC; 4, PC:CL (30 mol % CL). B,
percent fusion of SUV and LUV of lipid composition PC:PE:SM:CL
(1:1:1:1.5) in the presence of various peptides with increasing
peptide/lipid ratios. SV-208 is a fusion peptide derived from Sendai
virus F1 protein. The sequence of the peptide is
IKLTQHYFGLLTAFGSNGTIG.
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Effect of Lysolipid on Membrane Fusion and Involvement of Inner and
Outer Leaflets--
Hemifusion is an intermediate state where the
outer membrane leaflets mix to form a stalk structure (32-38).
Biological membrane fusion is presumed to proceed through this state.
Lysolipids stabilize positive curvature upon integration in the outer
leaflet because of their inverted cone geometry (38). Because
hemifusion is associated with curvature of the outer leaflet,
lysolipids are inhibitory to fusion before membrane merger (37). We
investigated whether peptide-mediated fusion involved a hemifusion
intermediate, and the ability of the peptides to convert the hemifusion
state to complete fusion wherein the inner leaflet lipids of the fusing membranes are also involved. The data presented in Fig.
3A indicate reduction of
fusion in the presence of LPC for all the five peptides that cause
fusion. This suggests hemifusion as a possible step in the fusion
process. To examine whether peptide-mediated fusion involved both
membrane leaflets, liposomes where the fluorescence of NBD present in
outer leaflet was quenched by dithionite treatment, were used in the
fusion assay (39). A considerable amount of resonance energy transfer
was observed even after the quenching of outer leaflet fluorescence by
dithionite (Fig. 3B). This shows that the peptides induced
lipid mixing in the inner leaflet along with the outer leaflet. The
efficiency of the inner leaflet mixing by these peptides appears to be
a little lower than the outer leaflet mixing.

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Fig. 3.
Characteristics of peptide-mediated model
membrane fusion. A, effect of the addition of LPC (10 µg/ml), an indicator for the existence of a hemifusion intermediate
on the fusogenic property of peptides. Percent fusion induced by
the palmitoylated peptides in the presence and absence of LPC at
peptide/lipid ratio 0.005 is shown. The liposomes were composed of
PC:PE:SM:CL (1:1:1:1.5). B, extent of inner leaflet mixing
upon bleaching of the outer leaflet NBD fluorescence by 10 mM dithionite is compared with the leaflet mixing in
untreated liposomes with composition as indicated in A. The
peptide/lipid ratio was 0.005. Inset, the initial NBD
fluorescence intensity with and without bleaching by dithionite. 60%
reduction in initial NBD fluorescence indicates complete bleaching of
outer leaflet.
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Environment of Trp in the Presence of Lipid Vesicles--
Because
the fluorescence of Trp is sensitive to environment (40), the
fluorescence of Trp in synthetic peptides (described in Fig. 1) in the
presence of lipid vesicles was monitored. All the peptides have a
single Trp residue. It was observed that the nonpalmitoylated peptides
when excited at 280 nm showed an emission maximum at 350 nm in buffer.
Addition of lipid vesicles did not result in change in the fluorescence
properties suggesting that the peptides do not bind to lipid vesicles.
This observation is also consistent with the inability of these
peptides to fuse lipid vesicles. Changes in fluorescence were observed
when lipid vesicles were added to the palmitoylated peptides (Fig.
4A). A linear increase in
F/Fo was observed for 14P2 with various
lipids. For 27P2, Sp27P4, and Sc27P4, the
F/Fo plateaued at a lipid/peptide ratio
of 20, whereas in 14P4 and 27P4, the plateau was observed at a ratio of
10. The data indicate that the tetrapalmitoylated peptides with
clustered cysteines bind strongly to lipid vesicles irrespective of the
nature of the head group. The accessibility of Trp to iodide was then
examined. The data in Fig. 4B indicate that while Trp is
solvent exposed in the nonpalmitoylated peptides, it is shielded for
the aqueous quencher in the palmitoylated peptides. The Trp is
relatively less accessible in the palmitoylated 27-residue peptides as
compared with the 14-residue peptides. Despite difference in the
fusogenic properties, the Trp location seems to be similar in 27P2,
27P4 and Sc27P4 and Sp27P4.

