From the Institut National de la Santé et de la Recherche
Médicale, Unité 464, Institut Jean Roche, Faculté de
Médecine Secteur Nord, Bd. Pierre Dramard, 13916 Marseille Cedex
20, France and the Mitsubishi Kasei Institute of Life
Science, Machida,Tokyo 194, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cysteine string proteins (Csps) are J-domain
chaperone proteins anchored at the surface of synaptic vesicles. Csps
are involved in neurotransmitter release and may modulate presynaptic
calcium channel activity, although the molecular mechanisms are
unknown. Interactions between Csps, proteins of the synaptic core
(SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor
P/Q-type calcium channels labeled with
125I--conotoxin MVIIC. However binding experiments
with 35S-labeled Csp1 demonstrated an interaction (apparent
KD = 700 nM at pH 7.4 and 4 °C) with
a fusion protein containing a segment of the cytoplasmic loop linking
homologous domains II-III of the
1A calcium channel
subunit (BI isoform, residues 780-969). Binding was specific as it was
displaced by unlabeled Csp1, and no interactions were detected with
fusion proteins containing other calcium channel domains, VAMP, or
syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus
located within the 200 residue synaptic protein interaction site that
can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as
a molecular chaperone to direct assembly or disassembly of exocytotic
complexes at the calcium channel.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cysteine string proteins (Csps)1 were initially discovered in Drosophila as neuronal antigens predominantly localized in synaptic regions (1). Subsequently Csps were identified in Torpedo (2) and mammals (3-5). Csps contain a fatty acylated cysteine-rich motif (6) and are associated with the cytoplasmic surface of secretory vesicles (7-10). The NH2-terminal region of Csps constitutes a J-domain with homology to bacterial DnaJ proteins that act as chaperones. Mammalian Csps cooperate with the heat shock protein (Hsc70), activating its ATPase activity and preventing aggregation of model protein substrates (11, 12).
The association of Csps with synaptic vesicles (7, 10) suggests that
they are involved in membrane trafficking and/or exocytosis of
neurotransmitters. Deletion of the cspc gene
in Drosophila causes temperature-sensitive failure of
synaptic transmission, resulting in paralysis (13) due to impaired
excitation-secretion coupling at nerve terminals (14, 15). Recent
findings suggest that the default involves either a deficit in calcium
entry or in the ability of calcium to trigger exocytosis (16). These data are thus consistent with earlier results indicating that Csps act
as positive modulators of -conotoxin GVIA-sensitive calcium channels
from Torpedo electric organ, heterologously expressed in
Xenopus oocytes (2). Fusion competent synaptic vesicles are
thought to be docked at the active zone in close proximity to the
voltage-gated calcium channels that trigger release. It has been
proposed that interactions between calcium channels and Csps may be
necessary for the channels associated with docked vesicles to open in
response to depolarization (7), although biochemical evidence in favor
of this interaction has not been reported. We have therefore examined
the molecular interactions of Csp with proteins of the synaptic core
(SNARE) complex (17) and P/Q-type calcium channels, which support a
major fraction of transmitter release in many synaptic fields of the
mammalian brain (18).
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant Proteins--
A maltose-binding protein (MBP)-Csp1
fusion protein was prepared (10) and purified by affinity
chromatography on an amylose column (New England Biolabs). Glutathione
S-transferase (GST) fusion proteins containing domains of
the rabbit brain calcium channel 1A (BI-2 isoform)
subunit (19), syntaxin 1A, VAMP 2, and synaptotagmin IV were generated
as reported previously (20, 21). A GST fusion protein, including
sequence from the III-IV loop of
1A, was constructed by
amplifiying base pairs 4561-4725 and subcloning into pGEXKG. Fusion
protein production was induced in cultures of the protease-deficient
Escherichia coli strain BL21, and lysates were purified with
glutathione-agarose. 35S-Csp1 was synthesized by coupled
transcription and translation in vitro, in the presence of
[35S]cysteine (>600 Ci/mmol, DuPont), using the
TNTTM system (Promega).
Immunoprecipitation Assays--
Polyclonal antibodies against
MBP-Csp1 (10) were purified on protein A-Sepharose Fast Flow beads
(Amersham Pharmacia Biotech). Immunoprecipitation of
125I--conotoxin MVIIC-labeled P/Q-type calcium channels
from CHAPS extracts of a rat cerebellar P2 fraction was performed as
described previously (22).
Binding Assays-- Binding assays were performed by incubating GST fusion proteins with in vitro-translated 35S-Csp in 300 µl of TBS containing 0.5% BSA at 4 °C in the presence of 30 µl glutathione-agarose beads (20, 21). Beads were washed three times by centrifugation with the same buffer, and 35S-Csp binding was quantified by scintillation counting.
