J. Biochem.
Calcium-Dependent
and
Synaptotagmin
-Independent
128,637--645
Hetero-Oligomerization
in
(2000)
the
Family1
Mitsunori
Fukuda*,2
and
Katsuhiko
Mikoshiba*,•õ,•ö
* Laboratory for Developmental Neurobiology Ch , Brain Science Institute, RIKEN (The Institute of Physical and emical Research), 2-1 Hirosawa, Wako , Saitama 351-0198; •õDepartment of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo , 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639; and •öCalciosignal Net Project, Exploratory Research for Advanced Technology (ERATO) 2 -28-8 Honkomagome , Japan Science and Technology Corporation (JST), , Bunkyo-ku, Tokyo 113-0021 Received
June
29, 2000;
accepted
Synaptotagmins
constitute
transmembrane
region
have
indicated
of function
that
totagmin cial
the
in
V, VI,
minal
and
Mikoshiba, show
K.
(1999)
moderate are
K.
(2000)
apparent
can
hetero-oligomerize that create
Key
words:
J.
Biol.
of Chem.
the
with
J. Biol.
Chem.
weak
VII
Ca2+-dependent a variety Ca2+
31421-31427].
C2
EC50
values
275,
activity,
of but and/or
presence (Syts
has about some
exocytosis,
This work was supported in part by grants from the Science and Technology Agency of Japan (to K.M.) and from the Ministry of Ed ucation, Science, Sports and Culture of Japan [to K.M., and M.F. (11780571 and 12053274)]. 2 To whom correspondence should be addressed . Tel: +81-48-4679745, Fax: +81-48-467-9744, E-mail:
[email protected] Abbreviations: cyto, cytoplasm; HRP, horseradish peroxidase; lum, lumen; Mr, molecular weight; N, amino; N-Gly, N-glycosylation; PAGE, polyacrylanvde gel electrophoresis; PCR, polymerase chain reaction; Syt(s), synaptotagmin(s). c 2000 by The Japanese Biochemical Society. 637
A Syts an
no
VIII,
N-ter E.,
IV, VIII, sets
-independent
self-oligomerization,
Ca2+
Group
homo
XI did
of synaptotagmin manner.
XI)
C syn
homo-oligomer
[Fukuda,
and
and
and
Ca2+-dependent
150ƒÊM Syts
(I, II,
or
at
Kanno,
of Ca2+. IX)
unique
hetero-oligomerization
The synaptotagmin (Syt) family comprises evolutionarily conserved proteins that are thought to be involved in vesic ular trafficking including synaptic vesicle exocytosis (re viewed in Refs. 1-3). They are characterized by a short amino (N)-terminus, a single transmembrane region, and two highly conserved C2 domains (named the C2A and C2B domains) in the large carboxyl terminus. The C2 do mains of Syt I, a well characterized isoform, are essential for synaptic vesicle exocytosis and endocytosis (4-9 and reviewed in Refs. 1-3) and this region is likely to be func tional in all synaptotagmin family proteins. To date, 12 syn-1
Vol. 128, No. 4, 2000
D)
28180-28185].
a Ca2+-dependent
domain,
of the
M.,
B Syts
all the proteins based on
Group
bonds
[Fukuda,
Group
Ca2+-dependent
and -independent of Ca2+-sensors.
sensor,
of Ca2+
disulfide
hetero
the synap an artifi
examined family groups
activity.
by
one
expression
about
study
oligomerization
presence
(Group
hetero-oligomerization in
for
is known
, and systematically among synaptotagmin family into four distinct
irrespective
Syt
studies
, little
hetero-oligomers
274,
by
IV).
by
biochemical
domain
and
irrespective
this
characterized
, we showed that (Nlumen/Ccytoplasm) by introducing
-independent
homo-
properties
any
gest may
and
strong
(Syt
oligomerization
In
are and
I is important
However
protein
N-terminal
characterized
activity
Mikoshiba,
family.
that genetic
(Syt)
vesicles.
homo-oligomerization
aptotagmins ization
the
Ca2+-dependent motif
Recent
of synaptotagmin of synaptic
at
proteins
domains.
of hetero-oligomerization the synaptotagmin
X) form
cysteine
of membrane C2
I membrane
site
possible combinations (Syts I-XI). We classified (III,
two
synaptotagmin
is a type
N-glycosylation
differences
a family and
exocytosis in
family
2 , 2000
oligomerization
during
oligomerization
August
M., not
and show
isoforms Our
data
sug
of synaptotagmins
synaptotagmin.
