JBC INTERFERin siRNA transfection reagent

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M310710200 on January 6, 2004

J. Biol. Chem., Vol. 279, Issue 13, 12574-12579, March 26, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/13/12574    most recent
M310710200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rickman, C.
Right arrow Articles by Davletov, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rickman, C.
Right arrow Articles by Davletov, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Synaptotagmin Interaction with the Syntaxin/SNAP-25 Dimer Is Mediated by an Evolutionarily Conserved Motif and Is Sensitive to Inositol Hexakisphosphate*

Colin Rickman{ddagger}, Deborah A. Archer§, Frederic A. Meunier{ddagger}||, Molly Craxton{ddagger}, Mitsunori Fukuda**, Robert D. Burgoyne§, and Bazbek Davletov{ddagger}{ddagger}{ddagger}

From the {ddagger}Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, §Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom, and **Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Received for publication, September 29, 2003 , and in revised form, December 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-cell communication relies on the regulated release of transmitter molecules from secretory vesicles. These vesicles fuse with the plasma membrane in a calcium-dependent manner to release the transmitter molecules (1, 2). Despite identification of the major players involved in intracellular membrane fusion (3, 4), the molecular steps leading to vesicle fusion are still not fully understood. Synaptotagmin I, a calcium-phospholipid binding protein, is essential for synchronous synaptic vesicle exocytosis, whereas membrane fusion itself relies on the three SNARE1 proteins: synaptobrevin, also known as vesicle-associated membrane protein or VAMP, on the vesicular membrane, and syntaxin and SNAP-25, on the target plasma membrane (5–8). The three SNARE proteins form a four-helical bundle that likely drives membrane fusion (9), with the syntaxin/SNAP-25 dimer (t-SNARE dimer) being an important intermediate in this process (10, 11).

Current models of calcium-triggered exocytosis depict the calcium sensor, synaptotagmin, being physically linked to the SNARE fusion machinery in anticipation of the calcium entry (12–15). Indeed, many independent studies have demonstrated that synaptotagmin I, the major brain isoform, can interact specifically with the neuronal SNAREs in the absence of calcium, as evidenced by pull-downs and affinity chromatography approaches followed by Coomassie staining (16–20). Using the brain-purified SNAREs, syntaxin1 and SNAP-25, we were recently able to show that synaptotagmin I binds specifically and with high affinity the t-SNARE dimer (but not the monomeric SNARE proteins) in a calcium-independent manner (18). Furthermore, we found that the majority of syntaxin and SNAP-25 in neuroendocrine cells likely exist as stable t-SNARE dimers on the plasma membrane (11), suggesting that interaction of vesicular synaptotagmin with this entity may take place during the SNARE-mediated fusion of the two membranes.

Because it is well established that synaptotagmin I plays a critical role in SNARE-mediated membrane fusion (3, 6, 7), the molecular basis for the observed calcium-independent physical link between synaptotagmin and SNAREs must be elucidated. All synaptotagmins possess tandem C2 domains connected to the transmembrane region through a variable length linker (21), and it is these domains in synaptotagmin I that are responsible for the calcium-independent SNARE binding (18). Here, we analyzed the synaptotagmin/SNARE link using genomic information of synaptotagmin amino acid sequences, the available structures of the individual C2 domains (22, 23), and biochemical/mutagenesis studies. We now show that lysine residues in the fourth {beta}-strand of the C2B domain of synaptotagmins I and II are essential for binding to the t-SNARE dimer. The almost complete conservation of this polybasic motif throughout evolution and between the two major members of the synaptotagmin family highlights its importance in exocytosis. Our functional analysis of the calcium-independent synaptotagmin/SNARE link suggests that this interaction serves a positive role in vesicle exocytosis and may be regulated by inositol polyphosphates.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Synaptotagmin Isoforms and Their Mutants—RT-PCR of rat brain mRNA was used to produce GST fusion clones in pGEX-KG corresponding to synaptotagmins I (amino acids 95–421) and II (amino acids 101–422). The individual C2 domains of synaptotagmin I and the synaptotagmin II C2B mutations were described previously (18, 24). For amperometry, synaptotagmin I C2AB construct (residues 96–421) in pcDNA3.1 was used. Site-directed mutagenesis was performed with the QuikChange system (Stratagene) to generate the K326A,K327A, D178N,D230N,D232N, and D363N,D365N mutations in the synaptotagmin I C2AB in pcDNA3.1. Similar levels of eukaryotic expression for synaptotagmin and its mutants were confirmed in HeLa cells by Western immunoblotting. To transfer the K326A,K327A mutation into a GST fusion, the wild-type Kpn-Pst fragment in synaptotagmin I pGEX-KG was substituted with the Kpn-Pst fragment from the mutant synaptotagmin I in pcDNA3.1.

