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INTRODUCTION |
Synapsins constitute a family of abundant synaptic vesicle
proteins that are peripheral membrane proteins, and they are attached to the vesicle by an unknown mechanism (1, 2). There are at least three
synapsin genes. Of these, the genes encoding synapsins Ia, Ib, IIa, and
IIb were characterized first (3), whereas the gene encoding synapsin
IIIa (no synapsin IIIb has been found) was identified more recently (4,
5). Synapsins Ia and Ib are the most abundant synapsin isoforms, and
synapsin IIIa is the least abundant synapsin isoform. All synapsins
share a conserved N-terminal phosphorylation site of unknown function
that is a substrate for cAMP-dependent protein kinase A and
Ca2+, calmodulin-dependent protein kinase I. In
addition, synapsins Ia and Ib and possibly other synapsins contain
multiple phosphorylation sites for a large number of kinases including
Ca2+, calmodulin-dependent protein kinase II
and proline-directed kinases (6-9).
Sequence analyses revealed that synapsins are composed of conserved
N-terminal and central domains that are shared between all synapsins
and variable C-terminal domains that differ between synapsins (3). The
largest domain of synapsins is the central C-domain, which measures
more than 300 residues and is the only domain that is conserved in
invertebrate synapsin (10). The C-domain is flanked on the N terminus
by short A- and B-domains; the A-domain contains the only
phosphorylation site that is present in all synapsins. On the C
terminus, the C-domain is followed by two to three variable domains
that differ between synapsins except for the E-domain, a shared domain
found at the very C-terminal domain of synapsins Ia, IIa, and IIIa but
not synapsin Ib or IIb (3-5).
Although synapsins bind to a number of proteins in vitro,
including all elements of the cytoskeleton (actin, microtubules, neurofilaments, and spectrin) and multiple Ca2+-binding
proteins (calmodulin and annexin VI), their functions have remained
obscure (reviewed in Refs. 11 and 12). Even the detailed analysis of
knockout mice lacking both synapsins I and II showed only that
synapsins perform essential functions in regulating exocytosis of
synaptic vesicles and that synaptic vesicles are destabilized without
synapsins (13-15). These functions agree well with the vesicular
localization and stoichiometric phosphorylation of synapsins but do
not tell us what synapsins actually do. A clue to the functions of
synapsins was obtained recently from an unexpected direction: the
crystal structure of the C-domain of synapsin I revealed that it is
highly homologous to bacterial ATP-dependent synthetases,
indicating an enzyme function (16). In support of an enzyme function of
synapsins, biochemical studies demonstrated that the C-domains of all
synapsins bind ATP with high affinity (3, 17). Interestingly,
ATP-binding to the three synapsin C-domains is differentially
regulated. Ca2+ is required for ATP binding to synapsin I
but inhibits ATP binding to synapsin III and has no effect on ATP
binding to synapsin II (4, 17). In synapsins I and II, the difference
in Ca2+ regulation was found to be due to a single,
evolutionarily conserved amino acid residue that differs between the
two synapsins (17). The biochemical and crystallographic studies
established that synapsins are evolutionarily conserved ATP-binding
proteins in which ATP binding is differentially regulated, providing a
rationale for the existence of multiple synapsins. However, the studies have not yet identified an enzyme activity associated with synapsins, which remains to be demonstrated.