aptotagmin isoforms have been described in rat and mouse (Ref 10 and references therein), and 7 and 5 isoforms respectively in Drosophila and Caenorhabditis elegans (11, 12). These isoforms can be divided into subclasses based on the phylogenetic relationship (10), gene structures (13) and differences in the biochemical natures of the C2 domains, such as Ca2+-dependent phospholipid or syntaxin binding to the C2A domain (14-16), and inositol 1,3,4,5-tetrak isphosphate binding to the C2B domain (17-19). Syts III, V, VI, and X form a small branch in the phyloge netic tree (10). This class of synaptotagmins form hetero oligomers through the N-terminal cysteine motif by disul fide bonding (10) and, except for Syt III, interact with Syt VII in a Ca2+-dependent manner at the cytoplasmic domain (20). Syts III, V, VI, and X are also characterized by weak or no inositol 1,3,4,5-tetrakisphosphate binding activity (19). Syts IV and XI are another class of synaptotagmins, char acterized by deficient phospholipid (phosphatidylcholine/ phosphatidylserine, 1/1 liposome) binding due to the substi tution of Ser for Asp at position 244 (or 247) of Syt IV (or XI) (14, 16). Syts I, II and IX also form a small branch in the phylogenetic tree (10). Although the functions of the other classes of synaptotagmins largely remain unknown,
638
M. Fukuda
Syt
I (or
II) is thought
probably the basis min
mutants
studies tion
(reviewed
(20, 22-26). properties:
erization the
to function
in
Syt
I (or
20),
mine
we
isoforms
21)
and
classes
(10,
20,
that
self-oligomeriza
just
multim
and
by the C2B also showed
domain (22Ca2+-depen
the
be
properties
interesting
would
In the
present
tematically
study,
we
determined
isoforms
examined
the
(Syts
homo-
properties of synaptotagmin two different epitope (T7
and
the
membrane
III-XI),
and
topo then
sys
EXPERIMENTAL
enzymes
were
CA, USA) Polyclonal
I-XI)
by using synaptotag
obtained
and and
tively.
from
other
from Sigma
Biosystems
from
chemicals
were
cel A10
(Millipore
System
Construction
of
Bedford,
the
T7-tag
underlined
in bold
letters]
was
MA,
template
(10) by
following
oligonucleotides:
tions
were
naturation extension
and
and
for
at 94•Ž
for 30s, for 30s.
gel by
PCR
sequencing
Other
plasmids
and
the
X, and full
were
length
excised
Madison, NotI
WI, site
pEF-T7(or
prepared DNA was
using
a Hitachi
in.
day
before
XI were
similarly
from
the by
of pEF-BOS
or a QIAGEN
Maxi
prep
then
substituted IX
pGEM-T NotI
DNA
Easy
(10).
30)
I-XI
verified
-Syts
previously (10, using Wizard-mini
by
this
method. tag
I-XI-cyto
dish) as
F
buffer
to
(5
was
described cells were
and
10%
and
cells
SDS-
immuno
of
pEF-T7-
x
105 cells,
carried
out
previously harvested,
by (10). and
with buffer containing 1% Triton 50mM HEPES-KOH, pH 7.2, 0.1 fluoride, 10ƒÊM leupeptin, and
pepstatin
by anti-T7 tag antibody-conjugated and immunoblotting analyses were
A at
4•Ž
previously
Membrane Synaptotagmin brane
for
1h.
Immunoprecipitation
of
agarose, SDSalso performed
(10).
Topology family
region
Syts
with
I and
of Mouse Synaptotagmins proteins have a single
a short
containing
N-terminus
tandem
II have
a single
N-terminus,
and
this
C2
the
N-terminal
domain
domain
of Syt
domains.
N-glycosylation been
has
carboxyl
In vertebrates, shown
to
F digestion (27, 29) In addition, antibodies I were
at
be
N-
or siteagainst into
hip
after membrane depolar of antibodies against the
squid
giant
presynapse
proteins, orientation.
showing Nlumen (or extracellular domain)/Ccytoplasm (Nlum/Ccyto) However, the Syt isoforms other than Syts I
II do
minus therefore
we
Gly-Ser)
the into
is no same
site
region
III-XI)
membrane -XI proteins
in such
cells
N-terminal
each
by
(Fukuda tagging
topology T7-Syts were transiently
immunoprecipitated
the
other To
N-ter
(10),
and
isoforms
address
this
site
(Asn-
N-glycosylation of
PC12
at
synaptotagmin
iso
(Fig . 1A). We first confirmed (T7 , T7-N-Gly or FLAG) to the N the subcellular localization of
of a short tag did not affect
isoforms so that
that
orientation.
artificial
N-terminus
ata),
evidence
Nlum/Cyto
the
several
the synap I membrane
N-glycosylation
direct an
results, type
transmembrane
(T7-N-Gly-Syts
III-
and
an
of the
introduced
were
the
contain
there
show
issue, form
not
upstream
on these to comprise
visualized
vesicles family
and
(4). Based is thought
incorporated
site
synaptic totagmin
that addition -terminus
20). The plasmid preps (Promega)
I XItransmem
a large
site
of Syt
I into
and
potential
glycosylated by N-glycosidase directed mutagenesis (31).
into
(Promega;
subcloned
sample
(PAGE)
COS-7
then
boiling
was added to for 1h at 37•Ž.