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Fig. 4.
Peptide association with model membranes
monitored by changes in tryptophan fluorescence. A,
relative increase in the tryptophan fluorescence of peptides upon
titration with SUV with lipid compositions PC ( ), PC:CL ( ), PC:PE
( ), PC:PE:SM:CL ( ). Lipid ratios were as described in the legend
to Fig. 2A. Nonacylated peptides did not show any increase
in tryptophan fluorescence, on titration with lipid vesicles.
B, accessibility of tryptophan to aqueous quencher
(I ). The normalized accessibility factors
(naf) were calculated as described under "Experimental
Procedures" from KSV of the peptides in the
presence of liposomes composed of PC ( ), PC:CL ( ), PC:PE ( ),
and PC:PE:SM:CL ( ).
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Conformation of Peptides--
The conformations of the peptides
were examined in buffer and lipid vesicles (Fig.
5). The palmitoylated peptides were not ordered in buffer and in the presence of lipid vesicles showed minima
at ~208 and 222 nm, which is characteristic of helical conformation
(41). The nonpalmitoylated peptides did not show ordered structure in
buffer and lipid vesicles. These differences could arise as a result of
the palmitoylated peptides binding to lipid vesicles and lipid-induced
conformational changes in 27P2 and 27P4.

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Fig. 5.
Secondary structure of peptides.
Circular dichroism spectra of the peptides recorded in water ( ) and
lipid ( ) vesicles composed of PC:PE:SM:CL (1:1:1:1.5).
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Leakage of Vesicle Contents--
Whether the fusion process is
accompanied by leakage of vesicle contents was next examined. The data
presented in Fig. 6A indicate
that a maximum release of 40% was observed with 27P4. No further
release was observed beyond a peptide/lipid ratio of 0.03. Release of
calcein as a function of lipid composition was examined for 27P4 (Fig.
6B). Unlike fusion, substantial release of calcein was also
observed with PC vesicles. Calcein release was less pronounced in the
presence of other palmitoylated peptides, which were less effective in
causing fusion. However, concentrations of the peptides at which there
was discernible calcein release were higher than the concentrations at
which they induced leaflet mixing.

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Fig. 6.
Membrane permeabilization measured by the
release of entrapped calcein. A, percentage of calcein
released by the palmitoylated peptides with an increasing peptide/lipid
ratio from liposomes with the composition PC:PE:SM:CL (1:1:1:1.5).
B, percentage of calcein release induced by peptide 27P4 at
a peptide/lipid ratio 0.04 from liposomes. 1, PC:CL;
2, PC; 3, PC:PE; 4, PC:PE:SM:CL. The
ratios of the various lipids are as described in the legend to Fig.
2A. Calcein release was measured 5 min after addition of
peptide. Beyond this time no further release was observed.
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Fusion of Natural Membranes--
The ability of the peptides to
fuse membrane vesicles with plasma membranes in vitro was
investigated using zymogen granules and pancreatic plasma membranes.
Their fusogenicity has also been compared with that of
Ca2+, which has been shown to stimulate fusion between
zymogen granules and plasma membranes in vitro (25, 28). The
data shown in Fig. 7A indicate
that 27-residue peptides have the ability to fuse the granules to the
plasma membrane. Ca2+ was more effective than the peptides
in inducing fusion. The extent of fusion caused by 27P4 and Sp27P4 was
similar. The shorter palmitoylated peptides as well as SV208 peptide
did not cause fusion. When the extent of fusion was normalized with
respect to Ca2+, 27P4 showed 50% fusion. Fusion by 27P4
was similar in the absence and presence of EGTA, indicating that
peptide-mediated fusion was Ca2+-independent. The
dependence of fusion on peptide concentration is shown in Fig.
7B. 27P4 and Sp27P4 showed similar extents of fusion at
various peptide concentrations used in the experiment.

