Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles

Abstract

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.

Full text

Synapsin Is a Novel Rab3 Effector Protein on Small
Synaptic Vesicles
II. FUNCTIONAL EFFECTS OF THE Rab3A-SYNAPSIN I INTERACTION*
Received for publication, April 14, 2004, and in revised form, July 8, 2004
Published, JBC Papers in Press, July 20, 2004, DOI 10.1074/jbc.M404168200
Silvia Giovedı`‡, Franc¸ois Darchen§, Flavia Valtorta¶, Paul Greengard储, and Fabio Benfenati‡储**
From the ‡Department of Experimental Medicine, Section of Human Physiology, University of Genova, Via Benedetto XV,
16132 Genova, Italy, §CNRS UPR 1929, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie,
F-75005 Paris, France, the ¶Department of Neuroscience, S. Raffaele Vita-Salute University, Via Olgettina 58,
20132 Milano, Italy, and 储Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University,
New York, New York 10021-6399
Synapsins, a family of neuron-specific phosphopro-
teins that play an important role in the regulation of
synaptic vesicle trafficking and neurotransmitter re-
lease, were recently demonstrated to interact with the
synaptic vesicle-associated small G protein Rab3A within
nerve terminals (Giovedı`, S., Vaccaro, P., Valtorta, F.,
Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F.
(2004) J. Biol. Chem. 279, 43760–43768). We have analyzed
the functional consequences of this interaction on the
biological activities of both proteins and on their sub-
cellular distribution within nerve terminals. The pres-
ence of synapsin I stimulated GTP binding and GTPase
activity of both purified and endogenous synaptic vesicle-
associated Rab3A. Conversely, Rab3A inhibited synapsin
I binding to F-actin, as well as synapsin-induced actin
bundling and vesicle clustering. Moreover, the amount of
Rab3A associated with synaptic vesicles was decreased in
synapsin knockout mice, and the presence of synapsin I
prevented RabGDI-induced Rab3A dissociation from syn-
aptic vesicles. The results indicate that an interaction
between synapsin I and Rab3A exists on synaptic vesicles
that modulates the functional properties of both proteins.
Given the well recognized importance of both synapsins
and Rab3A in synaptic vesicles exocytosis, this interac-
tion is likely to play a major role in the modulation of
neurotransmitter release.
Rab proteins, belonging to the Ras superfamily of small G
proteins, are implicated in the regulation of membrane traf-
ficking between subcellular compartments (1, 2). Rab3A–C are
expressed in brain where they specifically associate with syn-
aptic vesicles (SV)
1
(3). Rab3A, the most abundant and widely
distributed isoform in brain, cycles between a GTP-bound form
that associates with the SV membrane and a GDP-bound form
that becomes soluble upon formation of a complex with the
GDP dissociation inhibitor (RabGDI) (for review see Refs. 2, 4,
and 5). The cycling of Rab proteins is strictly linked to the
exo-endocytotic cycle of SV. Stimulation of neurotransmitter
release promotes GTP hydrolysis and dissociation of Rab3A
from the SV membrane (6, 7), whereas overexpression of
GTPase-deficient, constitutively active Rab3A inhibits neuro-
transmitter release (8–10).
Rab3 has been proposed to regulate vectorially SV trafficking
at various stages of the exo-endocytotic cycle through interac-
tions with specific effector proteins. Rab3 may participate in
the docking of SV to appropriate sites of the presynaptic mem-
brane by interacting with presynaptic effectors localized at
active zones, such as Rim (11), as well as in the following
priming and fusion events by interacting with a prefusion
complex and preventing fusion of primed SV with the presyn-
aptic membrane (for review see Refs. 4, 5, and 12). Whereas
most of the hypothesized roles of Rab3A has been confirmed by
studies on Rab3A knockout mice (13), its role in docking has
been questioned, as Rim knockout mice do not exhibit changes
in the number of docked SV (14). Under conditions of high
frequency stimulation, Rab3A knockout mice exhibit a de-
crease in the recruitment of SV at the presynaptic membrane
and a delayed recovery of release after stimulation (15),
whereas train and paired-pulse facilitations were increased
after injection of constitutively active Rab3A into Aplysia neu-
rons (10). These effects, together with the described interaction
between the Rab3 effector Rabphilin-3 and the actin bundling
protein
␣
-actinin (16), implicate Rab3 in the activity-dependent
trafficking of SV upstream of SV fusion, possibly by affecting
the dynamics of SV binding to the actin cytoskeleton.
After endocytosis of the fused SV, soluble Rab3A dissociates
from RabGDI, reassociates with the SV membrane, and under-
goes a GDP/GTP exchange that involves the intervention of a
guanine nucleotide-exchange factor. It has been demonstrated
recently that GTP/GDP exchange is not a prerequisite for
Rab3A binding to the SV membrane and that GDP-bound
Rab3A binds to a protein component of SV that competes with
* This work was supported by Italian Ministry of University Grants
Cofin 2001, 2002, 2003, and FIRB (to F. B. and F. V.), Fisher Founda-
tion for Alzheimer’s Disease Research (to F. B.), CNR (Progetto Strate-
gico Neuroscienze and Genomica Funzionale (to F. B. and F. V.), United
States Public Health Service Grants MH39327 and AG15072 (to P. G.),
and Telethon-Italy Grant 1131 (to F. B.). 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.
** To whom correspondence should be addressed: Dept. of Experi-
mental Medicine, Section of Human Physiology, University of Genova,
Via Benedetto XV, 3, 16132 Genova, Italy. Tel.: 39-10-3538183; Fax:
39-10-3538194; E-mail: benfenat@unige.it.
1
The abbreviations used are: SV, synaptic vesicles; BSA, bovine
serum albumin; CaMKII, Ca
2⫹
/calmodulin-dependent protein kinase
type II; cdk-1, cyclin-dependent protein kinase; DTT, dithiothreitol;
FRET, fluorescence resonance energy transfer; GAP, GTPase-activat-
ing protein; GDI, GDP dissociation inhibitor; GST, glutathione S-trans-
ferase; LRh-PE, N-[lissamine rhodamine B sulfonyl] L-
␣
-phosphati-
dylethanolamine; MAPK, mitogen-associated protein kinase Erk 1/2;
NBD-PE, N-[4-nitrobenzo-2-oxa-1,3-diazole] L-
␣
-phosphatidylethanol-
amine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI,
phosphatidylinositol; PS, phosphatidylserine; PKA, cAMP-dependent
protein kinase; RSV, synapsin-rebound synaptic vesicles; SSV, synap-
sin-depleted synaptic vesicles; USV, untreated synaptic vesicles;
GTP
␥
S, guanosine 5⬘-3-O-(thio)triphosphate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 42, Issue of October 15, pp. 43769–43779, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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soluble RabGDI for Rab3A delivery (17, 18). A GDI dissociation
factor has been identified recently for Rab5 and Rab9 (19, 20),
and a similar factor could be involved in the translocation of
GDP-Rab to SV.
