(Received for publication, September 11, 1996, and in revised form, November 22, 1996)
From the Department of Neurology and Neurosurgery, Synaptojanin is a nerve-terminal enriched
inositol 5-phosphatase thought to function in synaptic vesicle
endocytosis, in part through interactions with the Src homology 3 domain of amphiphysin. We have used synaptojanin purified from Sf9
cells after baculovirus mediated expression in overlay assays to
identify two major synaptojanin-binding proteins in rat brain. The
first, at 125 kDa, is amphiphysin. The second, at 40 kDa, is the major
synaptojanin-binding protein detected, is highly enriched in brain, is
concentrated in a soluble synaptic fraction, and co-immunoprecipitates
with synaptojanin. The 40-kDa protein does not bind to a synaptojanin
construct lacking the proline-rich C terminus, suggesting that its
interaction with synaptojanin is mediated through an Src homology 3 domain. The 40-kDa synaptojanin-binding protein was partially purified
from rat brain cytosol through a three-step procedure involving
ammonium sulfate precipitation, sucrose density gradient
centrifugation, and DEAE ion-exchange chromatography. Peptide sequence
analysis identified the 40-kDa protein as SH3P4, a member of a novel
family of Src homology 3 domain-containing proteins. These data suggest an important role for SH3P4 in synaptic vesicle endocytosis.
Synaptic vesicles are specialized organelles that neurons use to
secrete nonpeptide neurotransmitters. Following neurotransmitter release, synaptic vesicle membranes are retrieved by a process thought
to involve clathrin-coated pits and vesicles (1, 2), and recent data
suggest that this endocytic mechanism is active at both high and low
rates of exocytosis (3). We have identified a 145-kDa protein, referred
to as synaptojanin, which is enriched in nerve terminals and appears to
function in synaptic vesicle endocytosis (4-6). Synaptojanin is an
inositol 5-phosphatase that dephosphorylates inositol
1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and
phosphatidylinositol 4,5-bisphosphate at the 5 Synaptojanin was initially identified based on its ability to bind to
the Src homology 3 (SH3)1 domains of Grb2
(4). Cloning of synaptojanin revealed a 250-amino acid proline-rich
domain at its C terminus (6) that contains at least five sequences
forming potential SH3 domain-binding sites (11). A second, 170-kDa
isoform of synaptojanin is present in a wide variety of tissues
including neonate brain but is not detected in adult brain (6, 12). The
170-kDa synaptojanin isoform is generated by alternative splicing of
the synaptojanin gene leading to the presence of an additional
266-amino acid proline-rich domain with at least three additional SH3
domain-binding consensus sequences as compared with the 145-kDa isoform
(12).
Synaptojanin also binds to the SH3 domain of amphiphysin (6).
Amphiphysin was first identified in chicken synaptic fractions (13) and
mammalian amphiphysin, which is concentrated in presynaptic nerve
terminals, has been implicated in synaptic vesicle endocytosis (14). A
role for amphiphysin in endocytosis is supported by studies on its
yeast homologues, RVS 161 and RVS 167 (15-17). Mutations in these
genes cause an endocytosis defect characterized in part by an
impairment in Here, we have used purified synaptojanin in overlay assays to identify
its preferred binding targets in brain. In addition to amphiphysin, we
identified a 40-kDa synaptojanin-binding protein that is highly
enriched in brain, is concentrated in soluble synaptic fractions, and
co-immunoprecipitates with synaptojanin. Purification and peptide
sequence analysis revealed the 40-kDa protein as SH3P4, a novel SH3
domain-containing protein that was identified from a mouse library
screened with a Src SH3 ligand peptide (27). SH3P4, along with SH3P8
and SH3P13, define a family of similar proteins of unknown function
(27). Our data strongly implicate SH3P4, and perhaps other family
members, in synaptic vesicle endocytosis.