In addition to revealing that synapsin C-domains are ATP-binding
proteins, the crystal structure also demonstrated that the C-domain of
synapsin I forms a dimer in the presence or absence of ATP (16). The
contact surface between the two subunits in the dimer is very large,
indicating a stable interaction. Because the C-domains of different
synapsins are homologous to each other, questions arise as to whether
other synapsins also form homodimers and whether the three synapsins
associate into heterodimers. Such heterodimers would link together
synapsin isoforms that presumably perform similar functions via their
C-domains but that perform these functions in the context of distinct
regulatory properties. In the current study, we performed yeast
two-hybrid screens with the original intention of identifying possible
substrates for the C-domain ATPase. Upon analysis, however, we found
that the large majority of the specific prey clones isolated encode
synapsins, suggesting that synapsins are their own major binding
partners. We then identified the mechanism of multimerization and
showed that it occurs in vivo. Finally, we analyzed synapsin
I and II double knockout mice (which still express synapsin III) to
determine whether synapsin III changes in these mice. Our results
demonstrate that synapsins form homo- and heterodimers in
vivo and in vitro, thereby creating a multitude of
combinations of synapsins on the synaptic vesicle surface.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screens--
Yeast two-hybrid screens of a rat
brain cDNA library of postnatal day 8 in pVP16-3 were performed as
described previously (18-20) with a bait vector containing full-length
rat synapsin IIa fused to LexA cloned into the plasmid pLexN. 38 million transformants were screened, yielding 228 positive clones.
Analysis of 100 clones showed that 99 clones exhibited activation of
-galactosidase. Of these, 65 clones were sequenced. 45 clones
encoded synapsins, 9 clones encoded EST108711, 4 clones encoded 14-3-3 proteins, and the others encoded independent clones not identified in
the data banks (Table I).
Quantitative
-Galactosidase Assays of Yeast Two-hybrid
Interactions--
Full-length or partial coding regions of synapsins
Ia, IIa, and IIIa, synaptotagmin I, and lamin (as a negative control; a gift of Dr. S. Hollenberg, Vollum Institute) were cloned into the bait
and prey yeast expression vectors pLexN and pVP16-3 (18-20). The
following vectors were used containing the following inserts: 1)
pLexNSynI-C, residues 110-420 of rat synapsin Ia; 2) pLexN SynIIa,
residues 1-586 of rat synapsin IIa; 3) pLexNSynIII-C, residues 89-399
of rat synapsin IIIa; 4) pVP-SynI1-624, residues 1-624 of
rat synapsin I; 5) pVP-SynII91-479, residues 91-479 of
rat synapsin II; and 6) pVP-SynIII-C, residues 89-399 of rat synapsin
IIIa. Yeast strain L40 was co-transfected with bait and prey vectors
using lithium acetate. Transformants were plated on selection plates
lacking uracil, tryptophane, and leucine. After 3 days of incubation at
30 °C, colonies were inoculated into supplemented minimal medium
lacking uracil, tryptophane, and leucine and placed in a shaking
incubator at 30 °C for 48 h.
-Galactosidase assays were
performed on yeast extracts with protein concentrations of 20-40
mg/liter/assay (21).
Antibodies--
Polyclonal pan-synapsin antibodies (E028) were
raised against a peptide containing the N-terminal sequence found in
all synapsins (NYLRRRLSDSNFMANLPNGYMTDLQTPQP). Polyclonal synapsin III
antibodies (U549 and U551) were generated against a peptide with a
synapsin III-specific sequence (CATERRHPQPLAASF). All peptides were
coupled to keyhole limpet hemocyanin. The monoclonal antibody to
synapsin I (Cl10.22) was a gift from Dr. R. Jahn, and the NMDA receptor antibody was a gift from Dr. N. Brose.
Construction of Bacterial Expression Vectors and Expression and
Purification of Recombinant Proteins--
All vectors encoding
GST1-synapsins were described
previously (4, 17). For expression as a His6-fusion
protein, the C-domain of synapsin I was cloned from pGexrSynI-C (17)
into the BamHI-SalI sites of pET28a (Novagen) to
obtain pETrSynI-C. Various pET and pGEX constructs were co-transformed
into BL21 (DE3) cells, selected simultaneously with ampicillin and
kanamycin, and purified on glutathione-agarose or Ni2+-NTA
resin as suggested by the manufacturer.
COS Cell Transfections, Analysis of Antibody Specificity, and
Immunoprecipitations--
For expression of synapsins in COS cells,
the coding region of the bovine cDNA of synapsin Ia was cloned into
the EcoRI-HindIII sites of vector pCB1, yielding
pCMVbSynIa. Rat cDNAs of synapsin IIa and IIIa were cloned into the
BglII and ClaI sites or the EcoRI site
of pCMV5 to construct pCMVrSynIIa and pCMVrSynIIIa, respectively.