As Rab3 represents a clock for exocytosis, the identification
of Rab3 partners that modulate its cycle may be important for
the understanding of the fine mechanisms of neurotransmitter
release. By using phage display library analysis to uncover
high affinity synapsin-binding peptides, we have recently
found that Rab3A is one of the synapsin interactors in intact
nerve terminals (62).
Synapsins, a family of neuron-specific phosphoproteins, have
been demonstrated to regulate the supply of SV available for
exocytosis by binding to both SV and actin cytoskeleton in a
phosphorylation-dependent manner (for review see Refs. 21
and 22). Although these observations strongly support a pre-
docking role of the synapsins in the assembly and maintenance
of a large reserve pool of SV and in the regulation of short term
synaptic plasticity, recent results indicate that the synapsins
are also involved in some later step of exocytosis. Thus, the
kinetics of release was slowed in the squid giant terminal after
injection of a conserved synapsin COOH-terminal peptide (23),
as well as in Aplysia ganglion neuron terminals after neutral-
ization of endogenous synapsin by antibody injection (24).
As synapsins act as regulators of SV trafficking at both pre-
and post-docking stages of the SV cycle, we studied whether the
synapsin-Rab3 interaction plays some role in the multiple
stages of exocytosis. In this paper, we demonstrate that the
interaction between synapsin and Rab3A regulates the activi-
ties of both proteins. Thus, synapsin stimulated the Rab3A
cycle by increasing GTP binding, GTPase activity, and Rab3A
recruitment to the SV membrane, and conversely, Rab3A in-
hibited the actin binding and SV clustering activity of
synapsin.
EXPERIMENTAL PROCEDURES
Materials
[
␣
-
32
P]GTP, [
␥
-
32
P]GTP,
␥
-
35
S-GTP, [
3
H]GDP, and glutathione-
Sepharose were from Amersham Biosciences. Human recombinant
Rab3A was from Calbiochem; Bio-Spin 6 gel filtration columns were
from Bio-Rad; PEI-cellulose TLC sheets were from Merck; nitrocellu-
lose membranes and 0.45-
␮
m filters were from Schleicher & Schuell;
the Renaissance enhanced chemiluminescence detection system was
from PerkinElmer Life Sciences. The anti-synapsin and anti-Rab3A
polyclonal antibodies were raised in our laboratories. The other anti-
bodies were obtained from available commercial sources. Rat RabGDI
was expressed as His
6
-tagged protein in BL21(DE3) pLysS strain and
purified to homogeneity on nickel-nitrilotriacetic acid-agarose affinity
columns (Qiagen, Valencia, CA). Purified bovine synapsin I was stoi-
chiometrically phosphorylated in vitro by using purified protein kinase
A (PKA), Ca
2⫹
/calmodulin-dependent protein kinase type II (CaMKII),
mitogen-associated protein kinase Erk 1/2 (MAPK), or cyclin-dependent
protein kinase-1 (cdk-1) as described previously (25, 26). Bovine brain
phosphatidylcholine (PC), bovine brain phosphatidylethanolamine
(PE), bovine brain phosphatidylserine (PS), bovine liver phosphatidyli-
nositol (PI), N-[4-nitrobenzo-2-oxa-1,3-diazole]-L-
␣
-phosphatidyleth-
anolamine (NBD-PE), and N-(lissamine rhodamine B sulfonyl)-L-
␣
-
phosphatidylethanolamine (LRh-PE) were obtained from Avanti Polar
Lipids (Alabaster, AL), stored at ⫺20 °C in the dark, and used within 3
months. Bovine serum albumin (BSA) and all other chemicals were
from Sigma. SV were purified to homogeneity from rat forebrain
through the step of controlled pore glass chromatography (27). Mouse
forebrain homogenate was subfractionated through the step of sucrose
density gradient centrifugation as described (27) yielding purified SV
and synaptic membrane fractions (SG2 and SG4 fractions,
respectively).
Assays of the Biochemical Properties of Rab3A
Binding of
␥
-
35
S-GTP to Rab3A—The binding of
␥
-
35
S-GTP to Rab3A
was evaluated as described by Kikuchi et al. (28) with some modifica-
tions. Recombinant Rab3A was loaded with GDP (1
␮
M) by incubation
for 20 min at 30 °C in loading buffer (20 mMTris-Cl, 25 mMNaCl, 4 mM
EDTA) containing 1 mMdithiothreitol (DTT), and the nucleotide-pro-
tein complex was stabilized by chilling the samples on ice and adding
MgCl
2
to a final concentration of 20 mM. GDP-loaded Rab3A (2 pmol/
sample; final volume 50
␮
l) was then incubated with increasing con-
centrations (30–1000 nM)of
␥
-
35
S-GTP for 15 min at 30 °C in binding
buffer (20 mMTris-Cl, 10 mMEDTA, 5 mMMgCl
2
,1mMDTT, pH 7.5)
in the absence or presence of synapsin I (1
␮
M). Nonspecific binding was
defined as the binding in the presence of an excess unlabeled GTP (300
␮
M). The reaction was stopped by the addition of 2 ml of ice-cold
stopping solution (25 mMTris-Cl, 20 mMMgCl
2
, 100 mMNaCl, pH 7.4)
followed by rapid filtration on 0.45-
␮
m nitrocellulose filters. Filters
were washed five times with ice-cold stopping solution, and the retained
radioactivity was determined by liquid scintillation counting. For the
analysis of GTP binding to SV, synapsin-depleted SV (SSV) were first
loaded with GDP (500
␮
M) as described above, and the excess GDP was
removed by high speed centrifugation (400,000 ⫻gfor 20 min) after
addition of 20 mMMgCl
2
(17). GDP-loaded SV (10
␮
g of protein/sample)
were assayed for
␥
-
35
S-GTP binding as described above, followed by
filtration and scintillation counting.
Dissociation of [
3
H]GDP from Rab3A—Recombinant Rab3A or SSV
were loaded with [
3
H]GDP (1
␮
M) by incubation at 30 °C for 20 min in
20 mMTris-Cl, 4 mMEDTA, 1 mMDTT, pH 7.4, followed by cooling the
samples on ice and adding MgCl
2
to a final concentration of 20 mM. The
dissociation of [
3
H]GDP from recombinant Rab3A (2 pmol/sample) or
SSV (10
␮
g protein/sample) was initiated at time t⫽0 by the addition
of a 200-fold excess of unlabeled GTP in a mixture containing 20 mM
Tris-Cl, 200 mMNaCl, 2 mMMgCl
2
,1mMDTT, pH 7.5, in the absence
or presence of synapsin I (1
␮
M). The reaction was stopped by filtration
on 0.45-
␮
m nitrocellulose filters as described above at various times (15
min to 3 h) after the addition of GTP (28).