T7-Syts PAGE,
also III-
a T7-N-Gly
(pEF-T7-N-Gly-Syts and
for The
sequencer.
vector and
with
Syts by
with
digestion
(29,
kits.
was
constructed inserts
FLAG)Syts
as described prepared by
and
T7-N-Gly-tagged
synaptotagmin USA)
digested
by
of N-glycosidase
SDS
transfection/10cm method transfection,
of 20 CA-
A, and
subjected
electrophoresis elsewhere (10).
C2A
and from
were
unit
Germany) incubated
into
described
100ƒÊl IGEPAL
pepstatin
10ƒÊM
as described
as
in 0.5%
Procedures-Co-transfection
Reac
Pharma
UK),
one
were
pEF-FLAG-Syts
dish)
antibody-conju
denaturation
by adding
Proteins
of de
purified
(Amersham
SQ-5500
the
37•Ž,
COS-7
phenylmethylsulfonyl
10ƒÊM
pocampal neurons by endocytosis ization (32-34), and microinjection
for 30s,
products,
IX plasmid
encoding
the
5'-CGGA
consisting
of pGEM-T-T7-Syt
pGEM-T-T7-N-Gly-Syt
DNA
using
and
at 60•Ž
Column above)
insert
each
annealing The
(italics
site
IX as a
(PCR)
(sense)
cycles,
to
in
Novagen)
After
Mannheim, mixtures were
polyacrylamide gel blotting as described Syts
and
stopped
3
tag
0.1mM
microtubes.
were for
anti-T7
resuspended 0.1% SDS,
EDTA,
cooling
expressed
transfeetion/10cm
30ƒÊl,
leupeptin
two and
Reactions
the
(N-Gly)
reaction
Buckinghamshire, BamHI
III-
(antisense).
25
MicroSpin
ApaI-BamHI
reagent
pGEM-T-T7-Syt
chain
out
Biotech;
resulting
XI).
3min,
terminus
N-glycosylation
T7 primer
carried
of
[MASMTGGQQMG
from
polymerase
at 72•Ž
agarose
Apal
the
10ƒÊM into
II, T7-
RESULTS
water prepared and Milli-Q Bio
Synaptotagmins
TCCGTTGCGACCCATTTGCTGTCC-3'
The
fluoride,
20mM
I,
All
USA).
T7-N-Gly-tag
amplified
USA).
products
T7-N-Gly-Tagged
encoding
WI,
made up in deionized Purification System
RNGS;
VIII,
(Sigma),
volume
anti-T7
(Madison,
commercial
were Water
City,
(San Fran USA), respec
(HRP)-conjugated
Novagen
grade. Solutions with an Elix10
(Foster
(Tokyo), respectively (M2) against FLAG
before
Beads were pH 7.2,
proteins were solubilized X-100, 250mM NaCl, mM phenylmethylsulfonyl
andand polymerase restriction
Zymed Laboratories (St. Louis, MO,
peroxidase
was
XI-cDNA
PE
Toyobo Biochemicals monoclonal antibodies
Horseradish antibody
polymerase
III-XI
day
by
(wet
Miscellaneous
PROCEDURES
DNA
peptide were obtained cisco, CA, USA) and
the
630
hetero-oligomerization
family (Syts and FLAG)-tagged
the
the DEAE-dextran Three days after
Materials-AmpliTaq
cia
agarose
previously (10). mM HEPES-KOH,
(Roche Diagnostics, one tube, and the of
T7-Synaptotagmins III-XI-Recombinant
immunoprecipitated
for
variety
of
T7 N-Gly-Syts
other
a
Digestion
105 cells,
deter-
mins.