Rat brain homogenates were solubilized in 1% of the indicated detergent (see figure legends) in 10 mM Hepes, 0.15 M NaCl, 2 mM MgCl2, 1 mM EGTA, adjusted to pH 7.4 with NaOH, and incubated for 5 h at 4 °C with GST or GST fusion proteins bound to glutathione-agarose beads. Beads were then washed once with TBS containing 0.3% BSA and once with TBS, and bound Csp was detected by SDS-PAGE and immunoblotting (10).Surface Plasmon Resonance Spectroscopy-- Spectroscopy was performed on a BIAcore apparatus (Pharmacia Biosensor), with GST fusion proteins immobilized on the sensor chip via covalently linked anti-GST antibodies. 2 µM MBP-Csp or MBP were introduced at a flow rate of 5 µl/min at 22 °C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments were performed in order to explore the molecular mechanisms by which Csps may modulate exocytosis. First we performed co-immunoprecipitation experiments to examine the hypothesis that Csps may associate with components of the trimeric synaptic core (SNARE) complex and/or presynaptic P/Q-type calcium channels.
Co-immunoprecipitation Experiments Reveal Protein Complexes Containing Csp and VAMP-- Rat brain membranes were solubilized with CHAPS, and detergent extracts were incubated with non-immune immunoglobulins, polyclonal antibodies raised against Csp1 in the absence and presence of MBP-Csp1, and polyclonal anti-VAMP antibodies. Immune complexes were recovered on protein A-Sepharose beads, washed, and eluted with SDS-PAGE sample buffer. Western blots were prepared and probed with antibodies directed against Csp1, syntaxin 1, synaptosomal associated 25-kDa protein (SNAP-25) and VAMP. Anti-Csp antibodies were found to co-immunoprecipitate a small fraction of the total VAMP, but neither syntaxin 1 nor SNAP-25 was detected (Fig. 1). Immunoprecipitation was specific as an added excess of MBP-Csp1-blocked recovery of both Csp and VAMP. Conversely, when membrane extracts were incubated with an anti-VAMP antibody, Csp and the three SNARE proteins were all identified among the immunoprecipitated proteins (Fig. 1). These experiments suggest that anti-Csp antibodies trap protein complexes containing Csp and VAMP but not syntaxin 1 nor SNAP-25, whereas anti-VAMP antibodies recover both Csp/VAMP complexes and synaptic core complexes of VAMP, syntaxin 1, and SNAP-25. While this approach demonstrates that a small population of complexes containing Csp and VAMP does occur, it does not indicate whether these proteins interact directly or indirectly via unidentified protein partners. Cross-linking experiments were performed with the bifunctional reagent disuccinimidyl suberate, but failed to reveal complexes between Csp and VAMP or other proteins. Furthermore binding experiments with in vitro translated 35S-Csp1 and immobilized GST-VAMP 2 did not provide any support for a direct interaction between these two proteins (see below).
|
Immunoprecipitation Fails to Detect Complexes Containing Csps and
P/Q-type Calcium Channels--
A crude synaptosomal (P2) fraction from
rat cerebellum was incubated with 125I--conotoxin MVIIC
in conditions that allow specific labeling of P/Q-type calcium channels
and then extracted with CHAPS (22). As reported previously prelabeled
channels were immunoprecipitated by the monoclonal anti-syntaxin 1 antibody 10H5 (Fig. 2) (22). In contrast
anti-Csp antibodies failed to capture solubilized 125I-
-conotoxin MVIIC receptors. The amount of
radioactivity immunoprecipitated by anti-Csp antibodies was not
significantly different from that in control assays in which anti-Csp
antibodies were preincubated with a large excess of MBP-Csp1 or in
which non-immune rabbit IgG was used. As this approach did not detect
interactions between endogenous Csps and P/Q-type calcium channels,
experiments were performed with recombinant proteins.
|
Binding of 35S-Csp to the II-III Linker Region of the
Calcium Channel 1A Subunit--
35S-Csp1
was produced by coupled transcription and translation in
vitro in the presence of [35S]cysteine. SDS-PAGE and
autoradiography revealed a single major radioactive species migrating
at 28 kDa in accordance with the predicted molecular mass of Csp (Fig.
