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J Biol Chem, Vol. 274, Issue 26, 18290-18296, June 25, 1999
,§,,
From the Center for Basic Neuroscience, Department of
Molecular Genetics, and Howard Hughes Medical Institute, University of
Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 and
¶ Abteilung Neurobiologie, Max-Planck-Institut für
biophysikalische Chemie, 37075 Goettingen, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Multiple synaptotagmins are expressed in brain,
but only synaptotagmins I and II have known functions in fast,
synchronous Ca2+-triggered neurotransmitter release.
Synaptotagmin III was proposed to regulate other aspects of synaptic
vesicle exocytosis, particularly its slow component. Such a function
predicts that synaptotagmin III should be an obligatory synaptic
vesicle protein, as would also be anticipated from its high homology to
synaptotagmins I and II. To test this hypothesis, we studied the
distribution, developmental expression, and localization of
synaptotagmin III and its closest homolog, synaptotagmin VI. We find
that synaptotagmins III and VI are present in all brain regions in
heterogeneous distributions and that their levels increase during
development in parallel with synaptogenesis. Furthermore, we show by
immunocytochemistry that synaptotagmin III is concentrated in synapses,
as expected. Surprisingly, however, we observed that synaptotagmin III
is highly enriched in synaptic plasma membranes but not in synaptic
vesicles. Synaptotagmin VI was also found to be relatively excluded
from synaptic vesicles. Our data suggest that synaptotagmins III and VI
perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events,
indicating that the functions of synaptotagmins are more diverse than
originally thought.
Synaptotagmins represent a family of at least 12 proteins that are
thought to function in membrane traffic (reviewed in Ref. 1). All
synaptotagmins are composed of a short N-terminal sequence that is
intraluminal and/or extracellular, a single transmembrane region, and a
large cytoplasmic sequence that contains two C2-domains (1). In all synaptotagmins, the N-terminal sequence is translocated across the membrane during translation but lacks a cleaved signal peptide (2, 3), and the transmembrane regions contain multiple cysteine
residues that may be palmitoylated (4, 5). The C2-domains,
the most conserved feature of synaptotagmins (1), are separated from
the transmembrane region by a variable connecting sequence.
C2-domains are general Ca2+-binding motifs that
function as Ca2+-binding modules in most synaptotagmins
(reviewed in Ref. 6). In some synaptotagmins, however, the
C2-domains contain sequence changes that probably abolish
Ca2+ binding (e.g. synaptotagmins IV and XI;
Refs. 3 and 7), suggesting that C2-domains can also perform
Ca2+-independent functions.
Synaptotagmins I and II, the first molecularly characterized
synaptotagmins (8, 9), are abundant synaptic vesicle proteins that are
differentially expressed; synaptotagmin I is primarily present in
rostral regions, and synaptotagmin II is primarily present in caudal
brain regions (9, 10). Knockout experiments in mice revealed that
synaptotagmin I is essential for fast Ca2+-triggered
neurotransmitter release but is not required for exocytosis as such;
slow Ca2+-triggered neurotransmitter release and
Ca2+-independent release were still functional in the
knockout animals (11). These results indicated that synaptotagmin I
functions as a Ca2+ sensor for fast synaptic vesicle
exocytosis in upper brain regions. Synaptotagmin II probably performs
an equivalent role in lower brain regions because it is very homologous
to synaptotagmin I and is also enriched in synaptic vesicles (9,
11).
Similar to synaptotagmins I and II, synaptotagmins III-XII are
expressed primarily in brain (3, 12-17). However, the mRNAs of
most of these other synaptotagmins are detectable at low levels outside
of the brain (3, 12-17). Although the enrichment of synaptotagmins
III-XII in brain and their homology to synaptotagmins I and II led to
the notion that synaptotagmins III-XII may also reside on the synaptic
vesicle, their localizations and potential functions have not been
investigated in detail. Synaptotagmin III is particularly interesting
because it is the most abundant synaptotagmin after synaptotagmins I
and II and because it exhibits Ca2+-binding characteristics
that resemble the Ca2+ requirements of the slow component
of Ca2+-dependent neurotransmitter release
(18-20). These findings have led to the hypothesis that synaptotagmin
III may mediate the slow component of neurotransmitter release (18). In
support of this hypothesis, synaptotagmin III is enriched in synapses,
detectable in purified synaptic vesicles, and heteromultimerizes with
synaptotagmin I in vitro (10, 21). Furthermore, in
pancreatic In the current study, we have examined the subcellular localization of
synaptotagmin III and its closest relative, synaptotagmin VI.