Rab3A GTPase Activity—Assays for Rab3A GTPase activity were
performed as described previously (28, 29) with some modifications. For
the formation of the [
␣
-
32
P]GTP-Rab3A complex, recombinant Rab3A
(25 pmol/sample) or purified SV (5
␮
g of protein/sample) were incubated
for 10 min at 30 °C in loading buffer containing 50 nM[
␣
-
32
P]GTP (1
␮
Ci/sample). The mixture was placed on ice, and MgCl
2
was added to a
final concentration of 20 mMto stabilize the nucleotide-protein complex.
Excess unbound [
␣
-
32
P]GTP was removed by passage through a gel
filtration column Bio-Spin 6, previously equilibrated in reaction buffer
(20 mMTris-Cl, pH 7.4, 100 mMNaCl, 5 mMMgCl
2
,1mMDTT). For the
measurement of GTPase activity, GTP-loaded samples were incu-
bated at 30 °C in reaction buffer in the absence or presence of purified
synapsin I (25 pmol/sample). Aliquots (20
␮
l) of the reaction mixtures
were taken at various time intervals and stopped by the addition of
0.2% (w/v) SDS, 2 mMDTT, 2 mMEDTA, 0.5 mMGTP, 0.5 mMGDP
(final concentration) followed by incubation at 65 °C for 20 min. The
ratio of GTP and GDP bound to Rab3A was quantified by TLC as
described previously (30). Aliquots were applied to PEI-cellulose TLC
sheets, which were then immersed in methanol for 5 min, dried at
room temperature, and developed with 4 Mformic acid (brought to pH
3.5 with NH
4
OH). After completion of the run (about 1 h), chromato-
grams were immersed in methanol for 20 min, dried, and exposed at
⫺80 °C. Based on nucleotide staining, the radioactive spots corre-
sponding to [
␣
-
32
P]GTP and [
␣
-
32
P]GDP were scraped from the chro-
matogram and counted by liquid scintillation spectrometry. The hy-
drolysis of Rab3A-bound GTP was expressed as percent increase in
[
␣
-
32
P]GDP formation with respect to the amount of [
␣
-
32
P]GDP
present at time 0.
Rab3A-GTPase Activating Protein (GAP) Activity—Recombinant
Rab3A (10 pmol/sample), loaded with [
␥
-
32
P]GTP as described above,
was incubated with recombinant Rab3-GAP for 6 min at 25 °Cinthe
absence or presence of either Rabphilin-3 or synapsin I in a buffer
containing 20 mMTris-Cl, 100 mMNaCl, 1 mMMgCl
2
, pH 8.0. At the
end of the incubation, samples were diluted with ice-cold buffer and
filtered through 0.45-
␮
m nitrocellulose membranes, and the retained
radioactivity was determined by scintillation counting (31).
Assays of the Biochemical Properties of Synapsin I
Actin Binding and Bundling Assays—G-actin (5
␮
M) purified as
described previously (32) in G-buffer (0.2 mMATP, 0.2 mMCaCl
2
, 0.5
mM2-mercaptoethanol, 0.5 mMNaN
3
,2mMTris-Cl, pH 8.0) was poly-
merized for1hatroom temperature by the addition of 90 mMKCl, 2 mM
MgCl
2
and incubated at 25 °C for 1 h with synapsin I (0.5
␮
M), Rab3A
(0.5–5
␮
M), or synapsin I plus Rab3A in the presence of either GTP
␥
Sor
GDP. Identical samples were centrifuged either at low speed (10,000 ⫻
gfor 10 min) for recovery of actin bundles or subjected to high speed
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centrifugation in a Beckman TLA-100 rotor (400,000 ⫻gfor 30 min) for
recovery of total F-actin (33). The actin pellets and supernatant frac-
tions were solubilized in Laemmli sample buffer and subjected to SDS-
PAGE (34). The amounts of actin in the various fractions were detected
by Coomassie Blue staining, whereas the amounts of synapsin I bound
to actin or free in the supernatant were determined by either protein
staining of the gels or immunoblotting. Quantitative analysis of Coom-
assie-stained gels or immunoblots was performed either by laser scan-
ning densitometry (Ultroscan XL, Amersham Biosciences) by interpo-
lation of the density values into a suitable standard curve.
Vesicle Aggregation Assays—Phospholipid vesicles mimicking the
phospholipid composition of SV (PC:PE:PS:PI:cholesterol ⫽40:32:12:5:
10) or PC vesicles (PC:cholesterol ⫽90:10) were made by sonication as
described previously (35). Fluorescently labeled vesicles had the same
lipid composition with the addition of the appropriate amounts (2% of
the total lipid, w/w) of NBD-PE or LRh-PE either alone (single-labeled
liposomes) or in combination (double-labeled liposomes). Changes in
synapsin-induced vesicle aggregation in the presence of either Rab3A-
GDP or Rab3A-GTP were followed by analyzing the fluorescence reso-
nance energy transfer (FRET) between the energy donor NBD-PE and
the acceptor LRh-PE (35). Synapsin I (100 nM)-induced aggregation in
the presence or absence of GDP- or GTP-bound Rab3A (1
␮
M) was
followed by monitoring FRET at 22 °C using a PerkinElmer Life Sci-
ences LS-50 spectrofluorometer according to two distinct assays. In the
first case (aggregation/fusion assay), the fluorescence donor and accep-
tor were incorporated into separate vesicle populations. Two popula-
tions of vesicles (100
␮
g of phospholipid for each vesicle population,
containing 2% labeled phospholipid) were mixed, and FRET was meas-
ured by exciting the donor at 470 nm and following either the decrease
in NBD emission at 520 nm (NBD quenching) or the increase in LRh
emission at 590 nm (excitation and emission slits of 2.5 and 5 nm,
respectively). In the second assay (fusion assay), one population of
vesicles containing both fluorophores in equimolar amounts (50
␮
gof
phospholipid, 2% labeled phospholipids) was mixed with unlabeled
vesicles (150
␮
g of phospholipid). Under these conditions, pure aggre-
gation of vesicles not accompanied by fusion is silent per se but can by
evaluated as an enhancement in the rate and extent of vesicle fusion
induced by a subsequent addition of 3 mMCa
2⫹
. Membrane fusion,
leading to intermixing of labeled and unlabeled membrane components,
results in a decrease in the surface density of donor and acceptor
FIG.1.Synapsin I stimulates the GTPase activity of Rab3A. A, purified [
␣
-
32
P]GTP-loaded Rab3A (25 pmol) was incubated at 30 °C for the
indicated times in the absence (black bars) or presence (gray bars) of purified synapsin I (Syn I; 25 pmol). GTP hydrolysis was evaluated by TLC
(inset) and expressed as percent increase in [
␣
-
32
P]GDP formation versus time 0 (means ⫾S.E.; n⫽5). *, p⬍0.05; **, p⬍0.01, Student’sttest
versus Rab3A alone. contr., control. B, dose-dependent effect of synapsin I on Rab3A GTPase activity. Purified [
␣
-
32
P]GTP-loaded Rab3A (25 pmol)
was incubated for 60 min at 30 °C in the absence or presence of increasing amounts of purified synapsin I. GTP hydrolysis was evaluated as
described in A.Columns in the plot are means ⫾S.E. (n⫽5). **, p⬍0.01, Duncan’s multiple comparison test versus Rab3A alone. C, the
endogenous GTPase activity associated with SV is regulated by synapsin I. Five micrograms of untreated SV (black circles), synapsin I-depleted
SV (white circles), or synapsin I rebound SV (black triangles) were loaded with [
␣
-
32
P]GTP and incubated at 30 °C. Aliquots were taken at various
times (20–120 min) and analyzed for GTP hydrolysis by TLC (see A). Points in the plot are means ⫾S.E. (n⫽5). *, p⬍0.05; **, p⬍0.01, Duncan’s
multiple comparison test versus GTP hydrolysis in USV. D, effects of synapsin I and Rabphilin-3 on Rab3A-GAP activity. Purified [
␥
-
32
P]GTP-
loaded Rab3A (10 pmol) was incubated with recombinant Rab3A-GAP for 6 min at room temperature in the absence (control) or presence of either
Rabphilin-3 (black symbols) or synapsin I (white symbols). Samples were filtered through cellulose filters, and the retained radioactivity linked to
GTP was determined by scintillation counting. Data are expressed as percent changes with respect to the control samples (means ⫾S.E.; n⫽5).