3A, left panel). Furthermore radioactivity was immunoprecipitated by anti-Csp antibodies but not by
non-immune immunoglobulins (Fig. 3A, right
panel). SDS-PAGE and protein staining indicated that the
predominant forms of MBP-Csp1, MBP, GST-II-IIIA, and GST
(Fig. 3B) migrated at 70, 43, 48, and 27 kDa respectively,
consistent with apparent molecular masses of 27 kDa for Csp1, and 21 kDa for a polypeptide from the calcium channel II-III linker region
corresponding to residues 780-969 of the BI isoform of the
1A subunit.
|
Interactions between Recombinant and Endogenous Synaptic Proteins-- Preliminary surface plasmon resonance experiments indicated that the binding of Csp to the GST-II-IIIA fusion protein was diminished in the presence of CHAPS. The effect of a series of detergents (digitonin, cholate, Lubrol, Triton X-100, Nonidet P-40, CHAPS) on the interaction of 35S-Csp1 with the GST-II-IIIA was examined. All the detergents tested (at 1%, w/v) inhibited binding by 60-80% (not shown). As detergent concentrations within this range are required to solubilize native calcium channels from brain membranes, it is possible that dissociation of endogenous Csp/calcium channel complexes by detergents may account for our inability to detect interactions by co-immunoprecipitation (see Fig. 2). In order to examine this possibility the interaction of recombinant calcium channel domains with native Csp was studied. Detergent extracts of P2 membranes from rat brain were incubated with either GST-II-IIIA, GST-III-IVA, or GST immobilized on glutathione-Sepharose beads. After washing, beads were recovered by centrifugation, treated with SDS-PAGE sample buffer, and the eluted proteins analyzed by Western blotting with anti-Csp antibodies (Fig. 4A). This approach demonstrated that endogenous Csp associated specifically with the GST-II-IIIA fusion protein despite the presence of CHAPS, Nonidet P-40, or Triton X-100.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence points to an essential role for Csps in the synaptic release of neurotransmitters (13, 14) and suggests that they are involved in regulating the ability of presynaptic calcium channels to open in response to membrane depolarization (2, 16). We thus asked whether Csps interact with components of the trimeric synaptic core complex and/or the P/Q-type calcium channel. The synaptic core (SNARE) complex is composed of a synaptic vesicle protein: synaptobrevin (VAMP) and two proteins that are predominantly expressed in the synaptic plasma membrane: syntaxin 1 and SNAP-25 (17). This complex is thought constitute the hub of a sequence of protein/protein interactions that lead up to calcium-dependent exocytosis (reviewed in Refs. 23 and 24). SNARE complexes associate with P/Q-type calcium channels (22, 25) and co-expression of syntaxin 1 with P/Q-type channels in Xenopus oocytes modulates channel gating properties (26). It is therefore possible that Csp may regulate both exocytosis and calcium channel function by interacting with components of the SNARE complex. Co-immunoprecipitation experiments revealed complexes containing Csp and VAMP, but not syntaxin 1 or SNAP-25. Synaptophysin, which interacts with VAMP in synaptic vesicles (27), was not identified among proteins immunoprecipitated by anti-Csp antibodies (not shown). Direct binding between recombinant Csp1 and VAMP 2 was not detected. However in vitro translated and bacterially expressed Csps are not palmitoylated, and a direct interaction between Csps and VAMP could require fatty acylation. These data are thus consistent with either a direct interaction between native Csp and VAMP or an indirect association mediated by unidentified partners.
The calcium-conducting pore of P/Q-type calcium channels is formed by
the 1A subunit, which contains four homologous domains each composed of six helical transmembrane segments (19). A GST-fusion
protein containing a domain located in the cytoplasmic loop linking
homologous domains II and III of the
1A subunit displayed saturable binding to in vitro translated
[35S]cysteine-labeled Csp. The affinity of this
interaction is moderate with an apparent equilibrium dissociation
constant of 700 nM at 4 °C and pH 7.4. Csp thus binds an
approximately 200 residue segment on the
1A subunit
(residues 780-969 of the BI isoform). Specific interaction was
confirmed using surface plasmon resonance spectroscopy, which indicated
that MBP-Csp can bind to the same GST fusion protein immobilized on a
sensor chip.
In view of the molecular chaperone activity of Csp, it is important to
underline the specificity of binding. Hsp70 and DnaJ-like proteins act
as chaperones and bind polypeptides, but with different selectivity.