Surprisingly, we find that synaptotagmins III and VI are not enriched
in synaptic vesicles. Instead, synaptotagmin III is highly concentrated
on synaptic plasma membranes, and synaptotagmin VI is widely present in
membranes. Our data suggest that synaptotagmins may be functionally
more diverse than previously envisioned.
Production and Affinity Purification of
Antibodies--
Specificity tests revealed that our previously raised
antibodies against the C2-domains of synaptotagmins III and
VI (L181 and I471, respectively) weakly cross-react with other
synaptotagmins, presumably because their C2-domains are so
similar. Therefore we generated a new set of antibodies to the sequence
between the transmembrane region and the C2A-domain of
synaptotagmins III and VI (residues 88-296 and 86-229 of
synaptotagmins III and VI, respectively (3, 12)). Rabbits were
immunized with glutathione S-transferase-fusion proteins
(produced in the pGEX-KG vector (23)), and the resulting sera (S754 and
S756 for synaptotagmins III and VI, respectively) were
affinity-purified on immobilized maltose-binding protein fusion
proteins containing the same sequences of synaptotagmins (in pMal-C2;
New England Biolabs). The specificity of all antibodies was tested by
probing extracts from COS cells transfected with individual
synaptotagmins and by comparing their reactivities with brains from
wild type and knockout mice that lack either synaptotagmin III or
VI.1
Immunocytochemistry--
Cryostat sections (5-15 µm) from rat
and mouse brains or bovine retina were probed with affinity-purified
synaptotagmin antibodies followed by horseradish peroxidase-labeled or
biotin-labeled secondary antibodies and development of the signal with
the StreptABComplex/HRP kit (DAKO) or by metal enhancement essentially
as described previously (24-26). The specificity of the signals was
ascertained by two controls: 1) blocking of the signal with the
recombinant protein used for antibody production, and 2) analysis of
brains from knockout mice lacking synaptotagmin III.1
Subcellular Fractionations--
Rat brain homogenates were
subfractionated into crude synaptic vesicles, mitochondria, myelin, and
synaptic plasma membranes by the method of Jones and Matus (27). For
this purpose, whole brains including cerebella, olfactory bulbs, and
brain stem from 4-6-week-old rats were homogenized in ice-cold 0.32 M sucrose using a motorized glass-Teflon homogenizer. The
homogenate was centrifuged at 800 × g for 10 min to
remove nuclei and debris, and the resulting supernatant was
re-centrifuged at 9,000 × g for 20 min to obtain
synaptosomes and the synaptosomal supernatant. The synaptosomes in the
pellet were washed once in 0.32 M sucrose, lysed
hypotonically, and centrifuged at 25,000 × g for 20 min to isolate large synaptosomal membranes in the pellet, whereas the
free synaptic vesicles remained in the supernatant under these conditions. The synaptosomal membranes were resuspended in
dH2O, sucrose was added to 1.1 M, and the
solution was placed at the bottom of a Beckman SW28 rotor tube. The
sample was overlaid with 0.855 and 0.32 M sucrose solutions
and centrifuged for 2.5 h at 19,000 rpm, resulting in the
isolation of myelin (in the 0.32/0.855 M sucrose
interface), synaptic plasma membranes (in the 0.855/1.1 M
sucrose interface), and mitochondria (in the pellet). For the purification of synaptic vesicles, hypotonically lysed synaptosomes were used as a starting point for the isolation of crude synaptic vesicles that were further purified by velocity-density gradient centrifugation and controlled pore glass chromatography essentially as
described previously (28, 29). For organelle immunoisolation, beads
with immobilized monoclonal antibodies to synaptotagmin I (Cl41.1) or
synaptobrevin II (Cl69.1) and control beads containing only immobilized
glycine were prepared as described previously (30, 31). Rat brains were
homogenized in 5 mM HEPES-NaOH, pH 7.4, 0.32 M
sucrose, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 1 mg/liter pepstatin A, and 10 mg/liter aprotinin. The homogenates were centrifuged for 10 min at
7,800 × g to generate a postnuclear supernatant. Beads
were incubated for 30 min at 4 °C under rotation with the
postnuclear supernatant and washed four times, and bound proteins were
analyzed by quantitative immunoblotting using 125I-labeled
secondary antibodies.