A
BamHI-HindIII fragment from nucleotides 181 to
747, digested from a full-length synaptojanin cDNA (clone 9) (6),
was subcloned into the BamHI-HindIII sites of
pBluBac 4 (Invitrogen) (generating clone 1-18). A 5 Spodoptera frugiperda (Sf9; Invitrogen)
cells were grown at 27 °C in suspension cultures in Sf-900 II SFM
optimized serum-free medium (Life Technologies, Inc.) supplemented with
gentamycin. The baculovirus transfer vectors were co-transfected with
linear baculovirus into Sf9 cells, and recombinant baculovirus was
selected by plaque assay as described (28). Positive colonies were
confirmed by protein purification and Western blot, and high titer
stocks (108-109 plaque forming units/ml) were
generated as described (28). For purification of synaptojanin
constructs, 200-ml cultures of Sf9 cells (1.5 × 106
cells/ml) were infected with ~1 × 109 plaque
forming units of baculovirus. After 72 h of growth, cells were
washed with 4 °C phosphate-buffered saline (20 mM
NaPO4 monobasic, 0.9% NaCl, pH 7.4) and lysed in 30 ml of
buffer A (300 mM NaCl, 0.83 mM benzamidine,
0.23 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
aprotinin, 0.5 µg/ml leupeptin, 50 mM HEPES-OH, pH 8.0) by homogenization in a glass Teflon homogenizer and two passes through
a 255/8 gauge needle. Homogenates were spun at 12,000 × g for 10 min, and Triton X-100 (0.1% final) and imidazole
(20 mM final) were added to the supernatant before the
addition of 0.5 ml of Ni-NTA-agarose (Qiagen Corp.). The samples were
incubated overnight at 4 °C and washed three times in 20 ml of
ice-cold buffer A with 0.1% Triton X-100 and 20 mM
imidazole, and bound proteins were eluted with 4 × 1-ml
incubations in buffer A with 0.1% Triton X-100 and 200 mM
imidazole. Dynamin was purified from Sf9 cells after
baculovirus-induced expression as described (29) and was a generous
gift of Dr. Sandra Schmid (Scripps Research Institute).
Overlay assays using a glutathione
S-transferase/amphiphysin SH3 domain fusion protein were
performed as described (4). For synaptojanin overlay assays, protein
fractions on nitrocellulose membranes were blocked for 1 h in
blotto (phosphate-buffered saline with 5% (w/v) nonfat dry milk),
rinsed in water, and incubated overnight at 4 °C in buffer B (150 mM NaCl, 3% bovine serum albumin, 0.1% Tween 20, 1 mM dithiothreitol, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride, 20 mM
Tris-Cl, pH 7.4) containing approximately 10 µg/ml of purified
synaptojanin or the synaptojanin R-2 deletion construct. Transfers were
then washed and incubated with affinity purified anti-synaptojanin
antibody (Milo) (5) or 1852 (described below) in buffer B without
dithiothreitol for 1 h at room temperature. After washing,
transfers were incubated in goat anti-rabbit secondary antibody
conjugated to horseradish peroxidase for 1 h in blotto and
developed using the ECL kit (Amersham Corp.).
Various tissues were
dissected from adult male rats and were homogenized at 1:10 (w/v) in
buffer C (0.83 mM benzamidine, 0.23 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 20 mM HEPES-OH, pH 7.4) with a polytron or glass
Teflon homogenizer, followed by centrifugation for 5 min at 800 × gmax. The supernatant fractions were separated
on SDS-PAGE on 5-16% or 3-12% gradient gels. Subcellular
fractionation of brain homogenates to generate synaptic fractions was
performed as described (5).
Amphiphysin
immunoprecipitations were performed as described (14). For synaptojanin
immunoprecipitations, a rat brain was homogenized at 1:10 (w/v) in
buffer D (0.3 M sucrose, 0.83 mM benzamidine,
0.23 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 10 mM HEPES-OH, pH 7.4)
with a polytron. The homogenate was centrifuged at 800 × gmax for 5 min, and the supernatant was then
centrifuged at 180,000 × gmax for 2 h. The
soluble supernatant was precleared with protein G-agarose for 4 h
at 4 °C and the precleared extracts were incubated overnight at
4 °C with a monoclonal antibody against synaptojanin or a control monoclonal antibody raised against p75LNGFR precoupled to
protein G-agarose. Samples were washed five times in 1 ml of
buffer D and were eluted with SDS sample buffer.
Adult rat brains (typically four for
each preparation) were homogenized at 1:5 (w/v) in buffer C with a
polytron, and the extracts were centrifuged at 180,000 × gmax for 1 h. Ammonium sulfate powder was
added slowly to the soluble supernatant with stirring until 20%
saturation. After 45 min on ice, the sample was centrifuged at 2700 × gmax for 30 min, and the supernatant was
removed and precipitated with ammonium sulfate to 40% saturation. The
20-40% ammonium sulfate precipitate was resuspended in 16 ml of
buffer C and was loaded on four 40-ml 2.5-15% linear sucrose gradients prepared in buffer C. The gradients were centrifuged in a
Beckman VTi 50 rotor for 6 h at 45,000 rpm with slow acceleration and no brake. Gradient fractions (20 × 2 ml) were analyzed by synaptojanin overlay assay, and peak 40-kDa synaptojanin-binding protein fractions were pooled and passed over a 5-ml column of DEAE-Sephacel (Pharmacia Biotech Inc.) equilibrated in buffer C. Samples were recirculated over the column at a flow rate of 0.2 ml/min
for 16 h, and the column was then eluted into 20 4-ml fractions at
2 ml/min with an 80-ml linear gradient of 0-0.5 M NaCl
prepared in buffer C. Eluted fractions (80 µl/fraction) were analyzed
for the 40-kDa synaptojanin-binding protein by overlay assay.