an
II, and
boiling
of synaptotagmin
tag
(5 x
to
Ca2+-sensors. logy
I,
cells
divided
of differ
create
Syts
with
hetero-oligomerization
synaptotagmins
F
T7-N-Gly-Synaptotagmins
gated of
a Ca2+-de-
hetero-oligomerize
and
were
downstream
27, 28)
it would
N-Gyyeosidase
on
biochemical
Ca2+-independent
I or II can
whether of
in vitro two
state,
release synaptotag
homo-oligomerization
thought Syt
and
mediated isoforms
-independent
whether
ent
oligomerized
by a region
region
pendent oligomerization 26). Since several other (10,
an
11) shows
mediated
transmembrane
and
Ref
A SDS-resistant
probably
dent
in
as a Ca2+-sensor for neurotransmitter of the genetic analysis of Drosophila
and K. Mikoshiba
, M., unpublished d is unlikely to affect
I , II, and expressed
anti-T7
tag
T7-N-Gly-Syts III in COS -7 cells antibody-conju-
J. Biochem.
Ca2+-Dependent and -Independent Oligomerization of Synaptotagmins
Fig. 1. Determination of the membrane topology of synap totagmins I-XI. (A) Schematic representation of T7-N-Gly-tagged Syt IX.Top indicates the nucleotide and amino acid sequences around the T7-N-Gly-tag. T7-tag and N-glycosylation sites (N-Gly) are shown by open box and double lines, respectively. Non-coding regions and the major restriction enzyme sites are shown by small letters and underlining, respectively. Nucleotide and amino acid numbers are given on both sides. Bottom is a schematic representation of T7-N-Gly-Syt IX: T7-tag (black box), N-glycosylation (Y), transmembrane region (TM; open box), and two C2 domains (C2A and C2B; hatched boxes). (B) N-
gated showed
agarose. Although several isoforms heterogeneous bands which probably
translational
modifications,
(M,)
of
expressed
that
of T7-Syts
N-Gly-Syts
the
III-XI
III-XI
(data
were
were
then
treated
cleaves
the N-linked
sugar,
without
all
As
isoforms
cosidase
and
shown
decreased
when These
forms
via the
C2
co-expression
assay
T7-
and
the
association
these
immunoprecipitation vivo when
This
assay
system
T7-
and
in
the
FLAG-Syts
Vol. 128, No. 4, 2000
by
apparent
treated
Mr of
with
N-gly show
that
studies
because
on iso
all
isoforms
Hetero-Oligomeriza examine
the
Ca2+-de
hetero-oligomerization of synap used a T7- and FLAG-tagged Syts described
previously
co-expressed two
in the
interactions
with
synaptotagmin
meaningful
were of
isoform compared
indicate
-Independent
as
FLAG-Syts
which
synaptotagmins
Isoforms-To
and -independent isoforms, we
the
F,
topology.
and
of Synaptotagmin
pendent totagmin
Ca2+.
are
membrane
Ca2+-Dependent tion
were
between
domains
same
this,
1B, the
results
hetero-oligomerization
show
T7-
To confirm
was
of the
than that
the Mr of each
they all
weight
larger
N-glycosidase
Fig.
VII) post
suggesting
treatment in
that
Nlum/Ccyto orientation. the
with F
F, indicating
was
not shown),
N-glycosidase
immunoblotting.
molecular
III-XI
N-glycosylated.
beads or
apparent
T7-N-Gly-Syts
(e.g., Syt reflect
in
proteins
presence
has cells
were
was merits.
to
be
separately
Briefly, cells
and
evaluated
or absence
three seem
(10). COS-7
by
of 500ƒÊM
First, detected, expressed
only
in
because and
639
glycosidase F treatment of T7-Syts I, II and T7-N-Gly-Syts III-XI. Anti-T7 tag antibody immunoprecipitants were treated with or with out N-glycosidase F and analyzed by immunoblotting with HRP-con jugated anti-T7 tag antibody (1:1,000 dilution). Note that the apparent M, of all synaptotagmin isoforms was decreased by N-glycosi dase F treatment. A proportion of N-linked sugars of T7-N-Gly-Syts VIII and IX were further converted to a complex form because they showed broad bands and were resistant to endoglycosidase H treat ment (data not shown). The positions of M, markers (x10-3) are shown on the right.
mixed after solubilization, T7- and FLAG-Syts interactions were in many cases not detectable (10, 20). Second, since native synaptotagmins undergo posttranslational modifica tions such as palmitoylation (35, 36) or disulfide bond for mation (10), it would be better to use a mammalian rather than bacterial expression system. Third, significant levels of endogenous Syts I-XI are not expressed in COS-7 cells (Fukuda, M., unpublished data), so that the involvement of endogenously expressed Syts in oligomerization need not be considered. The only problem with this assay is that the expression levels of the two isoforms may be very different. The amounts of each immunoprecipitant from each isoform were different even when the same amount of plasmid was transfected (Fig. 2). For instance, Syts I, II, VIII, and IX were expressed at relatively high levels in COS-7 cells, Syts VII and XI were moderately expressed, but the expression levels of Syts III-VI, and X were at least 5-10 times lower than that of Syt I (Fig. 2A). To overcome this, we initially analyzed 1/10 the volume of immunoprecipitants by 10% SDS-PAGE and normalized them based on the immunore activity of anti-T7 tag antibodies in immunoblotting. Then, equivalent amounts of anti-T7 tag antibody immunoprecip itants were subjected to 10% SDS-PAGE and analyzed by immunoblotting using anti-FLAG rabbit antibody (Fig. 3, upper four panels and summarized in Table I). To confirm the amounts of anti-T7 tag antibody immunoprecipitants, the blots were stripped and reprobed with HRP-conjugated
640
M. Fukuda
anti-T7 tag antibody (Fig. 3, bottom panel). The results are summarized in Table I, and representa tive immunoblots are shown in Fig. 3. Based on the differ ences in oligomerization activity, we classified the synap totagmin family into four distinct groups. Group A Syts
and K Mikoshiba
(III, V, VI, and X) formed stable hetero-ohgomers with each other irrespective of the presence of Ca2+ (10). In addition, except for Syt III, group A synaptotagmins strongly associ ated with Syt VII (group D) in a Ca2+-dependent manner as described previously (Fig. 3, third and fourth panels) (20).