Plasmid DNAs were transfected into COS cells with various combinations
of synapsins using DEAE-dextran, and transfected cells were harvested
48 h after transfection as described previously (22, 23). COS
cells transfected with various synapsins were analyzed by
immunoblotting using the various synapsin antibodies. For
immunoprecipitations, transfected COS cells were solubilized in 20 mM HEPES/NaOH, pH 7.4, 0.1 M NaCl, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mg/liter
aprotinin, 2 mg/liter antipain, 1 mg/liter pepstatin, and 1 mg/liter
leupeptin. Insoluble material was removed by centrifugation (10,000 rpm
for 10 min at 4 °C). Soluble extracts were incubated in 1 ml of
solubilization buffer with 10 µl of polyclonal antibody or 2 µl of
monoclonal antibody under rotation at 4 °C; controls received no
primary antibody. 30 µl of a 50% slurry of protein A- or protein
G-Sepharose were then added, the reaction was incubated overnight at
4 °C, and samples were centrifuged, washed in solubilization buffer, and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting.
Protein Quantification--
Brain homogenates from wild type or
mutant mice lacking synapsins I and II (double knockouts) were analyzed
by 10% SDS-polyacrylamide gel electrophoresis and immunoblotting (10 µg of protein/lane) on the same gels to control for variations in
protein transfer. Blots were probed with antibodies to NMDA-receptor,
synaptophysin, and synapsin III using ECL detection or
125I-labeled antibodies (for quantification by a Molecular
Dynamics PhosphorImager). Eight mice of each genotype were analyzed
with two different antibodies to ensure reproducibility. The genotype was confirmed by immunoblotting for synapsins I and II. To standardize the immunoblotting signals, all blots were simultaneously probed for
NMDA receptors and either synaptophysin or synapsin III.
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RESULTS |
Yeast Two-hybrid Screens Reveal Synapsin Multimerization--
To
identify proteins that bind to synapsins, we screened a rat brain
cDNA library with full-length synapsin IIa as a bait by yeast
two-hybrid selection (18-20). Yeast two-hybrid methods were used
because the stickiness of synapsins as purified proteins, as documented
in their multitude of in vitro interactions, interferes with
clear-cut interpretations. Synapsin II was chosen instead of synapsin I
because synapsin I contains a positively charged C terminus with a
tendency for binding artifacts, and synapsin IIa was used instead of
synapsin IIb because synapsin IIa contains a C-terminal E-domain that
is also present in synapsins Ia and IIIa (3-5). More than 200 prey
clones were isolated. Sequencing of 65 of these revealed that only
three proteins were selected multiple times as independent overlapping
clones (Table I). 45 of the 65 sequenced
prey clones encoded synapsin I or synapsin II; thus, synapsins account
for the majority of the clones isolated. Nine clones encoded an
unidentified protein present as an EST sequence in the data banks
(EST108711). RNA blots indicated that the mRNA for this protein is
not enriched in brain but is ubiquitously distributed (data not shown),
and tests with irrelevant bait vectors suggested that the EST108711
clones were mildly autoactivating. Thus, it seems probable that their
interaction with synapsins is an artifact. Finally, four clones were
from two distinct isoforms of 14-3-3 proteins that we have frequently
isolated in yeast two-hybrid screens with other baits and may also
represent an artifact. Indeed, synapsin immunoprecipitations failed to
co-precipitate 14-3-3 proteins, indicating that they do not bind to
synapsins physiologically (data not shown).
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Table I
Yeast two-hybrid identification of Synapsin interacting molecules
Legend: 38 million transformants of a rat brain (postnatal
day 8) cDNA library in the prey vector pVP16-3 were screened with
a bait construct containing the full-length sequence of synapsin IIa
fused to LexA in pLexN. 100 positive clones from a total of 228 positives were analyzed; 99 were -galactosidase-positive, and the
plasmid DNA of 65 clones could be rescued and was sequenced. In
addition to those clones listed, we isolated seven independent single
isolates with no homology in the data banks and multiple transcription
factors that probably represent artifacts.