Alternatively, proteins from eluted fractions (1 ml/fraction) were
precipitated with 50% ice-cold trichloroacetic acid with 0.03% sodium
deoxycholate as a carrier and analyzed by Coomassie Blue staining of
protein gels. The peak 40-kDa synaptojanin-binding protein fraction was
concentrated, run on SDS-PAGE, and transferred to PVDF membranes. The
40-kDa protein was excised and subjected to Edman degradation but was
found to have a blocked N terminus. Therefore, the sample was treated
with cyanogen bromide (CNBr) to affect peptide bond cleavage at the
C-terminal side of methionyl residues (30). The PVDF membrane was
immersed in 100 µl of freshly prepared CNBr cleavage solution (70 mg
CNBr/ml in 70% formic acid), flushed with argon, sealed, and left
18 h at room temperature. The cleavage mixture was then dried in a
speed vacuum centrifuge, and the PVDF pieces were subjected to sequence
analysis in an Applied Biosystems model 470A protein sequencer equipped
with an on-line Applied Biosystems model 120A phenylthiohydantoin
analyzer (31) according to procedures as recommended by the
manufacturer. Sequence analysis revealed multiple sequencing signals
which were manually analyzed by overlaying sucessive high pressure
liquid chromatography traces. The strengths of the multiple signals
were ranked, and probable sequences were searched against protein data bases employing the Blastp algorithm.
A polyclonal anti-synaptojanin antibody (1852)
was prepared by injection of a rabbit with 200 µg of synaptojanin R-2
deletion construct in Titer-MaxTM adjuvant (CytRx Corporation) using
standard protocols. Serum was tested for immunoreactivity by Western
blot against brain extracts and purified synaptojanin. Antibodies were affinity purified from serum against purified synaptojanin on PVDF
membranes as described (5). A polyclonal antibody against synaptojanin
purified from rat brain (Milo) was described previously (5). Polyclonal
antibodies against amphiphysin were prepared as described (15) and were
a generous gift of Drs. Carol David and Pietro De Camilli (Yale
University). The monoclonal antibody against synaptojanin was raised
against a glutathione S-transferase fusion protein encoding
amino acids 1156-1286 of synaptojanin in the laboratory of Dr. Pietro
De Camilli and was a generous gift of Drs. Amy Hudson and Pietro De
Camilli (Yale University). The monoclonal antibody against
p75LNGFR was prepared as described (32) and was a generous
gift of Dr. Phil Barker (Montreal Neurological Institute) and Dr. Eric
Shooter (Stanford University).
To study the SH3 domain-binding properties of synaptojanin,
we generated full-length synaptojanin and a synaptojanin deletion construct (synaptojanin R-2) lacking the proline-rich C terminus for expression in Sf9 cells using baculovirus. The constructs had six
histidine residues introduced at the N terminus to allow for their
purification with nickel-agarose. Purification of nickel-binding proteins from Sf9 cell cultures infected with the full-length synaptojanin construct leads to the isolation of a 145-kDa protein (Coomassie, Fig. 1) that is strongly reactive
with a polyclonal antibody against synaptojanin (Milo
Western, Fig. 1). Infection of cultures with the synaptojanin R-2
construct leads to the production of a 120-kDa protein
(Coomassie, Fig. 1) that does not react with the polyclonal
antiserum raised against full-length synaptojanin (5) (Milo
Western, Fig. 1), indicating that the antibodies are directed
entirely against the last 231 amino acids of the proline-rich C
terminus of synaptojanin. However, a rabbit antiserum raised against
synaptojanin R-2 reacted strongly with both synaptojanin constructs
(1852 Western, Fig. 1).