Fig. 2. Expression of T7tagged Syts I-XI (A) and -Syts I-XI-cyto (B). T7tagged Syts were expressed in COS-7 cells as described previ ously (10). Cells were homoge nized in 1% SDS using a 27 gauge syringe. The solubilized proteins were boiled for 3min, subjected to 10% SDS-PAGE, and transferred to a polyvinyl idene difluoride membrane (Millipore). Expressed T7-Syts were detected by HRP-conju gated anti-T7 tag antibody (1/ 1,000 dilution). Since the expo sure time of (B) is less than half of that in (A), comparable amounts of Syt cytoplasmic domains were expressed in COS-7 cells. Notably, Syts IV and VI cytoplasmic domains were more highly expressed than full length proteins. Similar protein expression levels were observed in the case of FLAG-Syts markers (x 10-3) are shown on the left. Lanes 1-11, T7-Syts I-XI (or I-XI-cyto) and lane 12, vector
Fig.
3.
tion
properties
Ca2+-dependent
I-XI
and
pEF-FLAG-Syt
A), or -Syt combinant sence of followed were
and
VII
(Group
proteins 500ƒÊM by
detected
-independent
of synaptotagmins I (Group D) were
by
B), -Syt
co-transfected
were immunoprecipitated Ca2+ by anti-T7 tag
immunoblotting. exposure
hetero-oligomeriza
1, TV, V, and
VII.
IV (group into
C), COS-7
in the antibody-conjugated
Co-immunoprecipitated to X-ray
film
(X-OMATAR,
pEF-T7-Syts -Syt
V (Group
cells, presence
and
re
or ab agarose
FLAG-Syts Kodak)
for
2.5
I-XI (data (pEF-BOS)
not shown).The positions transfected control.
of Mr
min using an Enhanced chemiluminescence kit (Amersham Pharma cia Biotech). Bottom panel indicates the immunoprecipitated T7-Syts I-XI of the third panel from the top visualized by HRP-conjugated anti-T7 tag antibody Total (right most lane) includes 1/40 volume of reaction mixtures used for immunoprecipitation . Arrowheads indi cate the positions of the FLAG-Syt I , IV, V, or VII. Bars shown on the ri ght are Mr markers, corresponding to 97.4, 66.2, and 45.0 kDa from the top, respectively.
J. Biochem.
Ca2+-Dependent and -Independent Oligomerization of Synaptotagmins T7-Syt V was shown to interact well with FLAG-Syt II irre spective of the presence of Ca2+ (Table I) . In contrast, T7-S yt II-FLAG-Syt V interactions were difficult to detect unless a large amount of T7-Syt II immunoprecipitant was loaded on SDS-PAGE. This was probably due to the differ ence in expression levels of the two molecules; the amount of Syt II expressed was at least 10 times greater than that
TABLE
I. Summary
of Ca2+-dependent
Ca2+-dependent independent
(500ƒÊM) experiments
from
extracts
ray
the
cell
films
by
(Version "++++"
eye,
1.0) and
and (see
-independent immunoblots
co-transfected
but
and
in some
gomerization.
++,
weak
(2mM of Fig.
with
cases,
were
like
Syt
presence of Ca2+. +, very weak association, ing conditions, like Syt IV homo-oligomerization.
TABLE
II. Summary
Ca2+-dependent three
transfected in
some
Image)
with cases, as
dependent detected on
were
did
like
Syt
II
prolonged
exposure
-,
undetectable
under
show
Vol. 128, No. 4, 2000
apparent
to X-ray our
binding
hetero-oligomerization
-,
was
(Bio
very
conditions (see
weak weak like Table
and
were
quantified
association,
like
Ca2+-dependent association, Syt
when
IV homo-oligomerization.