Preparation of Proteins and Binding Assays—Recombinant synaptotagmin fragments, purified on glutathione-Sepharose beads (Amersham Biosciences) were washed with buffer A (20 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM EDTA) adjusted to 1 M NaCl and 1 mM dithiothreitol before elution with 15 mM reduced glutathione in buffer A. Proteins were bound to heparin-Sepharose (Amersham Biosciences), eluted using a linear gradient (0.1–1.2 M NaCl) in buffer A, and dialyzed against buffer A. Syntaxin and SNAP-25 were purified from bovine brain cortex, as described previously (25). All synaptotagmin/SNARE binding reactions were performed in the absence of calcium. Synaptotagmin domains (~3 µg each) attached to glutathione-Sepharose beads were incubated for 30 min at 22 °C in 100 µl buffer A in the presence of 1 µM SNAREs. Beads were washed three times with 1 ml of buffer A, and bound protein was analyzed by SDS-PAGE and Coomassie staining. For Western immunoblotting, 0.5% of bound material was run on SDS-PAGE followed by transfer to polyvinylidene difluoride membrane (Millipore). Protein bands were detected by using monoclonal antibodies to SNAP-25 (SMI 81, Sternberger Monoclonals) and syntaxin (HPC-1, Sigma) and enhanced chemiluminescence with exposure to x-ray film. For screening the synaptotagmin II C2B mutations, densitometric quantitation of bands on x-ray film was performed and normalized against the GST-C2B amounts used in each reaction. Phospholipid binding was performed as described previously (26), except that phosphatidylcholine/phosphatidylserine liposomes were labeled with 0.2% (w/w) DiO fluorescent dye (Molecular Probes), and bound fluorescence was measured using a microplate fluorimeter (Fluoroskan, Labsystems).

Genomic Synaptotagmin Sequences—Synaptotagmin I orthologs were predicted from genome sequences and correspond to the following reference nucleotide sequences: Homo sapiens, cDNA sequence m55047; Caenorhabditis elegans, cDNA sequence l15302; Anopheles gambiae, whole genome contig aaab01008964.1 (nucleotides 9168323–9168173, 9167627–9167347, 9167063–9166965, 9166862–9166668, 9166206–9166030, 9162205–9162088, 9161907–9161774, 9159169–9158981); Drosophila melanogaster, genome sequence contig ae003582.3 (259206–258966, 258905–258646, 258588–258493, 257994–257800, 257689–257513, 252486–252372, 250990–250857, 248592–248389); Ciona intestinalis, scaffold 152 (33210–33369, 33527–33708, 34123–34230, 34456–34530, 34853–34945, 35533–35706, 36206–36349, 36888–36992, 37206–37385).

Synaptotagmin/SNARE Immunoprecipitation from Rat Brain—All procedures were performed at 4 °C. One rat brain was homogenized in 12 ml of buffer A using a glass Teflon homogenizer, and membrane protein was solubilized by the addition of 2% Triton X-100 in the presence of Complete protease inhibitors (Roche Applied Science). The brain detergent extract was cleared by centrifugation at 100,000 x g for 1 h. Anti-SNAP-25 beads were prepared as described previously (18). The beads, with or without attached anti-SNAP-25 (SMI81, Sternberger Monoclonals), were incubated with the brain extract for 1 h and washed extensively in either 20 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, or in 20 mM HEPES, pH 7.0, 750 mM NaCl, 2 mM EDTA, 0.1% Triton X-100. The bound proteins were analyzed by Western immunoblotting using antibodies that specifically stain synaptotagmin I (clone 41.1, Synaptic Systems), synaptotagmin II (monoclonal antibody against the luminal part of the protein; Ref. 27), calcineurin (clone CN-A1, Sigma), and polyclonal anti-syntaxin and anti-SNAP-25. The immunoreactive bands were detected by enhanced chemiluminescence and visualized by using a Bio-Rad XRS detection system.

Catecholamine Release Assays—For amperometry analysis, chromaffin cells were transfected by electroporation with plasmids encoding the cytoplasmic part (C2AB) of synaptotagmin I and enhanced green fluorescent protein (EGFP), as described previously (28). Three days after electroporation, the transfected cells were identified with a Nikon TE300 inverted microscope. A carbon fiber electrode was positioned in contact with a cell, and stimulation was by pressure ejection of buffer B (139 mM potassium glutamate, 20 mM PIPES, 5 mM EGTA, 2 mM ATP, 2mM MgCl2, pH 6.5) containing 20 µM digitonin and 10 µM free Ca2+ for a 20-s pulse. Amperometric responses were monitored with a VA-10 amplifier (NPI Electronic), collected at 4 kHz, and digitized with a Digidata 1322A acquisition system. Data were analyzed by using the program Origin (Microcal) (28) and are shown as mean ± S.E. In each series of experiments, EGFP-negative control cells, in the same dishes, were recorded, using the same carbon-fibers. The permeabilization of chromaffin cells and fluorescent assay for catecholamine release were done as described previously (29). Briefly, chromaffin cells were incubated with 20 µM digitonin in buffer B for 5 min with or without IP6. The supernatant was discarded, and release was triggered for 5 min in buffer B containing 20 µM free Ca2+ in the absence or presence of IP6. Aliquots of the supernatant were assayed fluorometrically for catecholamine content (30). Released catecholamine was expressed as a normalized percentage to the release obtained in the absence of IP6.