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Fig. 7.
Fusion between zymogen granules and plasma
membranes. A, membrane fusion monitored by dequenching
of R18-loaded zymogen granules with pancreatic plasma membranes.
1, Ca2+; 2, 27P4; 3,
Sp27P4; 4, 27P2; 5, Sc27P4; 6, 14P4;
7, SV208; 8, 27C4; 9, control.
Inset, fusion induced by peptide 27P4 in the presence or
absence of 1 mM EGTA. Percent fusion was calculated
considering the fusion caused by Ca2+ as 100%. The
concentration of peptides and Ca2+ are 1 µM
and 1 mM, respectively. B, dependence of fusion
on peptide concentration. In both A and B, the
R18 fluorescence of zymogen granules was recorded for 2 to 3 min, after
which they were challenged with plasma membranes and other components.
The dip in the fluorescence intensity arises from the addition of
membranes and various components during the process of recording.
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The relation between Ca2+ and peptide-mediated fusion was
investigated (Fig. 8). Fusion mediated by
Ca2+ was observed to be independent from peptide-mediated
fusion. As shown in Fig. 8A, when fusion was initiated by
peptide, addition of Ca2+ resulted in an increase the
membrane mixing to an extent equal to that caused by Ca2+
alone. But when fusion was initiated by Ca2+, no further
increase in fusion was observed with the addition of peptides. A
possible explanation for this observation could be that the
Ca2+-induced lipid mixing is the maximum attainable for
this system under the conditions of the study and therefore addition of
peptides could not result in a further increase in the fusion. From the results shown in Fig. 8B, it was evident that
Ca2+-mediated fusion was inhibited by EGTA, whereas
addition of EGTA did not affect peptide-mediated fusion.
Ca2+ was unable to increase lipid mixing in the presence of
EGTA. Thus, peptide- and Ca2+-mediated fusion are
independent events. To examine whether peripherally bound proteins are
involved in the fusion process, the effect of KCl treatment on fusion
was investigated. The data shown in Fig.
9 indicate that salt wash abrogates
Ca2+-mediated fusion but not fusion caused by peptides.
This further confirms that Ca2+- and peptide-induced fusion
are independent events.

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Fig. 8.
Effects of EGTA and Ca2+ on
peptide-induced fusion between zymogen granules and plasma
membrane. A, effect of peptides on
Ca2+-induced fusion and effect of Ca2+ on
peptide-induced fusion for 27P4 and 27P2. B, effect of
addition of Ca2+ on fusion initiated by 27P2 and 27P4 in
the presence of 10 mM EGTA. In both A and
B, the Ca2+ and peptide concentrations are 1 mM and 1 µM, respectively. Control indicates
the basal level of fusion between the zymogen granules and plasma
membranes. The experimental conditions for recording R18 fluorescence
were same as those mentioned in the legend of Fig. 7. A second dip in
the fluorescence intensity seen here arises from the addition of
peptides/Ca2+ during the process of recording.
Arrows indicate the time at which the various components
were added.
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Fig. 9.
Requirement of peripheral proteins on the
plasma membrane for peptide-induced and Ca2+-induced
fusion. A, fusion induced by the peptides, 27P2, 27P4,
and Ca2+ prior to treatment of the plasma membrane with
KCl. The concentrations of the peptides were 1 µM and
that of Ca2+ and EGTA were 1 mM. B,
fusion after treatment of plasma membranes with 0.5 N KCl.
The amount of Ca2+, EGTA, and the peptides used was the
same as in A. Control indicates the basal level
of fusion between the zymogen granules and plasma membrane.
Experimental conditions for R18 fluorescence measurements were the same
as indicated in the legend to Fig. 7.
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The fusogenic ability of synthetic peptides on other membrane systems
like red blood cell ghost membranes was investigated. The data shown in
Fig. 10 indicate that the palmitoylated
27-residue peptides also cause fusion of red blood cell ghost
membranes. In this system, the SV208 peptide also causes fusion,
although to a lower extent.