I). a, some
fractions
mainly by
III,
and
Syt
Basic
V, VI,
"+++" like
and
from
Quantifier
software between
like
-,
undetectable
were
determined
Basic
Syt amount
ND,
of oligomerization
not
IX
from from X-ray
software
at cell films
in the our
least
bind-
because
Ca2+-independent.
two
by
co
eye,
but
1.0)
(Bio Ca2+-
which was the
or
extracts
moderate
homo-oligomerization
are
I homo-oli
(Version ++,
of immunoprecipitant determined
X-
I-XI.
judged
from
Quantifier
homo-oligomerization. like
Syt
under
of synaptotagmins
mainly
a large
determined
X). +++•},
"++",
immunoprecipitants
VII
at least two or three immunoprecipitants
IX homo-oligomerization
film.
T7-Syts-cyto
using
by Syt
association, only
"-",
to X-ray
and
activities
Image)
(Syts
domains
by
were
quantified
between and
exposure
determined
Oligomerization
+,
film.
prolonged
and A Syts
++•}, "--+"
of FLAG-Syts-cyto level
Ca2+-dependent
homo-oligomerization.
after
not
control.
Image)
VIII).
judged from by using the
activities
group
between
I -XI.
T7-Syts were was determined
the cytoplasmic
association
2000i/VGA
strong
after
between
background
Print
(Bio like
II and +-,
only
and level
2000i/VGA
(Syts
synaptotagmins
Oligomerization
association,
B Syts
detected
EGTA)
a vector Gel
+++,
control.
Print
strong
group
was
(2mM
by (20).
Gel
between
of FLAG-Syts background
a vector
homo-oligomerization.
FLAG-Syts-cyto and
captured
previously
association,
SDS-PAGE.
proteins
The
by
oligomerization
independent
pEF-FLAG-Syts-cyto bands
described
only
and
experiments.
like XI
which
of Ca2+-dependent
(500ƒÊM)
independent
association FLAG-Syts and
(20). ++++,
association
association,
EGTA) 3). The
captured
previously
of Syt V (Fig. 2A), and accordingly T7-Syt II homo-oligo mers were predominantly immunoprecipitated rather than T7-Syt II-FLAG-Syt V hetero-oligomers. Group B was com posed of Syts I, II, VIII, and XI, which form homo-oligomers irrespective of the presence of Ca2+. Syts I and II associated with each other irrespective of the presence of Ca2+ (Fig. 3, upper panel), consistent with the previous report that
oligomerization
pEF-FLAG-Syts
bands
(Bio Image) as described "+++" +++ , moderate
-independent
641
was loaded
full-length
642
M. Fukuda
Fig. 4. Summary -independent
and K Mikoshiba
of Ca2+-dependent oligomerization
and proper.
ties of synaptotagmins in COS-7 cells, Synaptotagmin isoforms are classified into four distinct groups according to their abil ity to self-oligomerize (Group A > D ? B > C; see text and Tables I and II). Isofonns that show strong or weak Ca2+-dependent homo-oligomerization mediated by cytoplasmic domains are indicated by red or green letters, respectively. Syt VII is further shaded because it shows robust Ca2+-depen dent oligomerization even when the fulllength proteins are used. Isoforms that do not essentially show hetero-oligomerization are indicated by blue letters (Syts VIII and XI). An isoform that does not essentially show homo-oligomerization is indicated by black letters (Syt IV). Lines indicate the het ero-oligomerization of two molecules and their thickness indicates the relative strength of the oligomerization, although the weak hetero-oligomerizations (e.g. "++" or "+" in Table I) are omitted in this figure. Hetero-oligomerization that was activated by Ca2+ is colored in red. Broken lines indi cate that the interaction of two isoforms was detected in only one way (e.g., T7-Syt VFLAG-Syt II interaction).
native
Syts
I and
other
(26).
these
associations
Compared ties
oligomerize, mer,
we
used
ing
activity
showed
of
weak
M Ca2+, acted
Syt
IX
not
Syts
Ca2+. VII
final
plasmic domains combinant Syt
were
Syt
the full of T7-(or
summarized Syts
of the
presence
equally
well
and
X in
mic
domain
ious
Syts
I protein
COS-7
evident
at
weakly
cytoplasmic
500ƒÊ
a
from
cells
II.