Quantitative Immunoblotting--
To quantitate the relative
levels of proteins in various fractions for the purification of
synaptic vesicles by subcellular fractionation or by immunobead
precipitation, we reacted immunoblots of the various fractions with
125I-labeled secondary antibodies and measured the
respective signals in a phosphorimager. All blots were probed
simultaneously with antibodies to the protein under study and
antibodies to synaptogyrin as an internal vesicle protein standard
(32). The synaptotagmin signals were then normalized for the
synaptogyrin signal by designating the ratio of the two signals
observed in starting brain homogenates or postnuclear supernatants as
1.0. For the immunobead experiments, the signals obtained in pulldowns
with glycine control beads were subtracted from the values from the
immunobead pulldowns before the ratios were calculated.
Miscellaneous Procedures--
SDS-polyacrylamide gel
electrophoresis and immunoblotting were performed using standard
procedures (33, 34). Immunoblots were developed by enhanced
chemiluminescence except for the quantitative experiments described above.
We raised multiple antibodies to synaptotagmins III and VI,
affinity-purified the antibodies, and tested their specificity using
COS cells transfected with various synaptotagmins (data not shown). Our
initial antibodies were directed against the C2-domains of
synaptotagmins III and VI. These antibodies, however, exhibited cross-reactivity with the more abundant synaptotagmins I and II, thereby limiting their usefulness. Therefore, we subsequently raised
more specific antibodies using the sequence that connects the
transmembrane region with the C2-domains and varies
considerably between different synaptotagmins (7-10, 12-17) as an
antigen. At least one of these antibodies for each synaptotagmin was
specific for that particular isoform, based on the following evidence: 1) as shown for synaptotagmin III in Fig.
1, the antibodies specifically reacted
with the cognate protein expressed in COS cells and recognized a single
band (synaptotagmin III) or two closely co-migrating bands
(synaptotagmin VI; see below) in total brain extract; and 2)
immunoblots of total brain homogenates from wild type mice and from
knockout mice lacking either synaptotagmin III or synaptotagmin VI were
analyzed (Fig. 2). The generation and
analysis of these knockout mice will be reported in a later, more
detailed report.1 The mouse immunoblots confirmed that the
synaptotagmin III antibodies reacted with a single band and that the
synaptotagmin VI antibodies reacted with a set of two closely spaced
bands in brain. In the knockouts that contain all other synaptotagmins,
the reactivity was abolished (Fig. 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-cells, synaptotagmin III is present on secretory
granules and may mediate Ca2+-triggered exocytosis of these
granules (22). If synaptotagmin III indeed functions as an exocytotic
Ca2+ sensor similar to synaptotagmins I and II, it should
also be on the secretory vesicles that undergo exocytosis. However, the localization of synaptotagmin III (or other synaptotagmins except for
synaptotagmins I and II) was not investigated systematically and
quantitatively, and its precise distribution is unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 1.
Characterization of synaptotagmin III
antibodies. Proteins from COS cells transfected with control DNA
(lane 1) or with a rat synaptotagmin III expression vector
(lane 2) and rat brain homogenates (lane
3) were analyzed by immunoblotting with affinity-purified
antibodies to synaptotagmin III. Signals were visualized by ECL. Note
that in brain only a single band is apparent; the low abundance,
smaller proteins in lane 2 probably represent proteolytic
breakdown products. Numbers on the left indicate
positions of molecular mass markers.
View larger version (21K):
[in a new window]
Fig. 2.
Definition of antibody specificity to
synaptotagmins III and VI by immunoblot analysis of brains from wild
type mice and from knockout mice lacking synaptotagmins III
(A) or VI (B). Brain homogenates
from the indicated mice were immunoblotted with affinity-purified
antibodies to synaptotagmins III (A) and VI (B).
Note that the immunoreactive bands are absent in the knockout mice,
indicating that the antibodies we raised are highly specific for those
particular isoforms.
Using these antibodies, we examined the regional distribution of
synaptotagmins I, III, and VI in rat brain (Fig.