To further characterize the baculovirus expressed synaptojanin, various
dilutions of dynamin and synaptojanin, both purified from baculovirus
infected Sf9 cells, were run on SDS-PAGE and overlaid (4) with a
glutathione S-transferase fusion protein encoding the SH3
domain of amphiphysin. The synaptojanin construct strongly binds the
SH3 domain of amphiphysin in this assay. Interestingly, when equal
amounts of the two proteins are compared directly, amphiphysin
demonstrates a greater relative affinity for synaptojanin than dynamin
(Fig. 2).
To identify the
major binding partners for synaptojanin in brain, we used purified
synaptojanin in overlay assays. Nitrocellulose transfers containing
proteins from brain extracts were incubated with purified synaptojanin,
and bound synaptojanin was detected with the antibody raised against
full-length synaptojanin (Milo). Control overlay assays were performed
with purified proteins from control infected Sf9 cells. Synaptojanin is
seen to bind to two major (stars, Fig. 3) and
two minor (arrowheads, Fig. 3) proteins in crude rat brain
extracts. In addition, synaptojanin itself is detected
(diamond, Fig. 3) and is the only protein seen in control
overlays (Fig. 3).
Based on previous results (6, 14), we predicted that
amphiphysin would be detected in the synaptojanin overlay assay. One of
the major synaptojanin-binding proteins migrates at approximately 125 kDa, consistent with the molecular mass of amphiphysin (13, 14). In
fact, amphiphysin and the 125-kDa synaptojanin-binding protein have an
identical mobility on SDS-PAGE (Fig. 4A). To
confirm the identity of this protein, we performed an
immunoprecipitation assay using two different amphiphysin antibodies
(CD5 and CD6). As seen in Fig. 4B, and in agreement with
previous data (14), both amphiphysin antibodies immunoprecipitate
amphiphysin from a rat brain extract, although CD5 is much more
effective than CD6. A synaptojanin overlay assay of the amphiphysin
immunoprecipitates demonstrates that the 125-kDa synaptojanin-binding
protein is amphiphysin.
A
protein at approximately 40 kDa is the strongest synaptojanin-binding
protein in brain (Figs. 3 and 4A). As determined by overlay,
the 40-kDa protein is enriched in brain, although it is also detected
in extracts from rat testis (Fig. 5A).
Synaptojanin is also enriched in adult brain (Fig. 5A)
although lower levels are seen in a wide variety of tissues (12).
Synaptojanin, which is concentrated in presynaptic nerve terminals (5),
is enriched in synaptic membrane fractions (Fig. 5B,
LP2). As determined by overlay, the 40-kDa
synaptojanin-binding protein is enriched in soluble fractions and is
concentrated in the LS2 fraction that corresponds to
cytosol isolated from lysed synaptosomes (Fig. 5B).
We used a monoclonal antibody
against synaptojanin to immunoprecipitate the protein from soluble
fractions of rat brain. Synaptojanin is enriched in the precipitated
material (Fig. 6). The 125-kDa synaptojanin-binding
protein, which we identified as amphiphysin, does not
co-immunoprecipitate with synaptojanin (Fig. 6; see "Discussion"). However, the 40-kDa synaptojanin-binding protein does
co-immunoprecipitate with synaptojanin and is enriched in the
synaptojanin immunoprecipitate as compared with the starting material
(Fig. 6). These data confirm the interaction between synaptojanin and
the 40-kDa synaptojanin-binding protein in the brain.
Rat
brain cytosol was fractionated using various concentrations of ammonium
sulfate, and the 40-kDa synaptojanin-binding protein was found
exclusively in the 20-40% ammonium sulfate precipitate (data not
shown). This fraction was then subjected to size fractionation on
2.5-15% linear sucrose density gradients, and the 40-kDa protein was
found in a narrow peak near the top of the gradient (data not shown).
Peak sucrose density gradient fractions were pooled and subjected to
anion exchange chromatography on DEAE-Sephacel. The column was eluted
with a linear gradient of NaCl from 0 to 0.5 M. A Coomassie
Blue-stained gel of the proteins eluted from the DEAE column is shown
in Fig. 7A. A band at 40-kDa was apparent that was strongly reactive in the synaptojanin overlay assay (Fig. 7A, synaptojanin overlay).
To further characterize the 40-kDa protein, partially purified samples
were overlaid with synaptojanin or synaptojanin R-2 deletion mutant
(Fig. 7B) using the antiserum that recognizes the N-terminal
domain of synaptojanin (1852 Western, Fig. 1). Synaptojanin,
but not synaptojanin R-2, binds to the 40-kDa synaptojanin-binding protein. This demonstrates that the interaction of the 40-kDa protein
with synaptojanin is mediated through synaptojanin's proline-rich C
terminus and suggests that the 40-kDa protein contains an SH3 domain.