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fluorophores and thereby in FRET that can be followed as an increase
in NBD emission at 520 nm (NBD dequenching) or a decrease in LRh
emission at 590 nm.
Rab3A-Synapsin I Pull-down Assays—The binding of glutathione
S-transferase (GST)-Rab3A to dephosphorylated synapsin I or to syn-
apsin I that had been stoichiometrically phosphorylated by either PKA,
CaMKII, MAPK, or cdk-1 was assessed by co-precipitation experiments
as follows. GST or GST-Rab3A loaded with GTP
␥
S was coupled to
glutathione-Sepharose (0.04 nmol of fusion protein per
␮
l of settled
beads) in binding buffer (10 mMHepes, 150 mMNaCl, 1% (v/v) Triton
X-100, 2 mg/ml BSA, pH 7.4) containing 5 mMMgCl
2
. After extensive
washing, protein-coupled beads (15
␮
l) were incubated with synapsin I
(0.1–0.5
␮
M)in500
␮
l of binding buffer for 3–5hat4°C. After the
incubation, the beads were pelleted by centrifugation, extensively
washed with binding buffer and detergent-free binding buffer, resus-
pended in Laemmli sample buffer, and boiled for 2 min. Binding was
analyzed by SDS-PAGE followed by quantitative immunoblotting.
Binding of Rab3A to Synaptic Vesicles
Purified SV containing endogenous synapsins (untreated SV (USV))
were quantitatively depleted of synapsin I by exposure to mild salt
treatment (synapsin-depleted SV (SSV)) and reassociated in vitro with
purified synapsin I (rebound SV (RSV)) as described previously (27).
Purified SV (USV, SSV, or RSV, 20
␮
g protein/sample) were analyzed
for the translocation of endogenous Rab3A and/or synapsin I between
the SV-bound and the soluble form. SV were loaded with GDP or GTP
␥
S
(500
␮
M) in loading buffer for 15 min at 37 °C, chilled on ice, and
supplemented with 20 mMMgCl
2
to stabilize the nucleotide-protein
complex. Samples were then incubated for 45 min at 37 °Cinthe
FIG.2.Synapsin I enhances GTP binding to Rab3A without affecting GDP dissociation. Aand B, GDP-loaded purified Rab3A (2 pmol)
or synapsin I-depleted SV (10
␮
g) was incubated with increasing concentrations (30–1000 nM)of
␥
-
35
S-GTP for 15 min at 30 °C in the absence
(black symbols or bars) or presence (white symbols or bars)of1
␮
Msynapsin I (Syn I). Nonspecific binding was defined as the binding in the presence
of an excess unlabeled GTP (300
␮
M). Incubation was stopped by rapid filtration on nitrocellulose filters, and the retained radioactivity was
determined by liquid scintillation counting. The average binding isotherm of GTP binding to purified Rab3A, calculated according to the one
ligand/one binding site model, is shown in A.Points in the plot represent means ⫾S.E. of six independent experiments. The calculated maximal
GTP binding (B
max
) to purified Rab3A or SSV is shown in Bin percentage of the values observed in the absence of synapsin I (means ⫾S.E.; n⫽
6). *, p⬍0.01, Student’sttest versus control. C, the increase in GTP binding induced by synapsin I is not due to reversal of EDTA-induced Rab3A
unfolding. GDP-loaded purified Rab3A was incubated with
␥
-
35
S-GTP (1
␮
M) as described above in the absence (black bars) or presence (white bars)
of BSA (2 mg/ml). Specific
␥
-
35
S-GTP binding is expressed in percentage of the values observed in the absence of synapsin I and BSA (means ⫾
S.E.; n⫽4). °,p⬍0.01; Student’sttest versus samples incubated in the absence of synapsin I. No statistical difference was found between the
two groups incubated in the absence or presence of BSA. Dand E, recombinant Rab3A (2 pmol, D) or SSV (10
␮
g, E) were loaded with [
3
H]GDP
(1
␮
M) as described under “Experimental Procedures.”GDP dissociation was triggered by an excess of GTP (300
␮
M) in the absence (black symbols)
or presence (white symbols)of1
␮
Msynapsin I. The residual amount of [
3
H]GDP bound to purified Rab3A or SSV was followed over time (15–180
min) and expressed in percent of the binding in the absence of GTP.
Effects of the Synapsin I-Rab3A Interaction43772
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presence of various concentrations of either synapsin I (0.25, 0.5, and 1
␮
M), purified RabGDI (0.05, 0.2, and 0.5
␮
M), or both. After the incu-
bation, samples were centrifuged for 20 min at 450,000 ⫻g, and the
resulting supernatant and pellet fractions were analyzed by SDS-PAGE
and immunoblotting.