3). As described previously (9),
synaptotagmin I was expressed in much higher levels in upper brain
regions than in hindbrain and spinal cord (which in turn synthesize
higher levels of synaptotagmin II). CASK as a general synaptic marker
was uniformly present in all brain regions (35, 36). The expression of
synaptotagmins III and VI, however, exhibited a different pattern: both
synaptotagmins were present in all brain regions but displayed large
regional variations. Synaptotagmin III was almost undetectable in the
cerebral cortex and olfactory bulb but was highly expressed in the
hippocampus and cerebellum. In addition, it was present at moderate
levels in spinal cord and hindbrain (Fig. 3). Synaptotagmin VI was
synthesized at rather low levels in all brain areas except for the
olfactory bulb, which contained exceptionally high levels of this
isoform.
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Previous data suggested that synaptotagmin III is localized to synapses
(10). This result was supported in the current study by two independent
observations. First, we examined the developmental expression profile
of synaptotagmins. The level of synaptic vesicle proteins goes up
dramatically postnatally in parallel with synaptogenesis (37). When we
analyzed brain proteins from rat forebrain as a function of age, we
found that synaptotagmin I levels increased postnatally, as expected
(10) (Fig. 4). By contrast, the general trafficking protein rab5 exhibited only a moderate developmental change. Synaptotagmins III and VI, similar to synaptotagmin I, experienced a striking postnatal increase in levels that paralleled the
time course of synaptotagenesis (Fig. 4). In adults, the levels of
synaptotagmin VI, but not synaptotagmins I and III, decreased slightly.
Two closely migrating bands were observed for synaptotagmin VI that may
be due to alternative splicing; the molecular basis for this
heterogeneity is unclear.
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The second observation confirming a synaptic localization of
synaptotagmin III was obtained by immunocytochemistry. Unfortunately, the quality of our synaptotagmin VI antibodies precluded a
morphological localization; even the signal for synaptotagmin III in
immunocytochemical stains was insufficient to allow
immunoelectronmicroscopy. At the light level, however, synaptotagmin
III was present in a pattern strongly suggestive of a synaptic
localization (Fig. 5; data not shown).
This was most clear in the retina, which contains two sharply
delineated synaptic layers, the thin outer plexiform layer composed of
photoreceptor synapses and the broader inner plexiform layer comprising
multiple sublayers of synapses (38). Synaptotagmin III was highly
concentrated in the two synaptic layers in the retina, similar to
synaptotagmin I (Fig. 5). The only difference between synaptotagmins I
and III was that in the inner plexiform layer, synaptotagmin I was
present throughout the synaptic zone, whereas synaptotagmin III was
enriched in a few sharply delineated sublayers (Fig. 5). These data
confirm that synaptotagmin III is highly enriched in synapses.
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Because synaptotagmins I and II are known stoichiometric components of
synaptic vesicles (1) and are highly homologous to synaptotagmins III
and VI (3, 12), a logical presumption is that synaptotagmins III and VI
are also vesicle proteins. This is supported by the finding that all
synaptotagmins are co-enriched in brain, where they appear to be
concentrated in the synapses, and that at least some synaptotagmin III
protein can be detected in the vesicles (10, 12). However, the relative
distributions of synaptotagmins between various brain fractions was
never quantitatively examined. Therefore, we studied this question
systematically by subcellular fractionation (Fig.
6). As expected, synaptotagmin I was
enriched in two fractions: 1) free synaptic vesicles (Fig. 6,
lane 6), and 2) synaptic plasma membranes that contain
active zones with firmly docked vesicles (lane 7).
Synaptotagmin III was also detectable in free synaptic vesicles but,
surprisingly, was not enriched in the vesicles (Fig. 6, lane
6). Instead, it was highly concentrated in synaptic plasma
membranes (lane 7). The pattern of synaptotagmin III closely
resembled that of neurexins, which are known components of the
presynaptic plasma membrane (39). Synaptotagmin VI was evenly present
in all fractions and was also not particularly enriched in synaptic
vesicles. The distributions of synaptogyrin I as an additional synaptic
vesicle marker (32) and of complexins as a marker for the cytosol (40)
were analyzed as controls in the same blots and found to be localized
in the appropriate fractions (Fig. 6).
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The subcellular fractionation data raised the possibility that
synaptotagmin III, although present in synapses, may not actually be a
synaptic vesicle protein. To test this hypothesis, we performed systematic studies on the enrichment of synaptotagmins with synaptic vesicles. We used two complementary techniques in these studies: 1)
purification of synaptic vesicles by
CPG2 chromatography (Fig.
7), which is widely accepted as the
method that yields the most highly purified vesicles (28, 29), and 2)
immunoprecipitation of synaptic vesicles by antibodies to synaptotagmin I or synaptobrevin II (Fig. 8), which
allows separation of the synaptic vesicles from similarly sized
organelles (30, 31).