Syt
II
but
not
V were
with
Syt
VII
a Ca2+-dependent showed (I, II, V, VI,
the
weakly
manner. broadest
IX, and
The
com-
greater domains
results
are
V, though
the
full-
The
Syts Syt
different
VII
and
III, VI,
IX,
cytoplas bound
affinities.
var
synaptotagmin
the
show
four
distinct
properties. associated with motif
by
the
with differing
previous
disulfide
XI were
I and
classed
as at
I itself
whereas
Syt (e.g.,
because
Syts
ally
equivalent
based
fact
that
tion ties
in brain (37), and of the C2 domains
I and
Syts
I only Syts on
the
were
the
20).
cytoplasmic very
Al-
N-terminus
weak
domain Ca-de-
conditions. In addition, we with a number of different
associated
with
, VII, and IX). I and II are thought high
B and (10,
at
II
II show
anal
1,3,4,5-tet
as group
showed
in our binding II can associate
in This
phylogenetic
to ƒÀ-mercaptoethanol as well
other
homo-oligomerization,
II hetero-oligomerize Syt
of isoforms
each
Ca2+these
affinities.
of inositol
Ca2+-independent
insensitive
Syts
with
with
and
by
Since
cysteine
weakness or absence binding ability (19).
I, II, VIII,
pendent activity found that Syt
17, 38).
into
hetero-oligomerize
is consistent
Syts
family
manner
(Ca2+ -dependent),
ber
isoforms
properties by using two dif FLAG). As illustrated in Fig. 4,
N-terminal
(Ca2+-independent)
surprising
irrespective
to hetero-oligomerize with
the
can
was
though
isoforms,
hetero-oligomerize
Syt
specificity
X) with
As or
that
(Nlum/Ccyto) at the N-
and accordingly their associations were At the cytoplasmic domain, however,
characterized which
synaptotagmin
their homo-oligomerization V, VI, and X) strongly
Ca2+-dependent
Syts cyto
(22-26).
associated
V seemed and
the
interaction of rewith detergent
2B).
could
strongly
Syt
via
ysis (10) and rakisphosphate
Cal-dependent
brain
(Fig.
of
I-XI-In the
by the bacteria
in
IX,
of Ca2+.
other
that
I membrane topology N-glycosylation site
synaptotagmin
on (III,
synaptotagmins
interdomain
demonstrated
and
the
based A Syts
bonding (10), independent.
of synaptotagmin
and
II and
classified
groups Group each
association
have
show type an artificial
domain,
its
Properties
examined
Table
I, VII,
length
IX also
the
we IV
or 250mM
length protein, comparable FLAG)-Syts I-XI cytoplasmic
in in
Syts
Syt at
Syt
we
complex hetero-oligomerization ferent epitopetags (T7 and
self-oligomeriz
Synaptotagmins
we
as demonstrated I or II expressed
expressed
with
was
properties
native to
which
study,
grouping
from
hetero-oligomerization
pared amounts
weak,
iso
manner.
of experiments,
solubilized
X-100
Hetero-Oligomerization
Domains
set
that
the
quite
250ƒÊM
Ca2+-Dependent Cytoplasmic
was
other
this
terminal
self-
as a mono
possibility
1% Triton
I, II, V, and
in a Ca2+-dependent
with
In
family proteins by introducing
expression
present
Although
Ca2+-dependency,
but
with
to
the
more affini
XI could
interact
out
solubilization.
in
DISCUSSION
Ca2+.
not detected
and
mainly
and
by
various
were
VIII
each
VII,
interacted
with
differences
essentially
is sensitive for
II
Syts
rule
Syt
activated
interactions
IV was
cannot
oligomerization
slightly
the
with
with
isoforms to
not
C Syt
associate
FLAG-Syt
In contrast,
did
group
though
NaCl
I,
due
2A). but
The
to be
these
probably Fig.
brain
associated
T7-tagged
I). However,
(see
forms.