Confocal Microscopy—Chromaffin cells seeded on polylysine-coated bottom glass coverslip dishes (Mattek) were permeabilized with 20 µM digitonin in buffer B for 5 min with GST-C2B of synaptotagmin I (130 nM) or GST (150 nM) in the absence or presence of 200 µM IP6. After a 15-min incubation, cells were washed, fixed with 4% paraformaldehyde for 20 min, and then blocked for 1 h in phosphate-buffered saline containing 3% goat serum and 0.05% Triton X-100. Incubation with anti-SNAP-25 monoclonal antibody was followed by a phosphate-buffered saline wash. SNAP-25 and bound exogenous GST were visualized using Alexa594 anti-mouse and Alexa488 anti-GST, respectively. Images were collected on a Bio-Rad 600 confocal microscope using an oil-immersion objective (63x/1.4 numerical aperture). Fluorescence intensity was quantified by using the LaserPix software (Bio-Rad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The absence of structural information for the synaptotagmin/SNARE complex currently hinders analysis of this potentially important interaction. We analyzed the molecular basis of this link by using functional assays that rely on the stoichiometric, high-affinity binding of synaptotagmin to the target SNARE dimer (Fig. 1A; Ref. 18). Binding of the cytoplasmic part of synaptotagmin, C2AB, to the target SNAREs was sensitive to ionic strength, indicating that it is of an electrostatic nature (Fig. 1A). Both syntaxin and SNAP-25 contribute acidic residues to a negatively charged surface on the ternary SNARE complex (9), suggesting that basic residues on synaptotagmin may be responsible for interaction with the t-SNARE dimer. Analysis of the surface charge of the two C2 domains of synaptotagmin I (22, 23) revealed a prominent patch of basic residues present on the C2B domain (Fig. 1B). This positively charged patch is due to a high concentration of lysines in the fourth strand of the C2B {beta}-sandwich structure (23). Comparison of synaptotagmin I sequences from worms, insects, a primitive chordate Ciona, and humans, assembled from genomic databases, revealed that the polybasic amino acid stretch is virtually identical in all animal species, despite the fact that conservation of the whole synaptotagmin sequence is in the range of only 50–65% (Fig. 1C and data not shown). This observation strongly suggests an essential role for the C2B polybasic motif.



View larger version (94K):
[in this window]
[in a new window]
  		FIG. 1.
Calcium-independent interactions of the cytoplasmic part of synaptotagmin I (C2AB) and the C2B domain with brain-purified syntaxin (Syx) and SNAP-25. A, binding of Syx/SNAP-25 to GST-C2AB of synaptotagmin I. 0.75 M NaCl disrupts the interaction of GST-C2AB with the t-SNARE dimer (shown in Coomassie-stained gel). B, electrostatic potentials of individual C2A and C2B domains of synaptotagmin I (-10 to +10 kT/e). Blue, positive charge; red, negative charge. The domains are orientated with their calcium-binding loops upwards. C, the polybasic motif is virtually identical among synaptotagmin I orthologs found in the animal kingdom. D, specific binding of the t-SNARE dimer to the C2B but not the C2A domain. The total input (left panel) and bound (center and right panels) proteins were analyzed by SDS-PAGE followed by Coomassie staining.

 
We investigated whether the C2B domain is indeed sufficient for binding the t-SNARE dimer. Recent reports demonstrated that the functional properties of the bacterially expressed C2B domain are altered by bacterial contaminants such as nucleic acids (23, 31). The C2B domain, isolated by a single affinity chromatography step from bacterial extracts, does not bind the t-SNARE dimer (18). Therefore, the recombinant C2A and C2B domains, although pure by SDS-PAGE, were thoroughly washed in 1 M NaCl to remove any contaminants. After washing, C2B but not C2A bound to the t-SNARE dimer in a calcium-independent manner (Fig. 1D). Importantly, neither C2 domain was able to interact with monomeric syntaxin or SNAP-25. Therefore, the C2B domain binds the t-SNAREs through the same mechanism as for native synaptotagmin I (18).

The C2B polybasic motif has been shown to bind inositol polyphosphates, including inositol hexakisphosphate (IP6) (24). Because IP6 can potently inhibit synaptic vesicle exocytosis (32), we analyzed whether IP6 may have an effect on the synaptotagmin/SNARE interaction. IP6 strongly suppressed synaptotagmin binding to the t-SNARE dimer with an IC50 of 6 µM (Fig. 2A), indicating that the conserved polybasic motif is responsible for SNARE binding. It is noteworthy that the binding of both target SNAREs decreased in parallel, confirming that they exist in a binary complex and that it is this entity which is capable of interacting with the C2B domain. To correlate the in vitro and in vivo IP6 effects, we investigated the action of IP6 on calcium-triggered exocytosis from digitonin-permeabilized chromaffin cells. The catecholamine release exhibited high sensitivity to acute application of IP6 with an IC50 of 16 µM, a value close to the IC50 for inhibition of the synaptotagmin/SNARE interaction (Fig. 2B). It has been shown previously that the cytoplasmic part of synaptotagmin I can bind to the plasma membrane in resting cells (33). The single C2B domain alone exhibited strong localization to the chromaffin cell plasma membrane carrying the t-SNAREs, and this localization was abolished in the presence of IP6 (Fig. 2C), suggesting that the C2B domain may interact with t-SNARE dimers on the plasma membrane.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 2.
Inositol hexakisphosphate (IP6) disrupts the synaptotagmin/SNARE link and inhibits catecholamine release. A, IP6 inhibits binding of the t-SNARE dimer to C2B of synaptotagmin I with an IC50 of 6 µM. Syntaxin and SNAP-25 binding to GST-C2B of synaptotagmin I was performed in the presence of the indicated concentrations of IP6. Inset, Coomassie-stained gel used to obtain densitometric values. B, inhibition of Ca2+-evoked catecholamine (CA) release from permeabilized chromaffin cells by IP6 (IC50 = 16 µM, n = 4). C, localization of GST-C2B of synaptotagmin I to the plasma membrane of permeabilized chromaffin cells is disrupted by IP6. Endogenous SNAP-25 and exogenous GST-C2B were visualized by double immunofluorescence. Bound fluorescence was quantified for GST-C2B or GST, used as a negative control (right, bar chart). *, p < 0.0001.