RESULTS
Effects of Synapsin I on the Biochemical Properties of
Rab3A—The rate of the Rab3 cycle within the nerve terminal is
regulated by the key events of GDP/GTP exchange, which
follows the association of Rab3 with SV, and GTP hydrolysis
that promotes Rab3 dissociation from SV during priming and
fusion. We employed biochemical assays to determine whether
the association between synapsin I and Rab3 modulates Rab3
GTPase activity or nucleotide binding. The intrinsic Rab3
GTPase activity was assayed by measuring the decrease in the
radioactivity associated with [
␣
-
32
P]GTP-loaded Rab3A and
the corresponding increase in [
␣
-
32
P]GDP formation as a func-
tion of time. Synapsin I significantly stimulated the GTPase
activity of both recombinant Rab3A (Fig. 1A) and endogenous
Rab3 associated with highly purified SV (Fig. 1C). Synapsin I
increased the GTPase activity of purified Rab3A in a time- and
concentration-dependent manner, with a ⬵5-fold increase in
the initial rate of GTP hydrolysis at a 1:1 synapsin:Rab3A
molar ratio (Fig. 1B). The GTPase activity of SV depleted of
endogenous synapsin (SSV) was decreased by ⬃50% in com-
parison to untreated SV (USV), whereas the in vitro reassocia-
tion of purified synapsin I with synapsin depleted SV (RSV)
restored GTP hydrolysis to the levels observed in untreated SV
(Fig. 1C). Rabphilin, another SV Rab3 interactor stimulating
Rab3 GTPase activity, is known to markedly inhibit in a dose-
dependent fashion GAP-stimulated GTPase activity of Rab3 by
binding to the Rab3 effector domain. However, at variance with
Rabphilin-3, synapsin I did not inhibit the effect of GAP on
Rab3 GTPase activity (Fig. 1D).
Equimolar concentrations of synapsin I significantly en-
hanced the binding of
␥
-
35
S-GTP to both purified and SV-
associated GDP-Rab3 by inducing a significant, 3-fold increase
in the B
max
of
␥
-
35
S-GTP (Fig. 2,Aand B). As the assay protocol
involved nucleotide stripping by EDTA, we wondered whether
the observed effects were simply attributable to Rab stabiliza-
tion by a chaperone-like activity of synapsin I. To test this
possibility, we repeated the assays in the presence of BSA and
found that the effect of synapsin I was fully preserved also
under these conditions (Fig. 2C), indicating that it was not
merely due a nonspecific chaperone-like effect. At variance
with GTP binding, the dissociation of [
3
H]GDP from either
recombinant Rab3A (Fig. 2D) or synapsin-depleted SV (Fig. 2E)
triggered by an excess of GTP was not affected by the presence
of synapsin I.
Modulation of the Synapsin-Rab3A Interaction by Synapsin
Phosphorylation—Synapsin I is a target of multiple protein
kinases, and site-specific phosphorylation of synapsin I is
known to affect its interactions with SV and the actin cytoskel-
eton and its compartmentalization within nerve terminals (22,
36, 37). Thus, we evaluated whether phosphorylation of synap-
sin by either PKA (site 1), CaMKII (sites 2 and 3), MAPK (sites
4–6), or cdk-1 (site 6) was able to affect its interaction with
Rab3A and the subsequent stimulation of Rab3A GTPase ac-
tivity. Rab3A pull-down assays revealed that the interaction
with synapsin I is poorly sensitive to its phosphorylation state,
whereas phosphorylation by CaMKII was totally ineffective,
phosphorylation by either PKA or MAPK/Erk induced only a
mild decrease in the binding at low synapsin concentrations
(Fig. 3A). Consistent with binding data, both phosphorylated
and dephosphorylated forms of synapsin I significantly stimu-
lated Rab3A GTPase activity, although PKA- or MAPK-phos-
phorylated synapsin I was less powerful than dephosphoryl-
ated synapsin I (Fig. 3B).
Effect of Rab3A on the Biochemical Properties of Synap-
sin I—Synapsin I binds to actin filaments via a major site
located in the central domain C and induces the formation of
actin bundles in vitro (33, 38). Thus, we investigated whether
the interaction with Rab3A was able to interfere with the
binding of synapsin I to F-actin and with the bundling of actin
filaments induced by dephosphorylated synapsin I. Rab3A
brought about a concentration-dependent inhibition of both
synapsin binding to actin filaments and synapsin-induced actin
bundling. Although the presence of up to 5
␮
MRab3A did not
alter actin assembly per se, it significantly inhibited the bind-
ing of synapsin I to F-actin and virtually abolished the synap-
sin I-induced actin filament bundling, as evaluated by high
speed and low speed sedimentation assays, respectively (Fig.
4A). Analysis of the dose-response curves using a 3-parameter
logistic function yielded IC
50
values for actin bundling of 1.49 ⫾
0.16 and 1.31 ⫾0.14
␮
M(means ⫾S.E.) for GDP-Rab3A and
GTP-Rab3A, respectively, and IC
50
values for synapsin binding
to F-actin of 3.58 ⫾0.28 and 3.14 ⫾0.48
␮
M(means ⫾S.E.) for
GDP-Rab3A and GTP-Rab3A, respectively. These values are in
FIG.3.Effects of site-specific phosphorylation of synapsin I on
the GTPase activity of Rab3A. A, dephosphorylated synapsin I (Syn
I) or synapsin I that had been stoichiometrically phosphorylated by
either PKA on site 1 (1P-Syn I), by CaMKII on sites 2 and 3 (2,3P-Syn
I), or by MAPK/Erk on sites 4–6(4,5,6P-Syn I) was incubated with
either GST or recombinant GST-Rab3A (20
␮
g) loaded with GTP
␥
S, and
the amounts of synapsin I bound to Rab3A were analyzed by quantita-
tive immunoblotting. B, effects of site-specific phosphorylation of syn-
apsin I on Rab3A-mediated GTP hydrolysis analyzed after 20 (black
bars)or60min(gray bars) of incubation at 30 °C. The results are
expressed as percent increase in [
␣
-
32
P]GDP formation versus time 0
(means ⫾S.E.; n⫽5). *, p⬍0.05; **, p⬍0.01, Dunnett’s multiple
comparison test versus control (NO Syn I). For further details, see
legend to Fig. 1. 6P-Syn I, cdk1-phosphorylated synapsin I; HI Syn I,
heat-inactivated synapsin I.