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First, we examined the levels of various synaptotagmins by quantitative immunoblotting in four fractions obtained during the isolation of highly purified vesicles. In this purification procedure, starting homogenate (Fig. 7, lane 1) is used to purify synaptosomes (lane 2) that yield free synaptic vesicles (lane 3; see also Fig. 6) that are further purified by CPG chromatography (lane 4). Coomassie Blue-stained SDS-gels revealed that the protein composition of synaptic vesicles changed dramatically upon purification of the vesicles by CPG chromatography (data not shown). We then measured the relative enrichment of various synaptotagmins by immunoblotting with 125I-labeled secondary antibodies and phosphorimager detection; all blots were probed for synaptogyrin as an internal standard. Data were calculated as the signal ratios of the protein of interest to synaptogyrin I. For the purpose of normalization, the ratio observed in the starting fraction was set as 1.0. As expected, the abundance of synaptotagmin I and synaptogyrin I, two known vesicle proteins, increased in parallel during purification; thus, their ratio remained constantly close to 1.0 (Fig. 7B). Synaptotagmins III and VI, however, did not increase with synaptotagmin I during synaptic vesicle preparation; their ratio to synaptogyrin decreased significantly in pure synaptic vesicles (Fig. 7B). These results suggest that synaptotagmins III and VI are not obligatory vesicle components but de-enrich from the vesicles.
To confirm this unexpected result by an independent method, we performed experiments in which organelles were immunoisolated from rat brain homogenates with beads coated with antibodies to either synaptotagmin I or synaptobrevin II, which are known synaptic vesicle proteins. As a control, incubations were performed with beads that were coated only with glycine (30, 31) (Fig. 8A). The resulting fractions were again analyzed by quantitative immunoblotting, and the signals obtained for glycine beads were subtracted from the synaptotagmin I or synaptobrevin II bead signals. As in the biochemical preparations, we normalized all results for synaptogyrin analyzed on the same gels. As before, immunoisolation dramatically increased the levels of synaptotagmin I and synaptogyrin I in the samples, and their ratio remained constant. In contrast, a quantitative loss of synaptotagmins III and VI from the immunoisolated vesicles was observed; their ratio to synaptogyrin decreased significantly in the immunisolated samples. (Fig. 8B). The two kinds of beads coated with the different antibodies gave identical results, thereby confirming each other.
Conclusion--
Synaptotagmins are a large family of homologous
proteins that are enriched in brain and thought to function in
Ca2+-regulated membrane traffic. Although synaptotagmins I
and II have known functions in synaptic vesicle exocytosis, little is known about the localizations, properties, and functions of the other
synaptotagmins. In the current study, we examined the localizations of
synaptotagmins III and VI. Unexpectedly, we have found that synaptotagmin III, although synaptic, is not on synaptic vesicles, and
that synaptotagmin VI also fails to co-purify with synaptic vesicles.
Our data suggest that the functions of nerve terminals must involve
other, as yet unidentified Ca2+-regulated membrane
trafficking pathways that involve these synaptotagmins. The high
concentration of synaptotagmin III in synaptic plasma membranes raises
the possibility that this isoform may, in fact, not be on an
intracellular vesicle but may be localized to the plasma membrane. One
possibility is that synaptotagmin III is a plasma membrane protein that
could regulate the expansion of presynaptic plasma membranes as a
function of activity; another possibility is that an as yet
unidentified organelle co-purifies with synaptic plasma membranes and
represents the true localization of this isoform. Future experiments
will have to test this and other possible hypotheses about what the
functions of these proteins might be in synapses that do not directly
involve synaptic vesicles.
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ACKNOWLEDGEMENTS |
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We thank A. Roth, I. Leznicki, and E. Borowicz for technical support and Drs. G. Lonart and S. Sugita for help with brain dissections.
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FOOTNOTES |
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* 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.
§ Supported by a fellowship from the Spanish Ministry of Education and Culture and by the Fulbright Commission.
To whom correspondence should be addressed. E-mail:
Tsudho{at}mednet.swmed.edu.
1 R. Fernandez Chacon, R. E. Hammer, and T. C. Südhof, unpublished observation.
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ABBREVIATIONS |
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The abbreviation used is: CPG, controlled pore glass.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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50 µM.