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Analysis of Interactions between Synapsins using Yeast Two-hybrid
Assays--
The isolation of synapsin I and II prey clones in yeast
two-hybrid screens with a synapsin IIa bait suggests that synapsin II
forms dimers by self-association and forms heterodimers by association
with synapsin I. Synapsin III may not have been isolated in the yeast
two-hybrid screens because it is present at low levels in brain. In the
synapsin I crystal structure, the C-domain forms a tight dimer with a
large contact area (16). Thus, it seemed likely that the interaction of
synapsins I and II with themselves and each other is mediated via their
C-domain.
To test this hypothesis and to correlate the crystal structure with
yeast two-hybrid methods, we measured the binding of synapsin IIa to
various fragments of synapsin Ib in yeast two-hybrid assays (Fig.
1). Full-length synapsin Ib and fragments
containing residues 117-451 interacted with synapsin IIa, whereas
fragments composed of residues 123-415 or less were inactive. Thus,
the full-length C-domain appears to be required for binding in the
yeast two-hybrid assays, confirming the correlation between the yeast
two-hybrid and structural approaches.

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Fig. 1.
Yeast two-hybrid analysis of the sequences of
synapsin Ib that are essential for heterodimerization with
synapsin IIa. The domain structure of synapsin Ib is shown
schematically at the top, and the locations of the sequences
encoded by the various prey clones used for analysis are depicted below
the domain structure. Yeast cells were co-transformed with a synapsin
IIa full-length bait construct and the indicated prey constructs. Cells
were selected on supplemented minimal plates lacking uracil,
tryptophan, and leucine. The -galactosidase activities and
resistance to THULL selection of the yeast strains harboring both
vectors were then estimated on selection plates that also lacked
histidine ( THULL) or on nitrocellulose filters soaked with
X-GAL as described previously (18-20).
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We next investigated whether synapsins I and II also interact with
synapsin III in yeast two-hybrid assays and whether the various
interactions exhibit similar strengths. For this purpose, we
constructed prey and bait vectors containing synapsins I, II, and III.
In the L40 yeast strain, interactions of bait and prey proteins that
are expressed in the yeast cells activate a
-galactosidase gene
(18). Therefore, as a quantitative measure of binding of the synapsins
to each other and to control proteins (lamin and synaptotagmin 1), we
determined the
-galactosidase activities in yeast cells that were
transformed with the various synapsin and control bait and prey
vectors. These experiments demonstrated that all three synapsins
interact with themselves and also bind to each other (Fig.
2).

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Fig. 2.
Quantitation of the interactions between
synapsins measured by yeast two-hybrid assays. Yeast cells
co-transfected with the indicated bait vectors in pLexN and with prey
vectors in pVP16-3 were grown in minimal medium lacking uracil,
tryptophan, and leucine. Cells were lysed, and their -galactosidase
activity was measured in quadruplicates as described previously (21).
Data are shown in nmol/min/mg protein ± S.D. (n = 4).
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Synapsin II bound equally strongly to itself and to synapsins I and
III. Similarly, the interactions of synapsin I with synapsin II and of
synapsin II with synapsin III were similar in magnitude to the
interactions of these synapsins with themselves. Only the binding of
synapsin I to synapsin III was significantly weaker (Fig. 2). No
binding of synapsins to control proteins or vectors was observed,
indicating that the observed interactions are specific (Fig. 2).
Characterization of Synapsin Antibodies--
The yeast two-hybrid
studies indicated that synapsins I, II, and III form homo- and
heterodimers via interactions between their C-domains, with the
heterodimerization between synapsins I and III being much weaker than
all other dimerization events. In preparation for an analysis of such
interactions on the protein level, we raised two antibodies to a
peptide from the N terminus of synapsin III and characterized the
relative specificities of these and other antibodies to synapsins. In
these experiments, we used COS cells transfected with the various
synapsin expression vectors and utilized three types of antibodies: 1)
a monoclonal antibody to synapsin I, 2) the two polyclonal antibodies
against synapsin III, and 3) a polyclonal antibody generated against a peptide from the N terminus of synapsin I with a sequence that is
similarly found in synapsins II and III.