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FIG.4. Rab3A inhibits the interac-
tions of synapsin I with actin. A, poly-
merized actin (5
␮
M) was incubated at
room temperature for1hintheabsence
or presence of synapsin I (Syn I; 0.5
␮
M),
Rab3A (5
␮
M), or both in the presence of
either GTP
␥
S or GDP. Identical samples
were centrifuged at either low speed
(10,000 ⫻gfor 10 min) for recovery of
actin bundles (actin bundling) or high
speed (400,000 ⫻gfor 30 min) for recov-
ery of total F-actin (actin binding). Pellets
and supernatant (sup) fractions were sub-
jected to SDS-PAGE and analyzed by Coo-
massie Blue staining. No effects of the
various treatments on the recovery of to-
tal F-actin were observed. No significant
actin bundling was seen in the absence of
synapsin I. Band C, the amounts of actin
bundles (B) and of F-actin-bound synap-
sinI(C) recovered in the low and high
speed pellets, respectively, were deter-
mined by densitometric scanning of the
stained gels and of the synapsin immuno-
blots. The densitometric readings, ex-
pressed in percent of the respective val-
ues obtained in the control samples
containing F-actin and synapsin alone,
were plotted as means ⫾S.E. (n⫽5)
versus the concentration of GDP-Rab3A
(black symbols) or GTP-Rab3A (white
symbols). *, p⬍0.05; **, p⬍0.01, Dun-
nett’s multiple comparison test versus
control samples. Dose-response curves
were analyzed using a 3-parameter logis-
tic function. IC
50
values for actin bun-
dling were 1.49 ⫾0.16 and 1.31 ⫾0.14
␮
M
(means ⫾S.E.) for GDP-Rab3A and GTP-
Rab3A, respectively. IC
50
values for syn-
apsin I binding were 3.58 ⫾0.28 and
3.14 ⫾0.48
␮
M(means ⫾S.E.) for GDP-
Rab3A and GTP-Rab3A, respectively.
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good agreement with the estimated K
d
of the synapsin-Rab3A
binding (1–1.5
␮
M) (62). The physiological significance of this
effect is also strengthened by taking into account the reduced
accessibility of synapsin molecules engaged in the thick actin
bundles and the observation that only 20% of recombinant
Rab3A is nucleotide-bound after the loading procedure.
Synapsin I binds to phospholipid vesicles mimicking the
phospholipid composition of SV, and its binding is associated
with vesicle clustering and stabilization of the lipid bilayer
(35). Thus, we investigated whether the association with
Rab3A was able to interfere with the synapsin-induced vesicle
clustering by using fluorometric assays sensitive to either ves-
icle aggregation or vesicle fusion (FRET aggregation/fusion
assay) or specifically sensitive to fusion (FRET fusion assay;
see “Experimental Procedures”). Both assays confirmed that
synapsin I induces vesicle clustering without triggering fusion,
as the addition of synapsin I readily increased FRET in the
aggregation/fusion assay (Fig. 5A), whereas it only potentiated
the fusogenic effect of a subsequent addition of Ca
2⫹
in the
fusion assay (Fig. 5B). Most interestingly, Rab3A in either the
GDP- or GTP-bound form virtually abolished the synapsin
I-induced aggregation of phospholipid vesicles (Fig. 5, A–C),
FIG.5.Rab3A inhibits the synapsin-
induced phospholipid vesicle aggre-
gation. A, equal amounts of two popula-
tions of phospholipid vesicles mimicking
the phospholipid composition of SV alter-
natively labeled with NBD-PE (donor) or
LRh-PE (acceptor) were incubated (ar-
row) in the absence or presence of synap-
sin I (100 nM), GTP-, or GDP-loaded
Rab3A (1
␮
M) or synapsin I plus Rab3A,
and the FRET was followed as a function
of time as the increase in the acceptor
fluorescence (aggregation/fusion assay;
see “Experimental Procedures”). FRET in
the presence of Rab3A alone, either in the
GDP- or GTP-bound form, did not differ
from FRET under control conditions (not
shown). B, phospholipid vesicles labeled
with both NBD-PE and LRh-PE were
mixed with unlabeled vesicles and prein-
cubated for 1 min in the absence or pres-
ence of synapsin I (100 nM), GTP-, or
GDP-loaded Rab3A (1
␮
M) or synapsin I
plus Rab3A (arrow) before the addition of
3m
MCaCl
2
to trigger fusion (arrowhead).
The decrease in FRET due to vesicle fu-
sion was followed as a function of time as
the increase in the donor fluorescence (fu-
sion assay; see “Experimental Proce-
dures”). C, for each experimental condi-
tion, the extent of FRET detected in the
aggregation/fusion assay was determined
6 min after protein addition and plotted in
percent of the FRET observed in the pres-
ence of synapsin I alone. **, p⬍0.01
versus synapsin I alone; Duncan’s multi-
ple comparison test (n⫽5).
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whereas it did not induce aggregation nor promote fusion when
incubated with vesicle suspensions in the absence of synapsin
I (Fig. 5C).
Effect of Synapsin I on the Association of Rab3A with Syn-
aptic Vesicles—We next analyzed whether the synapsin-Rab3
interaction plays some role in the association of either Rab3A
or synapsin I with the SV membrane. Incubation of purified SV
with GDP and RabGDI removes Rab3A from the membrane,
whereas the addition of the nonhydrolyzable GTP analogue
GTP
␥
S prevents Rab3A dissociation, consistent with the notion
that RabGDI solubilizes Rab3A from SV following GTP hydrol-
ysis (24, 39). To study whether synapsin I contributes to the
association of Rab3A with SV, highly purified SV depleted of
endogenous synapsin I or rebound to exogenous synapsin I at
various degrees of saturation were analyzed for the transloca-
tion of Rab3A between the SV-bound and the soluble forms
under conditions of GDP or GTP
␥
S loading. RabGDI promoted
a dose-dependent dissociation of Rab3A from synapsin-de-
pleted SV (SSV) in the presence of GDP (but not of GTP
␥
S) that
was markedly decreased by the reassociation of synapsin I with
the SV membrane (RSV; Fig. 6, Aand B). For a given concen-
tration of RabGDI, this effect was dependent on the degree of
saturation of the SV membrane with synapsin I (Fig. 6C). As
observed before, the effects of synapsin I were detectable only
when Rab3A was in the GDP-bound form and in the presence of
RabGDI. By preventing RabGDI-induced Rab3A solubilization,
synapsin I might thus stabilize the association of Rab3A with
SV. In contrast, Rab3A does not appear to contribute to the
binding of synapsin I to SV. In fact, incubation of untreated SV
containing endogenous synapsin I under conditions promoting
Rab3A dissociation (GDP loading and RabGDI) did not signifi-
cantly affect the association of synapsin I (or of the other SV-
associated Rab3 effector Rabphilin 3) with the SV membrane
(Fig. 6D).