To identify the 40-kDa protein, fraction 14 from the DEAE column
elution (Fig. 7A) was concentrated and transferred to PVDF membranes, and the 40-kDa protein band was subjected to peptide sequence analysis. The sample was refractive to automated Edman degradation, suggesting a blocked N terminus. Therefore, the sample was
cleaved at methionyl residues with CNBr and resubjected to sequence
analysis. The mixture resequencing revealed 2-3 major sets of
sequencing signals. The strengths of the multiple signals were ranked,
and the best guess sequence
(Met0-Glu1-Val2-Phe3-Gln4-Asn5-Phe6-Ile7-Asp8-Pro9-Asp10-Gln11-Asn12-Gln13-His14-His15-Ala16-Asp17-Leu18-Arg19)
was searched against the protein data base and was found to align
to internal sequences of mouse SH3P4, SH3P8, and SH3P13, three members
of a novel family of SH3 domain-containing proteins (27) (Fig.
7C). SH3P4 is the likely homologue of the 40-kDa synaptojanin-binding protein because its predicted protein sequence contains the required methionyl residue as well as 15 of 20 identities found (Fig. 7C). Further, a second major peptide that was
sequenced from the 40-kDa protein aligns with a peptide from SH3P4 that contains three of the five mismatches from the best guess sequence in
the proper position in relationship to the methionyl residue (Fig.
7C). In contrast, SH3P8 and SH3P13 lack the methionyl
residue at the start of the sequence, and only 12 and 9, respectively, of the above 20 residues are identical (Fig. 7C).
Furthermore, the major signals in the mixture resequencing data are
accounted for by CNBr cleavage at Met96,
Met121, Met133, Met201, and
Met207 of mouse SH3P4 (data not shown) whereas the
predicted sequences of the two SH3P4 homologues SH3P8 and SH3P13 (27)
code for only 3 (Met96, Met121, and
Met201) and 2 (Met96 and Met201) of
the required methionyl residues, respectively. On this basis, the
40-kDa protein is the rat homologue of mouse SH3P4.
Synaptojanin is an inositol 5-phosphatase implicated in synaptic
vesicle endocytosis (4-6, 14). Synaptojanin was initially isolated
based on its ability to bind to the SH3 domains of Grb2 (4), and a role
for Grb2 in endocytosis was recently demonstrated (26). Synaptojanin
also binds to the SH3 domain of amphiphysin (6, 14), and several pieces
of evidence implicate amphiphysin in endocytosis, including its SH3
domain-dependent interaction with dynamin (14). Thus, it
appears that SH3 domain-mediated interactions play a general role in
endocytosis. In an effort to better characterize the nature of SH3
domain-mediated protein-protein interactions with synaptojanin, we
generated synaptojanin and a synaptojanin deletion construct in Sf9
cells using the baculovirus system. The proteins were then purified on
nickel-agarose using a His6 tag engineered into the N
terminus of the constructs. To characterize the baculovirus expressed
synaptojanin, we compared the affinity of amphiphysin binding to
synaptojanin versus dynamin. When amphiphysin or Grb2 are
used as substrates for the purification of SH3 domain-binding proteins
from brain extracts, greater amounts of dynamin than synaptojanin are
isolated (5, 14), likely owing to higher levels of dynamin expression
in brain. However, as shown here, when equal amounts of purified
dynamin and synaptojanin are analyzed, amphiphysin shows stronger
binding to synaptojanin than dynamin. It has been proposed (14) that
amphiphysin may serve to target dynamin to sites of synaptic vesicle
endocytosis via its dual interactions with AP2 (14, 19) and dynamin.
Amphiphysin may also play a role in targeting synaptojanin to endocytic
sites. The higher affinity of synaptojanin than dynamin for amphiphysin binding may be important to allow for synaptojanin targeting in the
presence of high dynamin concentrations in the nerve terminal.
We used synaptojanin purified from Sf9 cells in a gel overlay assay to
identify two major synaptojanin-binding proteins with molecular masses
of approximately 125 and 40 kDa. The 125-kDa synaptojanin-binding
protein was identified as amphiphysin based on its co-migration with
amphiphysin on SDS-PAGE and its precipitation with amphiphysin
antibodies. The identification of amphiphysin as a major
synaptojanin-binding protein strongly suggests that the assay is
effective in identifying relevant synaptojanin-binding partners
in vitro and further suggests that amphiphysin and the 40-kDa protein are the major synaptojanin-binding proteins in vivo.