The synapsin I antibody and one of the synapsin III antibodies were
found to be specific for their respective isoforms, indicating that
these antibodies can be used to probe only these synapsins (Fig.
3). The second synapsin III antibody also
recognized synapsin I in addition to synapsin III (see below). The
antibody against the conserved N-terminal peptide of synapsins reacted
with all three synapsins, as expected, and therefore represents a
pan-synapsin antibody. On the immunoblots, synapsin I is the largest
protein, whereas synapsin III is the smallest protein, allowing an
unequivocal identification of each synapsin on immunoblots, even with
the pan-synapsin antibody (Fig. 3).

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Fig. 3.
Specificity of synapsin antibodies. COS
cells were transfected with expression vectors encoding synapsins I,
II, and III or with combinations of these expression vectors, as
indicated. Cells were analyzed by immunoblotting with the three
synapsin antibodies shown on the right.
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Analysis of Synapsin Heteromultimerization in Transfected COS
Cells--
To determine whether synapsin heteromultimerization occurs
in vivo and is not an artifact of yeast two-hybrid assays,
we co-transfected COS cells with synapsins I and II or with synapsin II
alone (Fig. 4A, lanes 1 and
4). We then immunoprecipitated extracts from the transfected
COS cells with the synapsin I monoclonal antibody and analyzed the
immunoprecipitates by immunoblotting with the pan-synapsin antibody
(Fig. 4A, lanes 2 and 5). As a control, identical
reactions were performed without the primary antibody (Fig. 4A,
lane 3). The synapsin I antibody co-precipitated synapsin II. The
ratio of synapsin II to I was lower in the immunoprecipitates than in
the COS cells, which is consistent with the notion that synapsin I is
complexed both into homodimers and into heterodimers. In the absence of
synapsin I, no synapsin II was precipitated by synapsin I antibodies,
demonstrating specificity (Fig. 4A, lane 5). Similarly, no
synapsins were brought down in control immunoprecipitations (Fig.
4A, lane 3). These data show that synapsins I and II
heterodimerize in transfected COS cells.

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Fig. 4.
Co-immunoprecipitation of synapsins from
transfected COS cells. COS cells transfected with a combination of
synapsins I and II (A) or II and III (B) were
extracted and subjected to immunoprecipitations using monoclonal
antibody to synapsin I (A, Cl10.22) and polyclonal antibody
to synapsin IIIa (B, 1, U549; 2,
U551). Control immunoprecipitations were performed without primary
antibodies. Starting fractions and immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting using the
polyclonal pan-synapsin antibody. In the experiment shown in
B, polyclonal rabbit antibodies were used for both the
immunoprecipitations and immunoblotting reactions, leading to a high
degree of background staining. Numbers on the
left indicate the positions of molecular weight
standards.
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We next performed similar experiments for synapsins II and III, using
the synapsin III-specific antibody for immunoprecipitation and the
pan-synapsin antibody for immunoblotting (Fig. 4B). However, in this case, the antibodies used for immunoprecipitation and immunoblotting are both polyclonal, leading to an increased background labeling of IgG. For this reason, we performed the immunoprecipitations with the two independent synapsin III antibodies. Despite the background reactivity for IgG, the two bands corresponding to co-precipitated synapsins II and III could be clearly identified in the
immunoprecipitates with synapsin III antibodies from co-transfected COS
cells but not in those from COS cells transfected with synapsin II
alone (Fig. 4B, arrows in lanes 3 and
6). However, when we attempted similar experiments for
synapsins I and III, we were unsuccessful in demonstrating an
interaction (data not shown). Therefore, synapsins II and III dimerize
not only in yeast two-hybrid assays but also in transfected COS cells,
whereas the binding of synapsin I to synapsin III is less certain.