The role of synapsin I in the subcellular compartmentaliza-
tion of Rab3A was also analyzed in mutant mice lacking syn-
FIG.6.Synapsin I contributes to the association of Rab3A with synaptic vesicles. A, highly purified SV depleted of endogenous synapsin
I (SSV, 20
␮
g) or reassociated in vitro with purified synapsin I (rebound SV or RSV, 20
␮
g) and loaded with either GDP or GTP
␥
S (500
␮
M) were
incubated for 45 min at 37 °C in the presence of increasing concentrations of RabGDI and subjected to high speed centrifugation. The amount of
Rab3A associated with the SV pellet was analyzed by immunoblotting with anti-Rab3A antibodies. B, the amount of Rab3A associated with
synapsin-depleted SV (SSV, black bars) or with synapsin I-rebound SV (RSV, white bars) loaded with GDP and incubated in the absence or
presence of RabGDI (0.5
␮
M) as described above was determined by quantitative immunoblotting. Data are expressed in percentage of the values
observed in SSV in the absence of RabGDI (means ⫾S.E.; n⫽5). *, p⬍0.01 versus SSV in the presence of RabGDI; °,p⬍0.01 versus SSV or
RSV in the absence of RabGDI; Duncan’s multiple comparison test. C, highly purified SV depleted of endogenous synapsin I (20
␮
g) loaded with
either GDP or GTP
␥
S (500
␮
M) were incubated for 45 min at 37 °C with increasing concentrations of synapsin I (Syn I;0–1
␮
M) in the absence or
presence of 0.5
␮
MRabGDI, and the amount of Rab3A associated with SV was analyzed as described in A.D, Rab3A does not contribute to the
binding of synapsin I to SV. Untreated highly purified SV (20
␮
g) were incubated as described in Aand simultaneously analyzed for the amounts
of synapsin I, Rab3A, and Rabphilin-3 associated with the SV membrane by immunoblotting with anti-synapsin I (
␣
Syn I), anti-Rabphilin-3
(
␣
Rph3), and anti-Rab3A (
␣
Rab3A) antibodies.
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apsin I, synapsin II, or both. Whereas the expression levels of
Rab3A in total brain homogenate were not greatly affected in
mutant animals, as reported previously (40), the levels of
Rab3A associated with sucrose gradient-purified SV (SG2 frac-
tion) were markedly decreased in synapsin knockout mice with
respect to wild-type littermates (Fig. 7A), suggesting that the
association with synapsin participates in the correct targeting
of Rab3A to the SV compartment. At variance with Rab3A, in
synapsin I knockout mice the levels of other integral SV pro-
teins such as SV2, synaptophysin, or synaptogyrin were de-
creased in both brain homogenate and purified SV to approxi-
mately the same extent, reflecting the decreased number of SV
observed in these animals (40, 41), whereas other proteins
predominantly associated with synaptic membranes, such as
CaMKII, Na
⫹
/K
⫹
-ATPase, GAP-43, or SNAP-25, were not af-
fected in both the homogenate and a sucrose gradient fraction
(SG4) enriched in synaptic membranes (Fig. 7B).
DISCUSSION
We have shown recently that Rab3A, the most abundant
monomeric GTPase associated with SV and involved in the
regulation of the exo-endocytotic cycle of SV, interacts with
synapsin I in purified SV and intact nerve terminals (62).
Synapsins have also been implicated in the regulation of SV
trafficking (35–37, 42, 43) and of the kinetics of the final steps
that immediately precede exocytotic fusion (23, 24). As shown
in Fig. 8, both proteins cycle between a cytosolic and an SV-
associated form and may represent clocks for the SV cycle by
ensuring directionality and reversibility to the process. Thus,
the interaction between synapsin I and Rab3A can be poten-
tially involved in various stages of the SV cycle.
The formation of a complex between synapsin I and Rab3A
affects the biochemical properties of both proteins. Synapsin I
enhances the intrinsic GTPase activity of Rab3A and the bind-
ing of GTP to GDP-Rab3A, suggesting that it can act as a
positive modulator of the Rab3A cycle. As synapsin I does not
affect GDP dissociation from either purified or SV-associated
Rab3A, the increase in GTP binding to Rab3A is likely to be
attributable to a conformational effect that makes Rab3A more
suitable for GTP binding. When compared with Rab3GAP,
synapsin I induces a rather weak stimulation of GTP hydroly-
FIG.7.The amount of Rab3A associ-
ated with SV is decreased in synapsin
I, synapsin II, and synapsin I/II
knockout mice. A, homogenate (HOM)
and purified SV (SG2 fraction) obtained
from synapsin I, synapsin II, and synap-
sin I/II knockout (KO) mice were analyzed
by SDS-PAGE and immunoblotting with
anti-synapsin (G143) and anti-Rab3A an-
tibodies. WT, wild type. B, brain homoge-
nate and fractions enriched in SV or syn-
aptic membranes (SG2 and SG4 fractions,
respectively) obtained from wild-type and
synapsin I knockout mice were assayed
for their content of the integral SV pro-
teins SV2, synaptophysin (SPYS), and
synaptogyrin (SGYR)(left) or of the syn-
aptic membrane-associated proteins
CaMKII, Na/K-ATPase, GAP-43, and
SNAP-25 (right) by immunoblotting with
specific antibodies.
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sis possibly by stabilizing the switch regions of Rab3A, as
already reported for Rabphilin-3 (44). However, at variance
with Rabphilin-3, the stimulation of GTP hydrolysis by synap-
sin I is not mutually exclusive with that promoted by
Rab3GAP, and the possibility exists that basic residues in the
COOH-terminal region of synapsin I involved in Rab3A binding
(syn-(618–652)) bind to the
␥
-phosphate of GTP, mimicking the
Rab3GAP arginine finger (31).
It is of interest to compare the respective properties of Rab-
philin-3 and synapsin I, the two major SV-associated Rab3
effector proteins. Both proteins are extrinsic membrane pro-
teins that bind SV through multiple sites (21, 46, 50), display
phospholipid binding, are phosphorylated by Ca
2⫹
-dependent
kinases that regulate their SV association, interact with actin
or actin-binding proteins, bind to the Rab3 effector domain, and
promote a weak stimulation of Rab3 GTPase activity (16, 21,
22, 44–47). Most interestingly, Rab3A binding inhibits both
the interaction of Rabphilin-3 with
␣
-actinin, an actin filament
cross-linking protein (16), and the binding/bundling of actin
filaments by synapsin I (this paper), indicating a role of these
interactions in the activity-dependent reorganization of the
actin cytoskeleton of the nerve terminal. However, important
differences exist. Rabphilin-3 displays a higher binding affinity
for Rab3A than synapsin I (48, 49), and its binding is entirely
specific for the GTP-bound form of Rab3A and strongly inhibits
Rab3-GAP activity (31, 44), which is not the case for synapsin
I binding. Although conflicting results were reported on
whether Rabphilin-3 recruits GTP-Rab3A to SV or is recruited
to SV by GTP-Rab3 (17, 51), it has been shown that the GDP-
bound form of Rab3A associates with SV before GTP/GDP
exchange (18) (Fig. 8, steps 4 and 5) and that synapsin I may
play a role both in recruiting GDP-Rab3A to SV and in the
subsequent GTP binding to Rab3A (this paper). Accordingly, no
changes in Rab3A compartmentalization have been observed in
Rabphilin-3 knockout mice (52), whereas a decreased SV tar-
geting of Rab3A is present in mutant mice lacking synapsins
(this paper). These observations suggest that synapsin I and
Rabphilin-3 can operate in series as it often occurs for Rab
effectors, with synapsin binding to Rab3A preceding Rabphi-
lin-3 binding to the SV membrane.