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Fig. 10.
Fusion between red blood cell ghost
membranes induced by peptides. A, peptides (2 µM) were added to a suspension of R18-labeled and
unlabeled ghost membranes. Time-dependent changes in R18
fluorescence are shown. 1, 27P4; 2, 27P2;
3, SV208; 4, 27C4, 5, 14P4;
6, 14C4, 7, control. Fusion (%) was calculated
as described under "Experimental Procedures." B, peptide
concentration-dependent fusion between red blood cell ghost
membranes.
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DISCUSSION |
The fusion events in eukaryotic cells are orchestrated events,
which are finely tuned by interaction with various proteins of the
SNARE family (1-7). SNAP-23/25, a crucial player in the fusion process
(8, 9) is a fairly hydrophilic protein but membrane-associated. The
protein does not have equivalents of "fusion peptide segments" that
exist in viral fusion proteins (42). However, it has multiple cysteine
residues, which are proximal to each other in a central domain.
Continuous stretches of cysteines are present in cysteine string
proteins, which are known to be involved in exocytosis (43). Most of
these cysteines are palmitoylated. In cysteine string proteins, the
cysteines and hence palmitoylation could be essential for membrane
targeting (43, 44). The role of cysteines and palmitoylation in the SNAP family proteins with respect to membrane attachment and fusion is
yet to be clearly elucidated. We have investigated the membrane binding
and the ability of peptides corresponding to the cysteine-containing SNAP segment to cause fusion of natural and model membranes.
Our studies indicate that palmitoylation is necessary for binding to
lipid vesicles. In the shorter 14-residue peptide, four covalently
linked palmitic acids are essential for fusion. The 27-residue peptide
where the four cysteines are clustered (27P4) caused maximum fusion.
When the amino acids after the cysteine cluster were scrambled, there
was a slight decrease in the extent of fusion of model membranes but
not natural membranes. The peptide Sc27P4 with relatively evenly spaced
out cysteines was less effective in causing fusion and the efficiency
was comparable with that of 27P2 where only two palmitic acids are
present at the N terminus. Hence, it appears that four palmitic acids
linked to a cluster of cysteines is an important determinant for
perturbation of the lipid layer so as to promote vesicle fusion. All
the acylated fusogenic peptides in this study fused vesicles that had
the lipid composition PC:PE:SM:CL with a higher efficiency compared
with lipid vesicles of other compositions. This observation could be relevant as there are reports that SNAREs might be clustered in sphingolipid cholesterol rafts in polarized apical sorting (45, 46).
This lipid composition is also very close to natural synaptic vesicle
membrane composition and the model membranes composed of these four
lipids have been shown to exhibit optimized fusogenic capabilities
(47).
The fusion process appears to proceed via a transient hemifusion state
as evident from sensitivity to lysolipid. Fusion also involves both
bilayer leaflets as indicated by bleaching of NBD in the outer leaflet
by dithionite. However, unlike fusion caused by peptide mimics of SNARE
(48) and vesicular stomatitis virus-G protein transmembrane
segments (23), Ca2+ does not potentiate the fusogenic
property of 27P4. Even in the case of fusion of zymogen granules to
plasma membranes, the process appears to be independent of
Ca2+ and also independent of peripheral membrane proteins.
Although proteins play crucial roles in the regulation of biomembrane
fusion, the process requires coming together and coalescence of lipid
bilayers. The role of lipids in membrane fusion has been addressed
(49-51). Defects in membrane lipid packing are necessary for
interaction between the hydrophilic regions of the bilayers. The 27- or
14-residue peptides are not intrinsically hydrophobic and are quite
different from viral fusion peptides (42). They do not associate with
lipid vesicles in the absence of palmitoylation. Even on
palmitoylation, the peptide chain does not appear to span the lipid
bilayer. Despite these differences, the fusion process seems to follow
the steps proposed in the Stalk hypothesis (32-38), involving binding,
formation of a hemifusion intermediate, and complete fusion.
A direct role for SNARE proteins in membrane fusion has emerged from
studies where isolated SNARE proteins have been reconstituted in model
membranes. Formation of SNARE complexes has been shown to be essential
for this fusion (52-54). Recently, the SNARE protein domains other
than those involved in ternary complex formation have been implicated
to be important for fusion (48). Palmitoylation of SNAP-25 in neurites
and PC 12 cells is dynamically regulated and the degree of
palmitoylation influences membrane binding (13). Our results indicate
that palmitoylated and nonpalmitoylated peptides exhibit distinctive
differences in membrane-binding properties and ability to promote
fusion of membranes. It is therefore possible that the palmitoylation
status of the cysteine cluster of SNAP proteins could play a crucial
role in regulating membrane fusion in cells.