Lack of Stable Heterodimerization between Synapsins I and
III--
The relatively weak interactions of synapsins I and III in
yeast two-hybrid assays compared with other synapsin pairs and our
failure to demonstrate synapsin I/III heterodimers in
immunoprecipitations raise the possibility that the binding of
synapsins I and III may be too weak to allow the formation of a stable
dimer. To test this directly, we expressed GST-synapsins I, II, and III
and His6-synapsin I singly or in combination in bacteria
and purified the recombinant proteins on glutathione-agarose and nickel
columns. The presence of GST- and His6-synapsins in the
purified protein fractions was then examined using Coomassie
Blue-stained SDS gels and immunoblotting (Fig.
5; data not shown). The results show that
the GST-fusion proteins exhibited much higher expression levels,
resulting in a very low abundance of heterodimers in
glutathione-agarose-purified recombinant proteins.
His6-synapsin I dimerization with GST-synapsin I and
GST-synapsin II but not with GST-synapsin III could be detected (Fig.
5, top panel). When we turned the experiment around and purified His6-synapsin I on nickel columns, a nearly
stoichiometric binding of GST-synapsins I and II was found, whereas
synapsin III was absent (Fig. 5, bottom panel). Because
GST-synapsin III was abundantly expressed in the bacteria, these
results indicate that synapsin III indeed does not stably dimerize with
synapsin I.

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Fig. 5.
Heterodimerization of bacterially expressed
synapsins. GST-synapsins I, II, and III and
His6-synapsin I were expressed either singly (lanes
1-3 and 7) or in combination (lanes 4-6)
in bacteria, and recombinant proteins were purified on
glutathione-agarose (top panel) or nickel columns
(bottom panel). Proteins were analyzed by Coomassie Blue
staining; numbers on the left indicate positions
of molecular weight markers. Note that the expression levels of
GST-synapsins vary, but they are much higher than the expression level
of His6-synapsin; as a result, heterodimerization between
synapsins I and II is readily apparent in the nickel column-purified
material but less apparent in the glutathione-agarose-purified material
(asterisks). Immunoblots confirmed that no GST-synapsin III
dimerization with synapsin I occurs (data not shown).
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Synapsin IIIa Levels in Synapsin I/II Double Knockout Mice Are
Decreased--
Knockout mice lacking synapsins I and II are viable and
fertile but exhibit severe changes in the regulation of
neurotransmitter release (13, 14). In addition, synaptic vesicles are
destabilized in these mice, and the levels of vesicle proteins are
decreased. At the time of the analysis of these knockout mice, however,
the existence of a third synapsin isoform (synapsin III) was unknown. Synapsin III is expressed only at low levels; it seems unlikely that it
could functionally substitute for synapsins I and II at physiological
expression levels. However, it is possible that synapsin III expression
may have been induced in the knockouts and obscured a more severe
synapsin knockout phenotype by compensating for the loss of synapsins I
and II. Conversely, some proteins that form heteromultimeric complexes
with other proteins become unstable when their binding partners are not
available (e.g. see Ref. 24). Thus, it is also possible that
synapsin III levels could actually be decreased in the double knockout
mice. To address these questions, we investigated the levels of
synapsin IIIa compared with the levels of a synaptic vesicle protein,
synaptophysin, and to a general synaptic marker, NMDA receptor, in wild
type and synapsin I and II double knockout mice.
Brain homogenates from wild type and double knockout mice were analyzed
by immunoblotting with four antibodies: the two independent antibodies
to synapsin III, an antibody to the NMDA receptor as a control protein
that does not change in the knockouts, and an antibody to synaptophysin
as a synaptic vesicle protein that was previously shown to decrease by
approximately 30% in the double knockouts (14). Because the endogenous
synapsin III levels in the brain are very low, synapsin III
immunoreactivity is weak, and peptide blocks were used to ensure that
the various bands were specific. One of the synapsin III antibodies we
raised cross-reacts with synapsin I; the corresponding band is absent
in the knockouts, whereas the lower synapsin III band is still
recognizable (Fig. 6, top
panel). Both synapsin III antibodies react with additional brain
proteins on immunoblots in a nonspecific reaction that cannot be
blocked with the peptide (Fig. 6, asterisks). Comparison of the patterns of reactivity between the two antibodies failed to uncover
a smaller specific band that could correspond to synapsin IIIb,
suggesting that the synapsin III gene only produces a synapsin IIIa
product (data not shown).