Further characterization of the 40-kDa synaptojanin-binding protein
demonstrates that it is highly concentrated in brain and is
predominantly a soluble protein that is enriched in cytosol isolated
from lysed synaptosomes. Proteins that function in clathrin-mediated endocytosis are often expressed at levels 10-50-fold higher in neuronal versus non-neuronal cells (33). For example, both
dynamin and synaptojanin are highly expressed in neurons, whereas these proteins or related isoforms are expressed at lower levels in non-neuronal cells (12, 34-36). The 40-kDa synaptojanin-binding protein is concentrated in brain but is also detected in testis, a
tissue with little or no expression of the 145-kDa isoform of synaptojanin (12). However, the testis does express the 170-kDa synaptojanin isoform (12), and this protein also binds strongly to the
40-kDa synaptojanin-binding protein (data not shown). An important role
for the 40-kDa synaptojanin-binding protein is also supported by the
observation that it co-immunoprecipitates with synaptojanin from rat
brain cytosol. This is in contrast to amphiphysin, which does not
co-immunoprecipitate with synaptojanin (Fig. 6). The reason for the
lack of amphiphysin/synaptojanin co-immunoprecipitation is unclear, but
it may be due to a technical reason such as steric interference of the
synaptojanin antibody with the site of amphiphysin binding. A more
interesting explanation may be that the binding of synaptojanin to the
40-kDa synaptojanin-binding protein excludes amphiphysin binding. Thus,
it is possible that the 40-kDa synaptojanin-binding protein could
regulate the ability of synaptojanin to bind to amphiphysin, and
this could play a key role in regulating the targeting of synaptojanin
to sites of endocytosis.
To identify the 40-kDa protein, we purified it from rat brain cytosol
and subjected it to peptide sequence analysis. The sequence analysis
identifies the 40-kDa synaptojanin-binding protein as SH3P4, a novel
SH3 domain-containing protein with a predicted molecular mass of 39,880 Da (27). The identification of the 40-kDa synaptojanin-binding protein
as an SH3 domain-containing protein is consistent with our observation
that the 40-kDa protein does not bind to a synaptojanin deletion
construct lacking the proline-rich C terminus. Further, the predicted
isoelectric point of 5.3 for SH3P4 (27) is consistent with its elution
from the DEAE ion exchange column in high salt. SH3P4, which was
identified from a mouse library screened with a Src SH3 ligand peptide,
is 75 and 63% identical to SH3P8 and SH3P13, respectively, two other proteins identified in the same screen (27). These three proteins define a novel protein family of unknown function. Our data strongly implicate SH3P4, and perhaps other family members, in synaptic vesicle
endocytosis. It will be of interest to determine if the interaction of
SH3P4 can regulate the ability of synaptojanin to bind to amphiphysin
and thus regulate the targeting of synaptojanin to sites of
endocytosis.
We thank Drs. Pietro De Camilli, Carol David,
and Amy Hudson (Yale University), Dale Warnock and Dr. Sandra Schmid
(Scripps University), Dr. Phil Barker (Montreal Neurological
Institute), and Eric Shooter (Stanford University) for providing
important reagents used in this study. We also thank Weihau Lu and
France Dumas for expert technical assistance and Drs. Pietro De
Camilli, Sandra McPherson, Sandra Schmid, Phil Barker, and Philippe
Segeula for support and discussion. Protein sequencing was performed at the Biotechnology Research Institute, Montreal.
Department of Anatomy and
Cell Biology, McGill University, Montreal,
Quebec H3A 2B4, Canada
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
position of the
inositol ring (6). Inositol phosphate metabolism has been implicated in
a variety of membrane trafficking events including endocytosis (7). In
addition, synaptojanin has an N-terminal domain that is homologous to
the cytosolic domain of the yeast SacI protein.
SacI mutants show genetic interactions with actin as well as
with the yeast secretory mutants sec6, sec9, and
sec14 (8, 9), and more recently SacIp has been
demonstrated to mediate ATP transport into the yeast endoplasmic
reticulum (10).
-factor receptor internalization (18). Further,
amphiphysin is known to interact with AP2 (14, 19), a component of the
plasma membrane clathrin coat (20). Evidence of a role for the SH3
domain of amphiphysin in synaptic vesicle endocytosis is provided by
its interaction with dynamin. A role for dynamin in endocytosis was
first determined based on its identity with the gene product of the
Drosphila shibire mutant (21, 22). Mutations in
Drosophila dynamin leads to a block in synaptic vesicle endocytosis (23), and recent data suggest that dynamin functions in the
nerve terminal by mediating the fission of endocytic vesicles (24, 25).