Hsp70 binds unfolded proteins recognizing short stretches of amino
acids with an extended conformation. In contrast, DnaJ-like proteins
such as Csp generally bind substrates exhibiting secondary and tertiary
structure, but exhibit very low affinity for polypeptides in extended
conformations (reviewed in Ref. 28). Our data demonstrate that
recombinant Csp does not associate nonselectively with bacterially expressed proteins irrespective of their sequence, as Csp did not bind
to GST alone, or GST fusion proteins containing syntaxin 1, VAMP 2, or
other regions of the 1A subunit. Furthermore it is
reasonable to assume that the folding of in vitro translated proteins provides a closer approximation to native conformation than
bacterially expressed proteins fused to large tag sequences. Although
the illustrated binding data were obtained using in vitro translated Csp and GST-II-IIIA, we have also verified that
in vitro translated [35S]methionine-labeled
II-IIIA interacts with MBP-Csp1 (not shown). Thus specific
binding occurs irrespective of which partner is generated by in
vitro translation and which is a fusion protein. Finally it is
significant that Csp binds selectively to a calcium channel domain
strongly implicated in excitation-secretion coupling. This region of
the
1A subunit constitutes a binding site for syntaxin 1 and SNAP-25 (BI residues 722-1036, Ref. 25) and synaptotagmin I (BI
residues 780-969, Ref. 21). The functional relevance of these
interactions is supported by evidence that injection of the equivalent
synaptic protein binding domain from the
1B subunit (29)
disrupts transmitter release in sympathetic neurons (30) and at the
amphibian neuromuscular junction (31).
In contrast to experiments with recombinant proteins, immunoprecipitation with anti-Csp antibodies failed to capture P/Q-type calcium channels from detergent extracts of nerve terminals. This could be due to the masking of Csp epitopes by protein/protein interactions or alternatively to the disruption of native complexes during membrane solubilization. All detergents tested inhibited GST-II-IIIA fusion protein binding to 35S-Csp. However the presence of detergents in membrane extracts did not prevent endogenous Csps from interacting with GST-II-IIIA fusion proteins. In contrast native calcium channels did not bind to immobilized MBP-Csp, suggesting that a limiting parameter may be the accessibility of the Csp binding site on the calcium channel. Syntaxin 1, SNAP-25, and synaptotagmin I bind to the same domain on the calcium channel as Csp, and these three proteins all display a higher affinity than Csp for binding to calcium channels (25, 21). Furthermore a large fraction of native P/Q-type channels are stably associated with complexes containing syntaxin I, SNAP-25, VAMP, and synaptotagmin I or II (21, 22). SNARE complexes associated with native calcium channels may therefore prevent interaction with endogenous or exogenous Csps.
If Csps promote calcium channel opening by binding to the II-III linker
region of the 1A subunit, how may intrinsic chaperone activity and cooperation with Hsp70 be involved? Apart from their role
in protein folding during translation, chaperones contribute to the
assembly and dissociation of multi-protein complexes. Hsp70 (DnaK) and
J-domain proteins (DnaJ) promote the assembly and stabilization of
glucocorticoid aporeceptor complexes (reviewed in Ref. 32), the
dissociation of complexes involved in bacteriophage
DNA replication
(reviewed in Ref. 33), and clathrin disassembly from coated vesicles
(34). It is thus possible that Csps act as chaperones to promote
assembly or disassembly of synaptic protein complexes associated with
the II-III linker region of the calcium channel. Co-expression of
syntaxin 1 with Q-type calcium channels decreases their availability
for opening by stabilizing an inactivated state (26). Csps could in
principle facilitate channel activation by promoting the dissociation
of syntaxin. Our data indicate that Csps can interact directly with the
P/Q-type calcium channel and also suggest an association with VAMP. We
can speculate that Csp may coordinate sequential protein/protein
interactions between syntaxin and two of its binding partners, calcium
channels and VAMP, and consequently modulate channel opening and
calcium-dependent exocytosis. It is interesting to note
that a strikingly similar pattern of evoked synaptic responses results
from either the disruption of SNARE protein interaction with calcium
channels in rat sympathetic neurons (30) or the deletion of the Csp
gene in motoneurones of Drosophila larvae (15). Both
manipulations produced a reduction in synchronous transmitter release,
while asynchronous release and paired pulse facilitation were
increased. The regulation by Csp of SNARE protein interactions with
calcium channels may thus be necessary to ensure synchronous synaptic
transmission. Further studies will be necessary to examine whether the
assembly of syntaxin 1-SNAP-25-VAMP-synaptotagmin complexes at the
synaptic protein interaction site on the calcium channel is regulated
by the chaperone activity of Csp and Hsp70.
![]() |
FOOTNOTES |
---|
* This work was supported by the International Human Frontiers Science Program and a grant (to S. P.) from the Institut Scientifique Roussel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 33-491698929; Fax: 33-491090506; E-mail: seagar.m{at}jean-roche.univ-mrs.fr.
1 The abbreviations used are: Csp(s), cysteine string protein(s); Hsc70 or Hsp70, heat shock proteins; MBP, maltose-binding protein; GST, glutathione S-transferase; VAMP, vesicle-associated membrane protein or synaptobrevin; TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SNAP-25, synaptosomal associated 25-kDa protein; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|