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Fig. 6.
Immunoblots of proteins in synapsin
knockouts. Brain homogenates from wild type mice (WT,
lanes 1 and 2) or double knockout mice lacking
synapsins I and II (DKO, lanes 3 and
4) were analyzed by immunoblotting. As indicated on the
right, immunoblots were probed with antibodies to synapsin
III in the presence and absence of the peptide used for raising the
antibody and with antibodies to the NMDA receptor and to synaptophysin.
The migration of synapsin III is shown on the left; one of
the synapsin III antibodies cross-reacts with synapsin I, which is
absent in the knockouts. In addition, both synapsin III antibodies
react with endogenous brain proteins in a manner insensitive to peptide
competition, indicating that this band is not immunologically related
to synapsin III (asterisks). Blots were visualized by
ECL.
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On the immunoblots, synapsin III can be clearly recognized in the
double knockout mice, but it seems to be decreased, not increased. This
suggests that synapsin III is not induced in the knockout but may
actually be destabilized (Fig. 6). To obtain a quantitative measure of
this, we performed immunoblots with 125I-labeled secondary
antibodies and detected the signal with a PhosphorImager. In agreement
with previous results, an analysis of multiple mice showed that
synaptophysin levels were decreased by approximately 30% in the double
knockouts compared with the NMDA receptor levels. Synapsin IIIa levels,
however, were depressed even further (by 50%; Fig.
7). Statistical analysis showed that the
decrease in synaptophysin and synapsin IIIa levels from wild type to
double knockout mice was highly significant. More importantly, the
relative decrease in synapsin IIIa levels compared with synaptophysin levels in the double knockouts was also statistically validated (p < 10
8). This result demonstrates that
the decrease in synapsin IIIa is not only due to the loss of synaptic
vesicles but represents a further decrease that cannot be accounted for
by the general destabilization of synaptic vesicles (Fig. 7).

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Fig. 7.
Level of synapsin III in synapsin I and II
double knockout mice. The relative levels of synapsin IIIa,
synaptophysin, and NMDA receptor were determined by quantitative
radioimmunoblotting with total brain samples from eight mice of each
genotype (WT, wild type mice; DKO, synapsin I/II
double knockout mice). Blots were reacted with monoclonal and
polyclonal antibodies against NMDA receptor, synaptophysin, and
synapsin III (see Fig. 6). Blots were probed with
125I-labeled secondary antibody, and signals were measured
on a PhosphorImager. All signals were normalized for the signal
obtained with the NMDA receptor antibody as an internal control.
Kruskal-Wallis one-way analysis on ranks was used followed by Dunn's
method for pairwise comparisons to determine statistically significant
effects as indicated.
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DISCUSSION |
Synapsins are peripheral membrane proteins of synaptic vesicles
that are stoichiometrically phosphorylated upon stimulation (1-5).
There are at least three synapsin genes (I, II, and III) directing the
synthesis of at least five synapsins (Ia, Ib, IIa, IIb, and IIIa). The
various synapsins are composed of similar N-terminal and central
domains followed by variable C-terminal domains (3). Their most
striking feature is the presence of a large central domain, the
C-domain, which binds ATP with high affinity and is evolutionarily the
most conserved domain of synapsins (3, 10, 16, 17). Although the
precise functions of synapsins are unknown, analysis of knockout mice
revealed an essential role for synapsins I and II in regulating
synaptic vesicle exocytosis and in stabilizing synaptic vesicles
(13-15). Together with the crystallographic and biochemical analysis
of ATP binding to synapsins, the current data suggest that synapsins
are enzymes that act on the synaptic vesicle in an unknown manner.