Thus, it appears likely that SH3 domain-mediated interactions of
amphiphysin with synaptojanin are important to the endocytic function
of synaptojanin in vivo. SH3 domain interactions involving
Grb2 have also been recently demonstrated to be important for
clathrin-mediated endocytosis of the epidermal growth factor receptor
in non-neuronal cells (26). Specifically, disruption of Grb2
interactions with the epidermal growth factor receptor blocks receptor
endocytosis, and epidermal growth factor can stimulate a transient
association of Grb2 with dynamin (26). Thus, SH3 domain-mediated
interactions appear to function widely in clathrin-mediated endocytosis.
Synaptojanin Baculovirus Transfer Vector Constructs
fragment encoding
the N-terminal of synaptojanin with a His6 tag was then
generated by PCR with Vent Polymerase (New England Biolabs) using the
forward oligonucleotide 5
-GCGGGATCCATGCATCACCACCACCACCACGCGTTCAGCAAGGATTTCG, which encodes a
BamHI site, and the reverse oligonucleotide
5
-ATGCGGTCCTCATCTGAAG, which corresponds to clone 9 at nucleotide
positions 470-489. The PCR product, which contains the
BamHI site at nucleotide 181, was digested with
BamHI, and the 67-base pair piece was subcloned into clone
1-18 after a complete BamHI digest (generating clone 1B7). A
HindIII-HindIII fragment from nucleotide
747-3970 of clone 9 was then subcloned into the HindIII
site of clone 1B7 (generating clone synaptojanin). For the deletion
construct, PCR was performed on clone 9 with Vent Polymerase using the
forward primer 5
-GGTGATGTCGACGACTAC corresponding to nucleotide
positions 3184-3195 spanning a SalI site and the reverse
primer R-2 (5
-GCGGTCGACAAGCTTTTATGCGGAGTACTCTGGTGC) corresponding to
nucleotide positions 3307-3325 encoding a SalI site
flanking a HindIII site. The PCR product was digested with SalI and subcloned into SalI-digested clone 9 generating clone Bluescript R-2, which was digested with
HindIII and the liberated fragment cloned into clone 1B7
generating clone synaptojanin R-2. At each step for both constructs,
the junctions of the clones were confirmed by sequence analysis.
Expression and Purification of Synaptojanin Constructs in Sf9
Cells
Fig. 1.
Purification of baculovirus expressed
synaptojanin constructs. Sf9 cells were infected with baculovirus
encoding full-length synaptojanin (synaptojanin) or a synaptojanin
deletion construct lacking the proline-rich C terminus (synaptojanin
R-2), and the synaptojanin proteins were purified with nickel-agarose.
Approximately 4 µg of protein from each sample were separated on
SDS-PAGE and stained with Coomassie Blue (Coomassie) or were
transferred to nitrocellulose and blotted with a polyclonal antibody
against synaptojanin purified from rat brain (5) (Milo
Western) or with a polyclonal antibody raised against synaptojanin
R-2 (1852 Western).
[View Larger Version of this Image (38K GIF file)]
Fig. 2.
Amphiphysin overlay of synaptojanin and
dynamin. Purified synaptojanin and purified dynamin (500-20 ng as
indicated) were separated on SDS-PAGE, transferred to nitrocellulose,
and overlaid with glutathione S-transferase/amphiphysin SH3
domain (amphiphysin overlay) as described (4). Transfers
were stained with ponceau S to ensure even electrophoretic transfer.
The arrows on the right indicate the migratory
position of the proteins detected on the blots.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Synaptojanin overlay of a rat brain
extract. Rat brain post-nuclear supernatant fractions were
separated on SDS-PAGE, transferred to nitrocellulose, and overlaid with
synaptojanin (synaptojanin overlay) or with protein purified
from mock infected Sf9 cells (control overlay). The
symbols on the right denote the migratory
positions of the two major (stars) and two minor
(arrowheads) synaptojanin-binding proteins detected on the
blot. The diamond denotes the migratory position of
synaptojanin, which is also detected in this assay.
[View Larger Version of this Image (21K GIF file)]
Fig. 4.