seemed
other
reverse,
level
from
II also
FLAG-Syt
with
(Table
in
I and
to
broadly
II proteins
Syts
sequence
almost
a limited
num
This finding is to be function-
similarity
complimentary
(10),
the
distribu
the fact that the biochemical proper tested so far are quite similar (15-
I and
II are
thought
to
be Ca2+-sensors
2 Biochem,
Ca2+-Dependent and -Independent Oligomerization of S
ynaptotagmins
643
for neurotransmitter release (1-3) , our results raise the modulate the function of other neuronal (I, II, V, X) and possibility that their hetero-oligomerization with other iso non-neuronal (VI, IX) isoforms without tethering at the Nforms having distinct biochemical activities may the variety of Ca2+-sensors produce terminal domain. While we were preparing this manu . In contrast, Syts VIII and XI script, Martinez et al. reported that Syt VII is involved in are thought to be present in the homo-oligomer or mono Ca2+-dependent exocytosis of lysosomes in fibroblasts (43). mer state. Therefore, it will be interesting to see whether Syt I-Syt Is Ca2+-dependent oligomerization of Syt I C2B domain VII hetero-oligomers are involved in neurotransmitter indeed essential for synaptic vesicle exocytosis in vivo? Al release in the future. though the importance of the Syt I C2B domain in synaptic Among the synaptotagmin isoforms, Syts I and II are vesicle exocytosis and endocytosis has been well demon known to be localized at synaptic vesicles as well as secre strated by antibody-loading experiments (5-7) and analysis tory vesicles of some endocrine cells (44). Recently, several of Drosophila syt mutants (21), there is no direct evidence synaptotagmin isoforms other than Syts I and II in brain that Ca2+-dependent oligomerization of the Syt I C2B do have been reported to have different subcellular localiza main itself is essential for synaptic vesicle exocytosis in tion: Syt III is mainly localized at the synaptic plasma vivo. Recently, however, we found that a Syt II mutant car membrane (45); Syt VI‡™TM, which lacks the transmem rying a Tyr-312 to Asn substitution in the C2B domain domain, is associated with various membrane frac , which c brane orresponds to the Drosophila AD3 mutation , had tions (45,46); and Syt IV is localized at the Golgi and distal completely impaired self-oligomerization activity but could part of neurites in nerve growth factor-differentiated PC12 weakly hetero-oligomerize with wild-type proteins in a cells and cultured hippocampal neurons (39, 40). These Ca2+-dependent manner (Fukuda , M., unpublished data). observations are consistent with our results that Syt I did This result is consistent with the fact that this syt allele not essentially interact with Syts III, IV, and VI even in the could complement another mutant phenotype , although it i presence of Ca2+. At this stage, we cannot completely rule s homozygous lethal (21) . Under our binding conditions , h out the possibility that certain adaptor proteins [e .g, syn owever, the Syt I cytoplasmic domain showed only very taxin IA, which binds Syt I in a Ca2+-dependent manner weak Ca2+-dependent self-oligomerization activity How (15)] bridges different Syt isoforms. However, this possibil ever, given that Syt I proteins are enriched on synaptic ves ity is unlikely, because we could only detect Syts and IgG icles at the preterminal (i.e., local concentrations of this used for immunoprecipitation, when immunoprecipitants protein are relatively high), it is possible ttt Ca2+-depen were visualized by Coomassie Brilliant Blue R-250 staining dddddddnt oligomerization of the two C2B domains that are pre (data not shown). Furthermore, the T7and FLAG-Syts co assembled at the N-terminal domain may occur in intact - transfection assay needs to be performed in other types of preterminals (20). cells, such as PC12 cells, in the future, because of the possi Group C synaptotagmins were characterized by weak bility that subcelluar localization of Syt isoforms may differ (Syt IX) or no homo-oligomerization activity (Syt IV) under between cell types. our binding conditions. Because of the weak oligomeriza In summary, we investigated the Ca2+-dependent and tion activity, we could not detect their SDS-insensitive -independent homoand hetero-oligomerization of synap homo-oligomerization on SDS-PAGE (10). Although the totagmin family proteins and showed that some isoforms Ca2+-independent homo-oligomerization activity of Syt IX can oligomerize via the N-terminal domain (Ca2+-indepen was very weak, Syt IX weakly associated with Syts I, II, V, dent) and/or the cytoplasmic domain (Ca2+-dependent). Our and VII in a Ca2+-dependent manner. In contrast, Syt IV is results and heterologous expression of synaptotagmin fam ily in brain (37, 47, 48) suggest that hetero-oligomerization probably present mainly as a monomer irrespective of the presence of Ca2+. When Syt IV is expressed in COS-7 cells, of a possible neuronal Ca2+-sensor, Syt I (or II), with other it is predominantly localized at the Golgi-like perinuclear isoforms that have different biochemical properties, may compartment, whereas other Syt isoforms are localized at create a variety of Ca2+-sensors. tubular or vesicular structures and/or plasma membranes (39,40 and Fukuda, M., unpublished data). Thus, this dif We thank Dr. Shigekazu Nagata for the expression vector, Dr. ferent compartmentalization of Syt IV to other isoforms in Masao Sakaguchi for helpful advice, Eiko Kanno for technical as sistance, Hiroyuki Kabayama, Keiji Ibata, and Fumiaki Hamazato intact cells may restrict the hetero-oligomerization of Syt for valuable discussions, and Akihiro Mizutani for critical com IV with other informs (Fig. 3, 2nd panel). In contrast to our ments on the manuscript. findings, however, Chapman et a). reported that Syts I and IV hetero-oligomerize in a Ca2+-dependent manner by REFERENCES using recombinant proteins from bacteria (25). This dis crepancy may be due to differences in binding conditions, 1. Sudhof, T.C. and Rizo, J. (1996) Synaptotagmin: C2-domain pro such as different recombinant proteins, amount of proteins teins that regulate membrane traffic. Neuron 17, 379-388 or incubation time. Alternatively, in vitro oligomenzation of 2. 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