The crystal structure of the C-domain of synapsin I uncovered two
striking properties of this large, autonomously folded domain: 1) its
structural homology to ATP-dependent synthases, and 2) its
association into tightly bound dimers (16). Biochemical analysis of the
C-domains from all synapsins showed that each C-domain constitutes an
ATP-binding module with strikingly different regulatory properties (4,
17). The homology between the synapsin C-domains and their similar ATP
binding properties now raise the questions of whether other synapsins
besides synapsin I form homodimers and whether synapsins associate into
heterodimers in addition to homodimers. This is a potentially important
question because homo- and heterodimerization of synapsins would imply
that synaptic vesicles are coated by a multitude of different synapsin
dimers with distinct regulatory properties. Because different synapsins are differentially expressed in neurons in a regulated manner (3,
25-28), the exact composition of the coat of synapsin dimers on the
synaptic vesicle surface could serve as a mechanism by which the
properties of the vesicles are regulated. Four findings reported in the
current study support the conclusion that all synapsins associate into
such homo- and heterodimers:
1) In yeast two-hybrid screens with full-length synapsin IIa as a bait,
synapsins I and II were isolated in 45 of 65 preys analyzed. Synapsin
III presumably was not found because of its low abundance. Because
yeast two-hybrid screens represent an unbiased approach to evaluating
protein-protein interactions, the dominance of synapsins in the preys
selected suggests that the major binding partner of a synapsin molecule
is another synapsin molecule.
2) Quantitative measurements using yeast two-hybrid assays confirmed
that all three synapsin C-domains homodimerize and heterodimerize in
strong, pairwise interactions. We observed an almost equal degree of
binding between synapsins I and II and between synapsins II and III. In
addition, a strong but less intense interaction was observed between
synapsins I and III. These findings agree well with the fact that ATP
binding is Ca2+-dependent for synapsin I,
Ca2+-independent for synapsin II, and
Ca2+-inhibited for synapsin III. Thus, by the strength of
the interactions, dimers between synapsins that are either induced by
Ca2+ or inhibited by Ca2+ would be least likely
to form.
3) In transfected COS cells, immunoprecipitation of synapsin I
co-precipitated co-transfected synapsin II. Similarly,
immunoprecipitation of synapsin III co-precipitated co-transfected
synapsin II. These data show that the synapsin I/II and II/III
heterodimers occur at the protein level. No synapsin I/III dimers were
observed. The heterodimerization of synapsins I and II and the lack of
stable synapsin I/III heterodimers were confirmed in bacterial
expression experiments that revealed that even in the presence of high
levels of synapsins I and III, no stable binding occurred.
4) Finally, in knockout mice lacking synapsins I and II, we found that
the levels of synapsin III were reduced beyond the general decrease
observed for synaptic vesicle proteins in these knockout mice. Previous
studies had shown that synaptic vesicles are destabilized in synapsin
I/II double knockout mice, leading to an overall reduction in vesicle
proteins of approximately 30% (14). This was confirmed in the present
study in which the level of the synaptic vesicle protein synaptophysin
was reduced by 30%. The level of synapsin III, however, was decreased
significantly more to approximately 50%. This result indicates that
synapsin III is disproportionately destabilized, which is consistent
with the notion that it is normally present in a dimer with other
synapsins and is destabilized in the absence of these synapsins.
In summary, our results demonstrate that synapsins homo- and
heteromultimerize with variable efficiency. All synapsins strongly homodimerize; in addition, synapsin II forms strong heterodimers with
synapsins I and III, whereas synapsins I and III interact only weakly,
if at all. These data suggest that synapsins exist on the vesicle
surface as obligatory dimers. As a result, up to 13 distinct
combinations of synapsin dimers may be formed by the five different
synapsins on the synaptic vesicle surface. The data portray synapsins
as a multigene family of synaptic vesicle proteins that are more
variable than previously imagined.