Identification of a major
synaptojanin-binding protein as amphiphysin. A, rat brain
post-nuclear fractions were separated on SDS-PAGE, transferred to
nitrocellulose, and overlaid with synaptojanin (synaptojanin
overlay) or were blotted with a polyclonal antibody against
amphiphysin (amphiphysin Western). B, a rat brain Triton X-100 soluble extract was subject to immunoprecipitation with
two different polyclonal antibodies against amphiphysin (CD5 and CD6)
or with control normal rabbit serum (NRS) conjugated to
protein A-Sepharose. Precipitated proteins were separated on SDS-PAGE
along with an aliquot of the soluble extract (starting material,
SM), transferred to nitrocellulose, and subjected to an
amphiphysin Western blot (amphiphysin; top panel) or a
synaptojanin overlay (bottom panel). The arrows
on the right indicate the migratory position of amphiphysin
detected on the blots.
[View Larger Version of this Image (18K GIF file)]
Fig. 5.
Characterization of the 40-kDa
synaptojanin-binding protein. A, post-nuclear tissue
extracts from a variety of tissues as indicated were separated on
SDS-PAGE, transferred to nitrocellulose, and were subjected to a
synaptojanin Western blot (synaptojanin, top
panel) or a synaptojanin overlay (bottom panel). The
arrows on the right indicate the migratory
positions of the proteins detected on the blots. B, proteins
of brain subcellular fractions were separated by SDS-PAGE, transferred
to nitrocellulose, and subjected to a synaptojanin Western blot
(synaptojanin, top panel) or a synaptojanin
overlay (bottom panel). Subcellular fractions were prepared
as described (5). H, homogenate; P, pellet;
S, supernatant; LP, lysed pellet; LS,
lysed supernatant; CPG, controlled pore glass. The eluted
fractions from the CPG column were pooled into three
fractions with purified synaptic vesicles in CPG-3. The
arrows on the right indicate the migratory
positions of the proteins detected on the blots.
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Co-immunoprecipitation of synaptojanin and
the 40-kDa synaptojanin-binding protein. A soluble fraction from
rat brain was subjected to immunoprecipitation with a monoclonal
antibody against p75LNGFR (anti-p75) or a
monoclonal antibody against synaptojanin
(anti-synaptojanin). Precipitated proteins were separated on
SDS-PAGE along with an aliquot of the soluble extract (starting
material, SM), transferred to nitrocellulose and subjected
to a synaptojanin Western blot (top panel) or a synaptojanin
overlay (middle and bottom panels). The
arrows on the right indicate the molecular masses
of the proteins detected on the blots.
[View Larger Version of this Image (28K GIF file)]
Fig. 7.
Partial purification and identification of
the the 40-kDa synaptojanin-binding protein. The 40-kDa
synaptojanin-binding protein was partially purified using a combination
of ammonium sulfate precipitation, sucrose density gradient
centrifugation, and DEAE anion-exchange chromatography. A,
the top panel shows a Coomassie Blue-stained gel
(Coomassie) of the proteins eluted from the DEAE column with
a 0-0.5 M NaCl gradient. A distinct protein band at 40 kDa
(arrow, top panel) binds synaptojanin by overlay
assay (synaptojanin overlay, bottom panel).
B, the partially purified 40-kDa synaptojanin-binding
protein was overlaid with synaptojanin or with the synaptojanin
construct lacking the proline-rich C terminus (synaptojanin R-2
overlay). C, the best guess sequence predicted from the
major mixture sequencing data is shown in bold. The aligned
sequences from SH3P4, SH3P8, and SH3P13 (27) are indicated and matches
to the best guess sequence are in bold. The sequence of a
second region of SH3P4, which was also identified in the sequencing
mixture, is indicated, and amino acids that align with three of the
five mismatches from the best guess sequence are in
bold.
[View Larger Version of this Image (46K GIF file)]
*
This work was supported by Grant MT-12046 (to W. S. S.) and
Grant MT-13461 (to P. S. M.) from the Medical Research Council of
Canada and by a Fonds De La Recherche En Santé Du Québec Establishment Grant (to P. S. M.).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.
§
Medical Research Council of Canada Scholars.
¶
Alfred P. Sloan Research Fellow. To whom correspondence should
be addressed: CBET Group, Montreal Neurological Inst., McGill University, 3801 University Ave., Montreal, PQ H3A 2B4, Canada. Tel.:
514-398-7355; Fax: 514-398-8106; E-mail: mcpm{at}musica.mcgill.ca.
1
The abbreviations used are: SH3, Src homology 3;
PCR, polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis; PVDF, polyvinylidene difluoride.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.