It is not yet clear whether GTP hydrolysis is important for
Rab3A to accomplish its function and the precise step at which
Rab3A acts. However, a large body of experimental data indi-
cate that GTP-bound Rab3A interacts with a prefusion complex
preventing fusion and that GTP hydrolysis removes this inhi-
bition (for review see Refs. 2, 4, and 53) (Fig. 8, step 2). Indeed,
in a variety of experimental systems overexpression of consti-
tutively active Rab3A inhibits neurotransmitter release (8–
10), whereas mutant mice lacking Rab3A exhibit an increased
number of SV fusion events per synapse and per impulse,
suggesting a role of Rab3A in regulating the efficiency of the
priming and/or fusion step(s) (13). Thus, it is tempting to spec-
ulate that the role of synapsin I in determining the kinetics of
SV fusion (23, 24) (Fig. 8, step 2) is linked to its interaction with
Rab3A.
Recent work has indicated that the reassociation of Rab3A
with SV after endocytosis occurs through delivery of GDP-
Rab3A to SV by RabGDI and involves a protein component of
SV that is not Rabphilin-3 (17, 18) (Fig. 8, step 4). Indeed,
synapsin I increases the association of GDP-Rab3A with the SV
membrane in the presence of RabGDI. This effect indicates
that synapsin I competes with RabGDI for GDP-Rab3A, exhib-
iting a GDI dissociation factor-like activity (19, 20). This inter-
pretation is strengthened by the following observations. (i)
Synapsin I can bind also to GDP-Rab3A, although to a lesser
extent than to GTP-Rab3A (62). (ii) Rab3A recruitment by SV
in the presence of RabGDI parallels the amount of synapsin I
bound to SV. (iii) In genetically altered mice lacking synapsins,
Rab3A appears to be mistargeted, as the amount of Rab3A
associated with SV is decreased, whereas the total amount of
Rab3A in brain is not significantly affected (Ref. 40 and this
paper).
The engagement of synapsin I in Rab3A binding alters its
interactions with actin and SV by inhibiting F-actin binding,
actin bundle formation, and vesicle clustering. As both the
bundling of F-actin and the phospholipid vesicle aggregation
FIG.8. Putative role of synapsin I-Rab3A in the exo-endocytotic cycle of synaptic vesicles. Evoked neurotransmitter release is a
multistep process in which SV, after being released from the actin cytoskeleton (step 1), dock to presynaptic membrane and undergo the sequential
steps of priming and Ca
2⫹
-triggered fusion (step 2). After fusion, SV are retrieved through a process of endocytosis (step 3) and become competent
for a new round of exocytosis (steps 4 and 5). Both synapsin I (S) and Rab3A (R) cycle between a cytosolic and an SV-associated form. Synapsin
I partially dissociates from SV and actin during step 1, making SV available for exocytosis, increases the rate of the post-docking events of priming
and/or fusion (step 2), and reassociates with SV after endocytosis (step 3). On the other hand, GTP-Rab3A (green square) is bound to SV through
an association with SV effectors such as synapsin I and Rabphilin-3 (not shown). After docking, membrane-bound GTP-Rab3A negatively
modulates priming/fusion and eventually dissociates from the SV membrane upon GTP hydrolysis by the action of GDI (step 2). After endocytosis,
GDP-Rab3 (green circle) is delivered to SV where it binds to synapsin I (step 4) and undergoes GTP/GDP exchange (step 5) to reenter the cycle. The
interaction between synapsin I and Rab3 may function in (i) accelerating priming/fusion by stimulation of GTP hydrolysis (step 2); (ii) promoting
the recruitment of GDP-Rab3A from the soluble RabGDI-associated store to the SV membrane (step 4) and the subsequent GTP/GDP exchange
(step 5); and (iii) increasing SV availability for release (step 1) by inhibiting the actin binding and SV clustering activities of synapsin I.
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induced by synapsin I are likely to be contributed by self-
association of synapsin molecules (35, 38, 54, 55) each binding to
single actin filaments or to phospholipid membranes, it is tempt-
ing to speculate that the effect of Rab3A on synapsin-induced
actin filament bundling and vesicle aggregation is attributable to
a change in the dimerization properties of synapsin I. Indeed,
preliminary data revealed that equimolar concentrations of
Rab3A markedly inhibit synapsin I dimerization.
2
The Rab3A-induced changes in synapsin function may alter
the maintenance of the reserve pool of SV by reducing recruit-
ment of SV in the actin-bound clusters and increasing their
availability for exocytosis (22, 36, 37, 43) (Fig. 8, step 1). Indeed,
Rab3A knockout mice also exhibited a decrease in the activity-
dependent recruitment of SV to the active zone and an incom-
plete recovery of the secretory response after exhaustive stim-
ulation (15). These effects, which become apparent under
conditions of high frequency stimulation and high probability
of release, are qualitatively similar to those observed in synap-
sin knockout mice and Aplysia neurons after synapsin neutral-
ization (24, 40, 41). A large body of experimental evidence
indicates that Rab GTPases, such as Rab6, Rab7, Rab11, or
Rab27, regulate intracellular vesicle trafficking by mediating
the interactions between organelles and the cytoskeleton (56–
61). The data presented here indicate that also Rab3A, by
interacting with synapsin I and modulating its actin binding
activity, can participate in the control of SV trafficking within
nerve terminals.
In conclusion, we have demonstrated that synapsin I is a
novel Rab3A effector. As shown in Fig. 8, this interaction may
function in accelerating the rate of the Rab3A cycle at multiple
levels by facilitating GTP hydrolysis (step 2), GDP-Rab3A re-
association with recycled SV (step 4), and GTP binding (step 5)
as well as in promoting an increased availability of SV for
exocytosis by down-regulating the pre-docking functions of syn-
apsin I (step 1). Further functional studies are required to
define in more detail the functional implications of the synap-
sin-Rab3A interaction in the fine-tuning of the exo-endocytotic
cycle of SV and in the regulation of the efficiency and kinetics
of neurotransmitter release.
Acknowledgments—We thank Dr. W. E. Balch (Scripps Research
Institute, La Jolla, CA) for the generous gifts of the rat RabGDI con-
struct and Dr. G. Cesareni for critical reading of the manuscript.
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S. Giovedı` and F. Benfenati, unpublished results.
Effects of the Synapsin I-Rab3A Interaction 43779
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Silvia Giovedì, François Darchen, Flavia Valtorta, Paul Greengard and Fabio Benfenati
FUNCTIONAL EFFECTS OF THE Rab3A-SYNAPSIN I INTERACTION
Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles: II.
doi: 10.1074/jbc.M404168200 originally published online July 20, 2004
2004, 279:43769-43779.J. Biol. Chem.
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