 
Synaptotagmin II is distributed in neuronal tissues in a complementary manner to synaptotagmin I, mainly in cerebellum and in neuromuscular junctions, and is likely to act through a similar mechanism in promoting vesicle exocytosis (21, 27, 34–36). To test whether synaptotagmin II can interact with SNAREs in vivo, we examined SNARE-interacting proteins in rat whole-brain extract using an anti-SNAP-25 antibody covalently linked to Sepharose beads. After incubation of these beads with the brain extract in the absence of calcium, the bound proteins were analyzed by Western immunoblotting. Staining for both syntaxin1 and SNAP-25 confirmed that these two proteins interact in brain. Monoclonal antibodies specific for synaptotagmin I (clone 41.1) and synaptotagmin II (27, 36) identified 65-kDa bands co-purifying with the SNAREs on anti-SNAP-25 beads but not on control beads lacking this antibody (Fig. 3A). Staining for a control protein, calcineurin, confirmed that co-isolation of the two synaptotagmin isoforms with the SNAREs is of a specific nature. Washing of the brain-purified synaptotagmin/SNARE assembly, using a salt solution, resulted in removal of both synaptotagmin I and II (Fig. 3A), demonstrating the electrostatic nature of the synaptotagmin/SNARE interactions for both isoforms. Together, these results support the view that syntaxin and SNAP-25 interact in vivo and, when assembled (11), these SNAREs can engage synaptotagmins I and II through an electrostatic mechanism.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 3.
Synaptotagmins I and II bind target SNAREs both in vivo and in vitro. A, Sepharose beads with (anti-SNAP-25) or without (control) covalently linked anti-SNAP-25 antibody were incubated in the absence of calcium with rat brain Triton X-100 extract for 1 h and, after extensive washing, were analyzed for bound proteins using Western immunoblotting. Staining for a control protein, calcineurin, indicates that co-isolation of the two synaptotagmin isoforms with syntaxin1 and SNAP-25 is of a specific nature. Washing with 0.75 M NaCl removes synaptotagmins I and II from the SNAREs. B, GST-C2ABs of synaptotagmins I and II attached to glutathione beads bind the t-SNARE dimer in the absence of calcium. The original beads (left panel), the total input of syntaxin and SNAP-25 (center panel), and bound proteins (right panel) were analyzed by SDS-PAGE followed by Coomassie staining.

 
To confirm that the calcium-independent interaction of the SNAREs with synaptotagmins does not require synaptobrevin (18), we analyzed the two major synaptotagmin isoforms in a reconstituted assay. The cytoplasmic part of synaptotagmin II was indeed able to bind the t-SNARE dimer as efficiently as synaptotagmin I, with an apparent equimolar stoichiometry (Fig. 3B; Ref. 18). The polybasic motif is fully conserved between mammalian synaptotagmin I and synaptotagmin II (34). To investigate the contribution of the basic residues in the evolutionarily conserved motif, we analyzed the t-SNARE binding by synaptotagmin II C2B domains carrying mutations of positively charged arginine and lysines to neutral glutamines (Fig. 4A). The single-point mutations 5 and 6 resulted in the largest decrease in the binding of the t-SNAREs (Fig. 4B). The double mutation 5,6 had a more dramatic effect, and the triple mutation 5,6,7 completely eliminated binding of the t-SNARE dimer (Fig. 4B). Notably, these mutations have a parallel effect on the binding of both syntaxin and SNAP-25 (Fig. 4C), again illustrating that synaptotagmins recognize the syntaxin/SNAP-25 dimer as a single molecular entity. We conclude that the t-SNARE dimer interaction with synaptotagmins involves the polybasic patch on the C2B domain.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 4.
Basic surface residues of the C2B domain are essential for binding the t-SNARE dimer. A, mutational screen of the synaptotagmin II C2B polybasic stretch. Mutated residues are numbered and shown on the ribbon diagram. B, binding of t-SNAREs is plotted as a percentage of the wild-type C2B (n = 3). The wild-type and mutant GST-C2B, immobilized on glutathione beads, were incubated with syntaxin and SNAP-25 for 30 min. After washing, bound SNAREs were analyzed by immunoblotting and densitometry. Binding was normalized to GST-C2B amounts in each reaction (lower panel, Coomassie-stained gel). C, mutations 5,6 and 5,6,7 have a parallel effect on the binding of both syntaxin and SNAP-25, thus demonstrating that C2B recognizes both t-SNAREs as a single molecular entity.

 
A recent D. melanogaster genetic study demonstrated that substitution of two lysine residues in the evolutionarily conserved motif of synaptotagmin I, corresponding to mutations 5 and 6 (Fig. 4A), results in a marked decrease of excitation/secretion coupling in neurotransmission (37). We decided to test the importance of the polybasic motif of synaptotagmin I in a model for mammalian exocytosis, namely in chromaffin cells from bovine adrenal medullae (38). We chose to study primary chromaffin cells rather than pheochromocytoma cell lines because PC12 cells exhibit normal exocytosis in the absence of synaptotagmin I, most likely because of the presence of other isoforms such as synaptotagmin IX (39, 40). In contrast, synaptotagmin I-deficient chromaffin cells exhibit reduced exocytosis, indicating a role for synaptotagmin I in calcium-triggered hormone release (41). We analyzed the effect of expression of the soluble synaptotagmin I cytoplasmic part in chromaffin cells. If, indeed, synaptotagmin I, which is resident on secretory vesicles, interacts with the syntaxin/SNAP-25 dimer on the plasma membrane to promote vesicle fusion, then expression of synaptotagmin I without its transmembrane part may affect catecholamine release through competitive inhibition. We co-transfected bovine chromaffin cells with the soluble synaptotagmin I C2AB and EGFP to tag the transfected cells. Carbon fiber amperometry was used to measure catecholamine release from single cells. Stimulation by a brief application of 0.003% digitonin in the presence of 10 µM Ca2+ was used to directly activate exocytosis, circumventing a requirement for calcium channels. Expression of synaptotagmin I C2AB led to inhibition of regulated catecholamine release compared with control non-transfected cells (Fig. 5A). Importantly, the amplitudes of single release events were indistinguishable between the two populations, whereas the number of spikes per cell was significantly decreased (Fig. 5A). This indicates that synaptotagmin regulates the release probability rather than the amount of transmitter releasable in a given fusion event, which is in agreement with genetic studies of neurotransmission (7, 42).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5.
C2AB inhibits exocytosis in chromaffin cells through its polybasic motif. A, inhibition of evoked exocytosis in cells transfected with the wild-type C2AB synaptotagmin I. Cumulative plot of amperometric spikes after stimulation (indicated by the arrow); inset, example traces from control and transfected cells. Bar chart at right shows normalized number of spikes per cell after stimulation as a percentage of control. *, p < 0.02. B, K327,328A mutation in synaptotagmin I C2AB disrupts binding of the t-SNARE dimer (n = 3) but not calcium-triggered phospholipid binding (n = 3). The wild-type and mutant GST-C2AB attached to glutathione beads were tested for binding the t-SNARE dimer or fluorescent phospholipid liposomes. Bound SNAREs were quantified with immunoblotting and expressed as a percentage of the wild-type C2AB binding. C, the mutated K327,328A C2AB is unable to inhibit evoked exocytosis. Cumulative plot of amperometric spikes after stimulation (indicated by the arrow; inset, example traces from control and transfected cells. Bar chart at right shows normalized number of spikes per cell after stimulation as a percentage of control.

 
We then introduced the double-point mutation K326A, K327A, which is identical to that used in the D. melanogaster study (37). Biochemical assays showed that this mutation severely disrupted the ability of C2AB to bind the t-SNARE dimer but did not compromise its calcium/phospholipid-binding property (Fig. 5B). The normal phospholipid binding indicates that the folding of the double C2 domain structure was unaffected by the K326A,K327A mutation. When expressed in chromaffin cells, synaptotagmin I C2AB carrying this mutation was no longer able to inhibit exocytosis (Fig. 5C), compared with the wild-type C2AB (Fig. 5A). Therefore, we conclude that synaptotagmin function in mammalian exocytosis involves the evolutionarily conserved motif, which can mediate coupling to the t-SNARE dimer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The remarkable conservation of the polybasic motif in the synaptotagmin C2B domain in Metazoan evolution indicates that this region carries an essential physiological function (Fig. 2A). Indeed, it was recently demonstrated that mutation of two lysine residues in the polybasic motif of D. melanogaster synaptotagmin I decreases calcium-triggered neurotransmitter release (37). Several independent lines of evidence indicate that the polybasic patch is essential for synaptotagmin interaction with the t-SNARE dimer, and that this interaction may be functionally important. First, mutations of specific lysine residues in the C2B domain of synaptotagmins I and II disrupt binding to the t-SNAREs (Figs. 4 and 5). Second, IP6, known to interact with the polybasic stretch (24), competitively inhibits the synaptotagmin/SNARE interaction, which is paralleled by the inhibition of exocytosis in chromaffin cells (Fig. 2). Third, inhibition of exocytosis by expressed soluble C2AB is abolished by mutations within the polybasic region (Fig. 5). The location of the polybasic stretch at a distance from the calcium/phospholipid binding region can now explain why synaptotagmin binding to the t-SNARE dimer is not linked to its calcium-binding properties. Our finding of a direct molecular link between the t-SNARE dimer and synaptotagmin, through the polybasic patch, provides a new explanation of how synaptotagmin mutations (37) can lead to a decrease in SNARE-mediated exocytosis. It is conceivable that vesicular synaptotagmin can position both syntaxin and SNAP-25 in close proximity to the vesicular membrane, thus increasing the probability of synaptobrevin engagement and thereby enhancing SNARE-mediated membrane fusion.

Our study focused specifically at a calcium-independent property of synaptotagmin, namely its proposed interaction with the SNAREs (12–15). For synaptotagmins I and II, the major neuronal isoforms, the SNARE-binding step may be essential for further calcium-dependent actions. These actions may include synaptotagmin binding to phospholipid membranes (23, 26, 42–44) or a recently proposed phospholipid-induced oligomerization (45). The in vivo competition assay used in our study could not test the calcium-dependent phospholipid binding by the endogenous synaptotagmin, most likely because of a large number of phospholipid-binding sites. Indeed, expression of C2AB carrying mutations of the calcium-coordinating residues in C2A (D178N,D230N,D232N) and C2B (D363N,D365N) results in a similar level of inhibition of catecholamine release, as for the wild-type C2AB (data not shown). It is possible that the calcium/phospholipid synaptotagmin action (42–44) is meaningful only when synaptotagmin has the ability to interact with the syntaxin/SNAP-25 dimer during exocytosis.

The present work sheds a new light on two early studies into the mechanisms underlying neurotransmitter release. First, Bommert et al. (46) demonstrated that injection of peptides, which include the conserved polybasic stretch, into squid giant nerve terminals reversibly blocked transmitter release without changing the vesicle population size. In the light of our results, it is possible that the polybasic peptide interferes with synaptotagmin/SNARE binding, resulting in a reversible blockade of neurotransmission. Second, it was shown that injection of IP6 in the squid giant nerve terminals decreases vesicle release probabilities by interfering with a late step of synaptic vesicle exocytosis (32). Llinas et al. (32) predicted that IP6 specifically disrupts synaptotagmin interaction with a molecular target preceding membrane fusion. We reproduced in mammalian cells the inhibition of vesicle exocytosis by IP6 and also demonstrated that IP6 disrupts the synaptotagmin interaction with the t-SNARE dimer over the same concentration range as for inhibition of exocytosis (Fig. 2). These parallel effects are likely due to the ability of IP6 to bind the polybasic patch (24), thus impeding coupling of synaptotagmin to the t-SNARE dimer. It now becomes necessary to investigate whether endogenous inositol polyphosphates and/or phosphoinositides can actually regulate synaptotagmin availability for the SNARE binding (47, 48).

Although we favor the idea that the direct link of vesicular synaptotagmin I to the target SNARE dimers on the plasma membrane plays an important physiological role, as suggested by current models (12–15), our present data do not exclude other synaptotagmin interactions. Chapman et al. (49) originally implicated the C2B polybasic motif in three independent functions: calcium-independent binding of the synprint peptide and AP-2, and calcium-triggered oligomerization of C2 domains. Recently, the calcium-triggered oligomerization has been brought into doubt, as synaptotagmin I recombinant fragments, after the removal of bacterial contaminants, no longer oligomerize, as observed in the original study (31, 45). It remains unclear, however, whether the reported synprint and AP-2 binding can be reproduced by using the pure C2B domain. It is unlikely that the in vivo data can be explained on the basis of a possible link between synaptotagmin and AP-2 or calcium channels that contain the synprint peptide. First, acute application of IP6 inhibits vesicle exocytosis at a late step, which is inconsistent with the possibility that replenishment of vesicle pools by endocytosis is responsible for this inhibition of exocytosis (Fig. 2; Ref. 32). Second, the use of calcium/digitonin in our assays likely circumvents a need for a link between synaptotagmin and calcium channels (Figs. 2 and 5). Importantly, exocytosis in chromaffin cells, investigated here, does not seem to rely on the association of secretory vesicles and calcium channels (50, 51). An alternative interaction involving the polybasic motif has recently been put forward by an electron microscopy study (45), which reported calcium-induced homooligomerization of recombinant synaptotagmin I fragments on phospholipid membranes. The temporal and spatial relationship between SNARE binding and synaptotagmin homo-oligomerization during exocytosis remains to be addressed.

The existence of preassembled t-SNARE dimers on the plasma membrane of chromaffin cells (11) suggests that they may be a molecular target not only for synaptobrevin, but also for synaptotagmin, in a sequence of events leading to exocytosis. We now present the first evidence that the high affinity interaction of synaptotagmin with the syntaxin/SNAP-25 dimer is driven by the hallmark signature sequence in the C2B domain and that this interaction is sensitive to inositol hexakisphosphate. Elucidation of the SNARE-binding motif on synaptotagmin provides a new interpretation for previous in vivo studies and will allow further experimentation on the molecular mechanisms of secretory vesicle exocytosis.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by a Wellcome Trust Prize studentship. Back

|| Present address: School of Biomedical Sciences, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 44-1223-402071; Fax: 44-1223-402310; E-mail: baz{at}mrc-lmb.cam.ac.uk.

1 The abbreviations used are: SNARE, soluble NSF attachment protein receptor; SNAP-25, synaptosomal-associated protein of 25 kDa; EGFP, enhanced green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione-S-transferase. Back

2 v1.0 genome assembly is available on the Internet at genome.jgi-psf.org/ciona4/ciona4.home.html. Back


   ACKNOWLEDGMENTS
 
We thank Yoko Shoji-Kasai for the monoclonal antibody against synaptotagmin II.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Katz, B., and Miledi, R. (1965) Proc. R. Soc. Lond. B Biol. Sci. 161, 496-503[Medline] [Order article via Infotrieve]
  2. Augustine, G. J., Charlton, M. P., and Smith, S. J. (1987) Annu. Rev. Neurosci. 10, 633-693[CrossRef][Medline] [Order article via Infotrieve]
  3. Sudhof, T. C., and Scheller, R. H. (2001) in Synapses (Cowan, W. M., Sudhof, T. C., and Stevens, C. F., eds) The Johns Hopkins University Press, Baltimore, MD
  4. Sollner, T. H. (2003) Mol. Membr. Biol. 20, 209-220[CrossRef][Medline] [Order article via Infotrieve]
  5. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Sudhof, T. C. (1990) Nature 345, 260-263[CrossRef][Medline] [Order article via Infotrieve]
  6. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Sudhof, T. C. (1994) Cell 79, 717-727[CrossRef][Medline] [Order article via Infotrieve]
  7. Yoshihara, M., and Littleton, J. T. (2002) Neuron 36, 897-908[CrossRef][Medline] [Order article via Infotrieve]
  8. Schiavo, G., Matteoli, M., and Montecucco, C. (2000) Physiol. Rev. 80, 717-766[Abstract/Free Full Text]
  9. Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353[CrossRef][Medline] [Order article via Infotrieve]
  10. Fasshauer, D., Antonin, W., Subramaniam, V., and Jahn, R. (2002) Nat. Struct. Biol. 9, 144-151[CrossRef][Medline] [Order article via Infotrieve]
  11. Rickman, C., Meunier, F. A., Binz, T., and Davletov, B. (2004) J. Biol. Chem. 279, 644-651[Abstract/Free Full Text]
  12. Koh, T. W., and Bellen, H. J. (2003) Trends Neurosci. 26, 413-422[CrossRef][Medline] [Order article via Infotrieve]
  13. Sutton, R. B., Ernst, J. A., and Brunger, A. T. (1999) J. Cell Biol. 147, 589-598[Abstract/Free Full Text]
  14. Augustine, G. J. (2001) Curr. Opin. Neurobiol. 11, 320-326[CrossRef][Medline] [Order article via Infotrieve]
  15. Hazuka, C. D., Foleti, D. L., and Scheller, R. H. (1999) in Neurotransmitter Release (Bellen, H. J., ed) pp. 81-125, Oxford University Press, Oxford
  16. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418[CrossRef][Medline] [Order article via Infotrieve]
  17. McMahon, H. T., Missler, M., Li, C., and Sudhof, T. C. (1995) Cell 83, 111-119[CrossRef][Medline] [Order article via Infotrieve]
  18. Rickman, C., and Davletov, B. (2003) J. Biol. Chem. 278, 5501-5504[Abstract/Free Full Text]
  19. Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T., and Takahashi, M. (1992) J. Biol. Chem. 267, 24925-24928[Abstract/Free Full Text]
  20. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259[Abstract/Free Full Text]
  21. Sudhof, T. C. (2002) J. Biol. Chem. 277, 7629-7632[Free Full Text]
  22. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Sudhof, T. C., and Sprang, S. R. (1994) Cell 80, 929-938
  23. Fernandez, I., Arac, D., Ubach, J., Gerber, S. H., Shin, O., Gao, Y., Anderson, R. G., Sudhof, T. C., and Rizo, J. (2001) Neuron 32, 1057-1069[CrossRef][Medline] [Order article via Infotrieve]
  24. Fukuda, M., Kojima, T., Aruga, J., Ninobe, M., and Mikoshiba, K. (1995) J. Biol. Chem. 270, 26523-26527[Abstract/Free Full Text]
  25. Hu, K., Carroll, J., Fedorovich, S., Rickman, C., Sukhodub, A., and Davletov, B. (2002) Nature 415, 646-650[CrossRef][Medline] [Order article via Infotrieve]
  26. Davletov, B. A., and Sudhof, T. C. (1993) J. Biol. Chem. 268, 26386-26390[Abstract/Free Full Text]
  27. Angaut-Petit, D., Juzans, P., Molgo, J., Faille, L., Seagar, M. J., Takahashi, M., and Shoji-Kasai, Y. (1995) Brain Res. 681, 213-217[CrossRef][Medline] [Order article via Infotrieve]
  28. Archer, D. A., Graham, M. E., and Burgoyne, R. D. (2002) J. Biol. Chem. 277, 18249-18252[Abstract/Free Full Text]
  29. Burgoyne, R. D. (1992) in Intracellular Messengers (Neuromethods, Vol. 20) (Boulton, A., Baker, G., and Taylor, C., eds) pp. 433-470, Humana Press Inc, Totowa, NJ
  30. Meunier, F. A., Feng, Z. P., Molgo, J., Zamponi, G. W., and Schiavo, G. (2002) EMBO 21, 6733-6743[CrossRef][Medline] [Order article via Infotrieve]
  31. Ubach, J., Lao, Y., Fernandez, I., Arac, D., Sudhof, T. C., and Rizo, J. (2001) Biochemistry 40, 5854-5860[CrossRef][Medline] [Order article via Infotrieve]
  32. Llinas, R., Sugimori, M., Lang, E. J., Morita, M., Fukuda, M., Niinobe, M., and Mikoshiba, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12990-12993[Abstract/Free Full Text]
  33. Fukuda, M., Moreira, J. E., Liu, V., Sugimori, M., Mikoshiba, K., and Llinas, R. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 14715-14719[Abstract/Free Full Text]
  34. Geppert, M., Archer, B. T., III, and Sudhof, T. C. (1991) J. Biol. Chem. 266, 13548-13552[Abstract/Free Full Text]
  35. Marqueze, B., Berton, F., and Seagar, M. (2000) Biochimie (Paris) 82, 409-420
  36. Juzans, P., Molgo, J., Faille, L., and Angaut-Petit, D. (1996) Pfluegers Arch. Eur. J. Physiol. 431, R283-R284[CrossRef][Medline] [Order article via Infotrieve]
  37. Mackler, J. M., and Reist, N. E. (2001) J. Comput. Neurol. 436, 4-16[CrossRef]
  38. Burgoyne, R. D., and Morgan, A. (2003) Physiol. Rev. 83, 581-632[Abstract/Free Full Text]
  39. Fukuda, M., Kowalchyk, J. A., Zhang, X., Martin, T. F., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 4601-4604[Abstract/Free Full Text]
  40. Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823[CrossRef][Medline] [Order article via Infotrieve]
  41. Voets, T., Moser, T., Lund, P. E., Chow, R. H., Geppert, M., Sudhof, T. C., and Neher, E. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11680-11685[Abstract/Free Full Text]
  42. Fernandez-Chacon, R., Konigstorfer, A., Gerber, S. H., Garcia, J., Matos, M. F., Stevens, C. F., Brose, N., Rizo, J., Rosenmund, C., and Sudhof, T. C. (2001) Nature 410, 41-49[CrossRef][Medline] [Order article via Infotrieve]
  43. Bai, J., Earles, C. A., Lewis, J. L., and Chapman, E. R. (2000) J. Biol. Chem. 275, 25427-25435[Abstract/Free Full Text]
  44. Mackler, J. M., Drummond, J. A., Loewen, C. A., Robinson, I. M., and Reist, N. E. (2002) Nature 418, 340-344[CrossRef][Medline] [Order article via Infotrieve]
  45. Wu, Y., He, Y., Bai, J., Ji, S. R., Tucker, W. C., Chapman, E. R., and Sui, S. F. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2082-2087[Abstract/Free Full Text]
  46. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165[CrossRef][Medline] [Order article via Infotrieve]
  47. Schiavo, G., Gu, Q. M., Prestwich, G. D., Sollner, T. H., and Rothman, J. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13327-13332[Abstract/Free Full Text]
  48. Osborne, S. L., Meunier, F. A., and Schiavo, G. (2001) Neuron 32, 9-12[CrossRef][Medline] [Order article via Infotrieve]
  49. Chapman, E. R., Desai, R. C., Davis, A. F., and Tornehl, C. K. (1998) J. Biol. Chem. 273, 32966-32972[Abstract/Free Full Text]
  50. Chow, R. H., Klingauf, J., and Neher, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12765-12769[Abstract/Free Full Text]
  51. Chow, R. H., Klingauf, J., Heinemann, C., Zucker, R. S., and Neher, E. (1996) Neuron 16, 369-376[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. ProteomicsHome page
S. L. Osborne, T. P. Wallis, J. L. Jimenez, J. J. Gorman, and F. A. Meunier
Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy
Mol. Cell. Proteomics, July 1, 2007; 6(7): 1158 - 1169.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
A. Maximov, O.-H. Shin, X. Liu, and T. C. Sudhof
Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release
J. Cell Biol., January 1, 2007; 176(1): 113 - 124.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
C. A. Loewen, S.-M. Lee, Y.-K. Shin, and N. E. Reist
C2B Polylysine Motif of Synaptotagmin Facilitates a Ca2+-independent Stage of Synaptic Vesicle Priming In Vivo
Mol. Biol. Cell, December 1, 2006; 17(12): 5211 - 5226.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Z. P. Pang, O.-H. Shin, A. C. Meyer, C. Rosenmund, and T. C. Sudhof
A Gain-of-Function Mutation in Synaptotagmin-1 Reveals a Critical Role of Ca2+-Dependent Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Binding in Synaptic Exocytosis
J. Neurosci., November 29, 2006; 26(48): 12556 - 12565.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Li, O.-H. Shin, J.-S. Rhee, D. Arac, J.-C. Rah, J. Rizo, T. Sudhof, and C. Rosenmund
Phosphatidylinositol Phosphates as Co-activators of Ca2+ Binding to C2 Domains of Synaptotagmin 1
J. Biol. Chem., June 9, 2006; 281(23): 15845 - 15852.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
G. Nagy, J. H. Kim, Z. P. Pang, U. Matti, J. Rettig, T. C. Sudhof, and J. B. Sorensen
Different Effects on Fast Exocytosis Induced by Synaptotagmin 1 and 2 Isoforms and Abundance But Not by Phosphorylation
J. Neurosci., January 11, 2006; 26(2): 632 - 643.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
C. Rickman, J. L Jimenez, M. E. Graham, D. A. Archer, M. Soloviev, R. D. Burgoyne, and B. Davletov
Conserved Prefusion Protein Assembly in Regulated Exocytosis
Mol. Biol. Cell, January 1, 2006; 17(1): 283 - 294.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. R. L. Constable, M. E. Graham, A. Morgan, and R. D. Burgoyne
Amisyn Regulates Exocytosis and Fusion Pore Stability by Both Syntaxin-dependent and Syntaxin-independent Mechanisms
J. Biol. Chem., September 9, 2005; 280(36): 31615 - 31623.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
T.-i. Nishiki and G. J. Augustine
Dual Roles of the C2B Domain of Synaptotagmin I in Synchronizing Ca2+-Dependent Neurotransmitter Release
J. Neurosci., September 29, 2004; 24(39): 8542 - 8550.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. K. Rao, C. Huynh, V. Proux-Gillardeaux, T. Galli, and N. W. Andrews
Identification of SNAREs Involved in Synaptotagmin VII-regulated Lysosomal Exocytosis
J. Biol. Chem., May 7, 2004; 279(19): 20471 - 20479.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow