From the Center for Biochemistry II, Medical Faculty,
Joseph-Stelzmann-Strasse 52, University of Cologne, Cologne D-50931,
Germany, the ¶ Institut de Biologie Physico-Chimique, Unité
Propre de Recherche CNRS 1929, 13 rue Pierre et Marie Curie,
Paris 75005, France, and the ** Max-Planck-Institute of
Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden
D-01307, Germany
Received for publication, August 21, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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We have characterized mammalian
endophilin B1, a novel member of the endophilins and a representative
of their B subgroup. The endophilins B show the same domain
organization as the endophilins A, which contain an N-terminal domain
responsible for lipid binding and lysophosphatidic acid acyl
transferase activity, a central coiled-coil domain for oligomerization,
a less conserved linker region, and a C-terminal Src homology 3 (SH3)
domain. The endophilin B1 gene gives rise to at least three splice
variants, endophilin B1a, which shows a widespread tissue distribution,
and endophilins B1b and B1c, which appear to be brain-specific.
Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits
lysophosphatidic acid acyl transferase activity, and interacts with
dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to
endophilins A, endophilin B1 does not bind to synaptojanin 1 and
synapsin 1, and overexpression of its SH3 domain does not inhibit
transferrin endocytosis. Consistent with this, immunofluorescence
analysis of endophilin B1b transfected into fibroblasts shows an
intracellular reticular staining, which in part overlaps with that of
endogenous dynamin. Upon subcellular fractionation of brain and
transfected fibroblasts, endophilin B1 is largely recovered in
association with membranes. Together, our results suggest that the
action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.
The endophilins, originally named SH3p4/p8/p13 (1-3),
SH3GL1-3 (4), and SH3d2a-c (5), are a family of proteins identified in
search for SH3 domain-containing proteins. The most extensively studied
isoform of the mammalian endophilins, the neuron-specific endophilin A1
(2, 6), is essential for synaptic vesicle endocytosis (7-12). Via its
C-terminal SH3 domain, endophilin A1 binds to proline-rich domains of
amphiphysin (13), dynamin (2), and synaptojanin (2, 3), three proteins
also involved in synaptic vesicle endocytosis (14).
In addition to interacting with other proteins, endophilin A1
has been shown to bind lipids. Thus, endophilin A1 binds to lysophosphatidic acid (LPA)1
and fatty acyl-CoA and, via its intrinsic lysophosphatidic acid acyl
transferase (LPA-AT) activity, condenses them to phosphatidic acid (8).
Moreover, endophilin A1 binds to liposomes and alone is sufficient to
deform them into narrow tubules (15). The latter observation provides
direct evidence in support of the hypothesis that the role of
endophilin A1 in synaptic vesicle endocytosis is related to its ability
to generate membrane curvature (6, 8, 9, 15, 16).
In contrast to the neuron-specific isoform endophilin A1,
another member of the endophilins A, endophilin A2, shows a ubiquitous tissue distribution (2, 4). Accordingly, the endophilins A have been
implicated not only in synaptic vesicle endocytosis but in various
membrane traffic events, most of which, however, occur at the plasma
membrane (7-12, 17-20). We report here the molecular and cell
biological characterization of a novel endophilin that is a
representative of a new subgroup of endophilins, the endophilins B (6).
While sharing many structural and functional properties with the
endophilins A, endophilin B is distinct in that it does not appear to
operate in endocytosis at the plasma membrane but, rather, is
associated with intracellular membranes. While this work, which focuses
on the brain-specific splice variant endophilin B1b, was prepared for
publication, other investigators independently reported on other
members of the endophilins B, which show a widespread tissue
distribution (15, 21, 22).
Yeast Two-hybrid Screen
The experiments were performed using the Matchmaker 2 two-hybrid system (Clontech). The
full-length PACSIN 1 open reading frame was cloned in-frame with
the GAL4 DNA-binding domain in the pAS2-1 vector and confirmed by
sequencing. The pAS2-1 PACSIN 1 plasmid was sequentially cotransformed
with the Matchmaker mouse brain cDNA library into the yeast strain
Y190. Transformed yeast cells expressing interacting Gal4 fusion
proteins were selected by the ability to grow on minimal synthetic
dropout medium lacking L-tryptophan,
L-leucine, and L-histidine and checked for
their ability to express the lacZ gene by
Isolation of Clones and DNA Analysis
Four filters with dotted cDNA derived from a 9-day
postcoitum murine embryo cDNA library (no. 559) were
obtained from the Resource Center of the German Human Genome
Project. Filter hybridization was performed in 50% formamide,
5× Denhardt's solution (0.1% bovine serum albumin, 0.1% Ficoll 400, and 0.1% polyvinylpyrrolidone), 5× SSPE (0.75 M NaCl, 50 mM sodium phosphate (pH 7.6), and 5 mM EDTA),
1.5% SDS, and 300 µg/ml salmon sperm DNA with a 1831-bp probe
specific for murine endophilin B1. The latter cDNA fragment was
derived from the two-hybrid clone by EcoRI/XhoI
digestion and radiolabeled by random priming (TaKaRa). The filters were finally washed with 0.1× SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.5) and 0.1% SDS at 65 °C for 20 min and subjected to autoradiography. Positive clones and protein
sequence analyses were performed as described above.
RT-PCR and Southern Blot Analysis
Reverse transcription of poly(A)+ RNA isolated from
various mouse tissues was performed with Superscript II (Promega)
according to the manufacturer's protocol. For each tissue tested 500 ng of poly(A)+ RNA was converted into first-strand cDNA
using oligo(dT) primers in a 10-µl reaction mix. Endophilin B1
fragments were amplified by PCR using AmpliTaq DNA Polymerase
(PerkinElmer Life Sciences) and specific primers (sense: 5'-AGA CTG GAT
TTG GAT GCT GC-3', antisense: 5'-AGG TCA TTG AGG TTA GAA GG-3'). The
resulting PCR fragments were separated by electrophoresis on a 4% TBE
polyacrylamide gel and blotted onto a Hybond XL membrane (Amersham
Biosciences) by electrophoretic transfer (200 mA, 4 h) in 0.5×
TBE (24). Hybridization was performed in a formamide mix with a
radiolabeled probe corresponding to nucleotides 1-1831 of mouse
endophilin B1b generated by using a labeling kit (TaKaRa). The blot was
stringently washed and analyzed using autoradiography.
Antibodies
A GST murine endophilin B1b fusion protein was produced by
cloning the cDNA corresponding to the C-terminal 201 amino acids (residues 186-386) into the pGEX-4T1 vector (Amersham Biosciences) followed by expression in Escherichia coli (BL21). The
fusion protein was purified by affinity chromatography on
glutathione-Sepharose 4B (Amersham Biosciences) and used as an antigen
to immunize rabbits. Polyclonal antibodies against amphiphysin
2/BIN1 (EVA) were a generous gift of Dr. Pietro De Camilli (Yale
University, New Haven, CT), and antibodies against synaptojanin 1 (1852) were a generous gift from Dr. Peter McPherson (McGill
University, Montreal, Canada). Polyclonal antibodies against synapsin 1 were purchased from Sigma, monoclonal antibodies against dynamin (clone
41) were from Transduction Laboratories, a monoclonal antibody against
huntingtin was from Chemicon, a monoclonal antibody against amphiphysin
1 (clone 2) was from Oncogene Research Products, and a monoclonal
antibody against the hemagglutinin (HA) tag was from Roche Molecular
Biochemicals. All monoclonal antibodies were derived from mouse except
for anti-HA, which was from rat. For visualization of primary
antibodies Alexa 488- and Cy3-conjugated goat antisera against rabbit
and mouse immunoglobulins (IgG) were purchased from Molecular Probes
and Jackson ImmunoResearch Laboratories, respectively.
Immunoblotting
Proteins were extracted by mechanical disruption of freshly
prepared mouse tissues or NIH 3T3 cells in radioimmune precipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 50 mM Tris/HCl, pH 7.5) containing
protease inhibitors (Sigma, 50 µl per gram of tissue) and subsequent
centrifugation to remove detergent-insoluble material. Aliquots of the
extracts corresponding to 80 µg of protein were resolved on 10%
SDS-polyacrylamide gels followed by transfer to PVDF membranes
(Millipore). Blots were blocked at room temperature for 1 h with
5% skim milk in TBS containing 0.1% Tween 20 (TBST), rinsed with
TBST, and incubated at room temperature for 1 h with diluted
antibodies. After three 5-min washes with TBST, bound antibodies were
detected using peroxidase-conjugated anti-rabbit IgG antibodies (Dako)
and the ECL kit (Amersham Biosciences).
Protein Binding to Endophilins
Bead-immobilized Endophilins--
GST fusion proteins of
full-length murine endophilin B1b, full-length murine endophilin A1
(8), and a C-terminal fragment of murine endophilin A3 (2) were bound
to glutathione-Sepharose 4B (Amersham Biosciences) in PBS. Mouse brains
were homogenized on ice in 2.5 ml/g wet weight PBS containing 1%
deoxycholate supplemented with a protease inhibitor mixture (Sigma, 50 µl per gram of tissue) using a Dounce homogenizer and centrifuged for
30 min at 21,000 × g. The supernatant was decanted and
recentrifuged. After addition of Triton X-100 to a final concentration
of 0.05% to the supernatant, it was dialyzed against PBS buffer
lacking deoxycholate for 48 h. Glutathione-Sepharose 4B beads
saturated with GST-endophilins were incubated overnight with
supernatant containing 1 mg of the protein extract in 400 µl at
4 °C with end-over-end rotation. The beads were washed extensively
with PBS/1% Triton X-100. Bound proteins were eluted with
double-concentrated SDS-PAGE sample buffer, separated by SDS-PAGE on a
4-10% gradient gel, and transferred to a PVDF membrane for subsequent immunodetection.
Endophilin Overlay--
PVDF membranes containing rat brain
proteins were prepared as described above for immunoblotting, blocked,
rinsed with TBST, and incubated for 12 h at 4 °C with GST
fusion proteins containing the murine endophilin B1 and rat endophilins
A1-A3 SH3 domains. The latter were produced by cloning the cDNAs
corresponding to the C-terminal SH3 domains into the pGEX-6p1 vector
(Amersham Biosciences) followed by expression in E. coli
(BL21) and purification by affinity chromatography on
glutathione-Sepharose 4B (Amersham Biosciences). Following incubation
with the GST fusion proteins, the membranes were washed three times for
5 min, and bound GST fusion proteins were detected using an anti-GST
antibody (Amersham Biosciences), peroxidase-conjugated anti-rabbit IgG
secondary antibodies (Dako), and the ECL kit (Amersham Biosciences).
Subcellular Fractionation
The complete open reading frame of murine endophilin B1b was
cloned into the eukaryotic expression vector pHA-CMV
(Clontech). NIH 3T3 cells were transfected with
HA-tagged endophilin B1b using FuGENE 6 (Roche Molecular Biochemicals)
according to the manufacturer's protocol, and subcellular fractions
were prepared essentially as described previously (25). All steps were
performed at 4 °C. The cell pellet was homogenized in 3 ml of
ice-cold 0.32 M sucrose in 5 mM HEPES (pH 7.4)
supplemented with a protease inhibitor mixture (Sigma), using nine
strokes of a glass homogenizer. The homogenate was centrifuged for 10 min at 1000 × g to produce a pellet (P1), which was
washed by resuspension in an equal volume of homogenization buffer and
recentrifuged for 10 min at 1000 × g. The original
supernatant and wash were combined (S1) and then centrifuged at
10,000 × g for 20 min yielding pellet (P2) and
supernatant (S2). The S2 fraction was centrifuged at 105,000 × g for 60 min to give a high speed pellet (P3) and a high
speed supernatant (S3). The subcellular fractions obtained by this
method were analyzed by Western blotting.
For the isolation and analysis of membrane fractions derived from mouse
brain, a discontinuous sucrose gradient was used (26). All steps were
performed at 4 °C. Finely chopped mouse brains were homogenized in 3 ml per gram of wet weight of ice-cold 0.25 M sucrose in 10 mM Tris-HCl (pH 7.4) supplemented with a protease inhibitor
mixture (Sigma), using ten strokes of a glass homogenizer. Larger cell
debris and nuclei were removed by centrifugation for 10 min at 500 × g. After adjusting the post-nuclear supernatant to 1.4 M sucrose and 1 mM neutralized EDTA, 3 ml of
the post-nuclear supernatant were underlaid with 2.5 ml of 1.6 M sucrose/10 mM Tris-HCl (pH 7.4) and overlaid
with 4 ml of 1.2 M sucrose/10 mM Tris-HCl (pH
7.4) and 2.5 ml of 0.8 M sucrose/10 mM Tris-HCl
(pH 7.4). After centrifugation at 110,000 × g for
2 h, the various sucrose phases, interfaces, and the pellet were
collected for analysis by immunoblotting.
Immunofluorescence
NIH 3T3 fibroblasts plated on 12-mm glass coverslips were
transfected with the pHA-endophilin B1b expression vector using FuGENE
6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. 24 h after transfection, cells were fixed in 2%
paraformaldehyde in PBS for 10 min, permeabilized by incubation in
0.2% Triton X-100 in PBS for 1 min, and blocked for 15 min in 1%
bovine serum albumin and 5% horse serum in PBS. All antibody
incubations and washing steps were performed in PBS containing 0.02%
Triton X-100. Samples were analyzed by confocal laser scanning
microscopy (Leica).
Endocytosis Assay
For flow cytometric analysis (FACS) of transferrin uptake, NIH
3T3 cells were transfected with constructs containing the C-terminal SH3 domain of either murine endophilin A1, A2, or B1 fused to red
fluorescence protein (DsRed), and with DsRed as negative control (27).
48 h after transfection, cells were incubated for 20 min in
serum-free medium followed by a 30-min incubation in medium containing
5% fetal calf serum and 25 µg/ml FITC-conjugated human transferrin
(Molecular Probes), washed three times in PBS, trypsinized with 1%
trypsin, and finally washed once with PBS. A FACSCalibur (Becton
Dickinson) was used to detect red fluorescence proteins (FL2) and
FITC-transferrin (FL1) in 0.5-1.0 × 104 cells.
Palmitoyl-CoA Binding Assay
NIH 3T3 fibroblasts were transfected with HA-tagged endophilin
B1b and harvested after 48 h. Cells were lysed in ice-cold PBS
containing 0.1% Triton X-100, followed by short sonication, and the
lysates were centrifuged 20 min at 20,000 × g in a
cooled centrifuge. The supernatants were incubated with 50 µl
of palmitoyl-CoA-agarose (Sigma) overnight by end-over-end rotation at
4 °C, centrifuged for 5 min at 500 × g to remove
unbound proteins, washed three times with PBS/0.1% Triton X-100, and
eluted with 4 mM free palmitoyl-CoA in PBS/0.1% Triton
X-100 or with 1% Triton X-100 in PBS. Aliquots of the lysates, bound
and unbound material, supernatants, and the eluates were analyzed by
SDS-PAGE followed by immunoblotting using HA high affinity antibodies
coupled to horseradish peroxidase (Roche Molecular Biochemicals).
LPA-AT Assay
Recombinant murine endophilin B1b was produced by cloning the
cDNA corresponding to the full-length protein into the pGEX-6P1 vector (Amersham Biosciences) followed by expression in E. coli (BL21) and purification according to the manufacturer's
instructions with the following modifications. The bacterial pellet was
resuspended in PBS, incubated on ice for 20 min in the presence of
lysozyme (20 mg/ml), sonicated, and incubated at 4 °C for another 20 min after adding Triton X-100 to a final concentration of 1%. After centrifugation of the lysate at 20,000 × g for 15 min
and of the resulting supernatant at 226,000 × g for
1 h, glutathione-Sepharose 4B was added to the high speed
supernatant followed by incubation for 4 h at 4 °C. GST fusion
protein bound to the Sepharose was washed twice with PBS containing 1%
Triton X-100 followed by three additional washes using PBS without
detergent. For the LPA-AT assay, either the GST fusion protein or
endophilin B1b without GST tag was used. In the first case,
GST-endophilin B1b and, as control, GST were eluted according to the
manufacturer's protocol. In the second case, endophilin B1b was
liberated from GST by proteolytic cleavage with the PreScission
protease (Amersham Biosciences). Recombinant His-tagged mouse
endophilin A1 was expressed and purified by poly(L-proline)
affinity chromatography from the 280,000 × g
supernatant obtained from bacteria lysed by freezing/thawing in the
absence of detergent, as previously described (8).
For the data shown in Fig. 5B, the LPA-AT activity of
endophilins was determined as described previously (8). For the data shown in Fig. 5C, the LPA-AT activity of endophilins was
determined by modifying the previously described assay (8) as follows. Aliquots of [14C]oleoyl-CoA (100 nCi, 56 mCi per mmol,
PerkinElmer Life Sciences) in 0.01 M sodium acetate buffer,
pH 6, were dried in a SpeedVac and dissolved by adding 100 µl of
gel-filtered cytosol (GGA buffer) followed by sonication for 2 min. The tubes were then transferred to ice and brought to a final
reaction volume of 150 µl by sequential additions of GGA buffer, LPA,
and recombinant proteins. LPA was added from a 1 mM stock
prepared in LPA buffer (20 mM HEPES-KOH, pH 7.4, 10 mM sucrose, 1 mM EDTA, 2.5 mg/ml fatty
acid-free bovine serum albumin (Roche Molecular Biochemicals)) to a
final concentration of 10 µM. For the incubations that
did not contain LPA, an equivalent volume of LPA buffer was added.
Recombinant endophilins A1 and B1 were added at a final concentration
of 500 nM. Reactions were carried out for 20 min at
37 °C and terminated by addition of 150 µl ice-cold 0.8 M KCl, 0.2 M H3PO4.
Samples were mixed with 450 µl of chloroform/methanol (2:1) and
centrifuged for 5 min at 10,000 × g. The lower phase
was collected, dried, redissolved in 35 µl of
chloroform/methanol/water (2:1:0.1, v/v), subjected (along with a
[14C]phosphatidic acid standard, PerkinElmer Life
Sciences) to thin layer chromatography using Silica Gel 60 and
chloroform/pyridine/formic acid (50:30:7, v/v) as solvent, and analyzed
by phosphorimaging.
Isolation and Characterization of Endophilin B1 cDNAs--
In
a search for PACSIN 1/syndapin 1 (28, 29) interaction partners using
the yeast two-hybrid system, we identified one false-positive clone
(see explanation below) that represented a novel endophilin distantly
related to the endophilins A. We therefore further characterized this
protein and named it endophilin B1 to distinguish it from the
previously known members, which we propose to refer to as endophilins
A1-A3 (6). Using the two-hybrid clone as a probe, we obtained three
clones on screening a mouse embryo cDNA library. Two contained the
entire coding region (nucleotides 271-1431) and comprised a cDNA
of endophilin B1 with a length of 4012 bp. The first in-frame ATG is
located 271 nucleotides from the 5'-end and fulfills Kozak's criteria
for a translation initiation site, showing conservation of 9 out of 10 bases in the consensus (30). In the mouse mRNA this putative
initiator is not preceded by any in-frame stop codons, which in the
two-hybrid system leads to the translation of additional 90 amino
acids, including several prolines. These were sufficient to interact with the PACSIN 1 SH3 domain, resulting in the false-positive interaction (data not shown). In human endophilin B1, whose
cDNA sequence was obtained from data base searches and sequencing
of EST clones, the putative translation initiation site is located at
nucleotides 151-153 and, in contrast to the murine homolog, is
preceded by an in-frame stop codon (nucleotides 70-72).
The predicted protein products encoded by the open reading frames range
from 365 to 386 residues in length, with calculated molecular masses of
40,855 to 43,239 Da. Reasons for the differences in length include the
existence of splice variants such as endophilins B1a and B1b (Fig.
1A), which are addressed
further below. The human and murine endophilin B1 sequences show 96%
amino acid identity to each other, and the murine protein is 58%
identical to the Drosophila melanogaster homolog CG9834
(EMBL accession number AJ437142) (11) and 48% identical to the
Caenorhabditis elegans homolog F35A5-8
(GenBankTM accession number U46675). The comparison of
murine endophilin B1 to endophilins A1-3 revealed an average overall
identity of only 25%, showing that endophilin B1 is only distantly
related to the endophilin A subfamily and, together with endophilin B2, which originally was referred to as SH3GLB2 (21), belongs to a novel
endophilin subfamily whose overall domain organization is, however, the
same as that of the endophilins A. Specifically, we noticed the highest
similarity of endophilin B1 to the endophilins A in (i) the N-terminal
domain (38-42%), being the region of LPA acyl transferase activity,
liposome binding, and tubulation (8, 15), (ii) the central coiled-coil
region (41-52%), and (iii) the C-terminal SH3 domain (46-48%).
Following our deposition of the mouse and human endophilin B1 sequences
in the data base in 2000 as cited in a previous study (6), three other
groups have independently reported sequences identical to endophilin
B1a (15, 21, 22), the short splice variant of endophilin B1, which, in
contrast to the brain-specific longer splice variant endophilin B1b
studied here, shows a widespread tissue distribution (see below).
Expression and Genomic Organization of the Endophilin B1
Gene--
To determine the expression of the endophilin B1 gene, we
performed Northern blot analysis on mRNA isolated from several
adult mouse tissues. This revealed a similar tissue distribution of endophilin B1 mRNAs as previously reported (22) (data not shown). Four mRNA transcripts were detected, with sizes of 2.6, 5.0, 6.5, and 8.0 kb. Most of these can be attributed to the alternative use of
polyadenylation signals, as can be concluded from the existence of
various endophilin B1 expressed sequence tags (ESTs) and numerous potential polyadenylation sites in the murine and human 3'-noncoding region (data not shown).
A rabbit antiserum raised against the GST-endophilin B1 fusion protein
recognized a band of 40 kDa in most tissues tested, referred to as
endophilin B1a (Fig. 1B, filled arrowhead) but also reacted with a larger, 42-kDa form of the protein specifically detected in brain tissue and referred to as endophilin B1b (Fig. 1B, open arrowhead). In addition, an
immunoreactive band of 43 kDa was detected in lung and to lesser extent
in spleen (Fig. 1B, asterisk). The identity of
these different forms of endophilins B will be discussed below.
To explore whether endophilins B1a and B1b represented alternatively
spliced variants, we performed a sequence search against the
high throughput genomic sequences data base and identified the human
endophilin B1 gene-containing genomic clone J612B15, which was mapped
to the chromosomal region 1p22.2-1p31.1 by the Sanger Centre. By
comparing it with the human and murine cDNAs, we defined the
exon/intron boundaries of all 11 exons of the endophilin B1 gene,
except for the 5'-end of exon 1. This showed that the ubiquitously
expressed endophilin B1a (Fig. 1B, filled
arrowhead) represents a splice variant lacking exons 6 and 7 (Fig.
1C). Using RT-PCR analysis with primers in exons 5 and 10, we verified the existence of two additional transcripts, endophilins
B1b and B1c, that appear to be brain-specific (Fig. 1D).
Endophilin B1b uses a donor splice site within exon 6 and therefore
contains the shorter exon 6s and exon 7 (Fig. 1C).
Endophilin B1c, which exists as a brain-derived EST clone
(GenBankTM accession number BF470089), contains the longer
exon 6l and exon 7. The extended exon 6l leads to the insertion of 16 additional amino acids. Here, the alternative splice site within exon 6 is not used (Fig. 1C). This indicates that in brain, exons
6s, 6l, and 7 are alternatively used. Presumably, the 43-kDa endophilin B1 variant in lung and spleen (see Fig. 1B,
asterisk) represents yet another splice variant not detected
by the oligonucleotides used.
Protein Interactions of Endophilin B1--
To gain insight into
the function and mechanism of action of endophilin B1, we analyzed its
possible interaction with dynamin, huntingtin, synaptojanin, and
amphiphysin, which were previously shown to bind to endophilins A (2,
13, 31). For this purpose, a mouse brain detergent extract was
incubated with bead-immobilized GST fusion proteins containing
full-length mouse endophilin B1b, endophilin A1, a C-terminal
endophilin A3 fragment (2), or, as a control, GST alone. Brain was
chosen because this tissue expresses several splice variants of
endophilin B1 (see Fig. 1, B and D) and therefore
is likely to contain relevant interaction partners. Endophilin B1 was
able to bind dynamin, huntingtin, and both amphiphysins as detected by
immunoblotting (Fig. 2A). Compared with endophilins A1 and A3, endophilin B1
preferentially bound the larger splice variant of amphiphysin 2. Interestingly, endophilin B1, in contrast to endophilins A1 (2) and A3,
did not bind to synaptojanin 1, which has been implicated in synaptic vesicle uncoating (32). We also investigated the possible interactions of endophilins A and B with synapsin 1, which has been proposed to
anchor the reserve pool of synaptic vesicles via binding to actin
(reviewed in Ref. 33). Remarkably, synapsin 1 was detected among the
proteins bound to GST-endophilin A1 and A3 but not GST-endophilin B1
(Fig. 2A).
Endophilin B1, like the endophilins A, contains a single SH3 domain,
which is likely to be responsible for these protein interactions. We
therefore further analyzed some of the interactions in an overlay assay
by incubating blots containing brain proteins with purified GST-SH3-domain fusion proteins (Fig. 2B). Interestingly, the
SH3 domain of endophilin B1, like that of endophilins A1-3, recognized dynamin (Fig. 2B, filled circles), amphiphysin 1 (Fig. 2B, filled squares), and amphiphysin 2 (Fig. 2B, open circles), whereas synaptojanin 1 was only recognized by the SH3 domain of endophilins A but not endophilin B1 (Fig. 2B, open squares). The
interaction of the SH3 domains of endophilins A with synapsin 1 in the
overlay assay was at the limits of detection (data not shown). These
observations confirm the results obtained with the bead-immobilized
GST-endophilin fusion proteins (Fig. 2A) and show that the
binding of dynamin and amphiphysins 1 and 2 to bead-immobilized
endophilin B1b involves the direct interaction of its SH3 domain with
the former three proteins.
Using the yeast two-hybrid system, we could verify the direct
interaction of endophilin B1b with dynamin and huntingtin and show that
its SH3 domain was sufficient for this interaction (data not shown). In
these studies, we also noticed that full-length endophilin B1b can self
interact, in contrast to an endophilin B1b deletion construct lacking
the N-terminal LBM (lipid binding and modifying) domain and the
coiled-coil region (data not shown). The ability of the coiled-coil
region to mediate homo-oligomerization of the endophilins B has also
been described by others (21).
Intracellular Localization of Endophilin B1b--
The subcellular
localization of endophilin B1b was studied by both subcellular
fractionation and immunofluorescence (Fig. 3). For this purpose, we engineered a
hemagglutinin (HA) epitope-tagged version of endophilin B1b. Upon
differential centrifugation of transfected NIH 3T3 cells, virtually all
of HA-endophilin B1b was recovered in the microsomal pellet
(P3) and was not detectable in the crude nuclear pellet
(P1), the crude mitochondrial fraction (P2), nor
the cytosol (S3) (Fig. 3A, upper
panel).
To complement these results, a postnuclear supernatant of a mouse brain
homogenate was subjected to floatation/sedimentation using a
discontinuous sucrose gradient (26) (Fig. 3A, lower panel). The vast majority of endophilin B1 floated from the 1.4 M sucrose load to fractions of lower density, indicating
its association with membranes. The splice variant endophilin B1c,
which was only detected in brain by RT-PCR (Fig. 1D), was
detected only at the 0.8 M/1.2 M sucrose
interface (Fig. 3A, lower panel,
diamond). Endophilin B1b, like endophilin B1c brain-specific
(Fig. 1, B and D) and the major splice variant in
this tissue (Fig. 1B), peaked at the 0.8 M/1.2
M sucrose interface but was also found in the 1.2 M sucrose fraction and in the 1.4 M sucrose
load (Fig. 3A, lower panel, open
arrowhead). Consistent with the immunoblotting results using total
brain extract (Fig. 1B), only a weak signal was obtained for
the shortest endophilin B1 splice variant, the ubiquitous endophilin
B1a, which was detected at the 0.8 M/1.2 M
sucrose interface and in the 1.4 M sucrose load (Fig.
3A, lower panel, filled arrowhead).
To obtain a first indication as to the membranes with which endophilin
B1 is associated, HA-tagged endophilin B1b expressed in NIH 3T3 cells
was analyzed by confocal immunofluorescence microscopy. The transfected
endophilin B1b showed a reticular pattern throughout the cytoplasm,
indicating its association with intracellular membranes rather than the
plasma membrane (Fig. 3B). Part of the HA-endophilin B1b
staining appeared to overlap with that of endogenous dynamin (presumably dynamin 2) (Fig. 3B).
Lack of Influence of Endophilin B1 Overexpression on
Endocytosis--
The SH3 domain of endophilin A1 was previously shown
to inhibit transferrin endocytosis (17). We analyzed the SH3 domain of
endophilin B1 fused to the red fluorescent protein DsRed for a similar
activity by transiently overexpressing it in NIH 3T3 cells, with the
SH3 domains of endophilin A1 and A2 fused to DsRed as positive controls
and DsRed alone as negative control. Cells showing DsRed fluorescence
were isolated by FACS, and their uptake of FITC-labeled transferrin was
quantified (Fig. 4). In contrast to cells
expressing the SH3 domain of endophilins A1 and A2, which showed an
inhibition of transferrin uptake as expected from previous studies
(17), no inhibition of endocytosis was observed in cells expressing the
SH3 domain of endophilin B1 (Fig. 4).
Endophilin B1 Binds Activated Fatty Acids and Exhibits LPA Acyl
Transferase Activity--
We examined whether endophilin B1, like
endophilin A1 (8), binds fatty acyl-CoA and exhibits LPA-AT activity.
NIH 3T3 cells were transiently transfected with HA-tagged murine
endophilin B1b. As revealed by Western blotting (Fig.
5A), HA-endophilin B1b present
in a detergent extract prepared from the cells efficiently bound to
palmitoyl-CoA agarose and was eluted by addition of excess free
palmitoyl-CoA.
To analyze its potential LPA-AT activity, we expressed murine
endophilin B1b as a GST fusion protein in bacteria. Because GST-endophilin B1b was purified from bacteria lysed in the presence of
detergent, we first compared its LPA-AT activity with that of GST
alone, purified in parallel by glutathione-Sepharose affinity chromatography. The latter control should provide an indication of the
amount of endogenous bacterial LPA-AT activity that was nonspecifically
present in the glutathione-Sepharose eluate because of the use of
detergent. Indeed, the GST control sample showed some background LPA-AT
activity (Fig. 5B). However, the LPA-AT activity of
GST-endophilin B1b was substantially higher than that of the GST
control and in the range of His-tagged mouse endophilin A1 purified by
poly(L-proline) affinity chromatography from bacteria lysed
in the absence of detergent (8) (Fig. 5B). The LPA-AT activity of purified endophilin B1b liberated from GST by specific proteolytic cleavage, like that of purified His-tagged mouse endophilin A1, was totally dependent on the presence of exogenously added LPA,
with the level of LPA-AT activity of the two endophilins being very
similar (Fig. 5C).
The present characterization of endophilin B1, a representative of
the B subgroup of the endophilins (6), suggests that the role of the
endophilins in membrane dynamics is broader than previously
assumed. Endophilin B1 has the same overall domain organization
as the previously characterized endophilins A, with the hallmarks of an
N-terminal lipid binding and
modifying domain (LBM domain (11)), and a C-terminal SH3
domain mediating protein interaction (see below). Like endophilin A1
(8, 15, 16), endophilin B1 binds lipids, exhibits LPA-AT activity, and
tubulates liposomes, properties that have been shown or are likely to
reside in its LBM domain (Fig. 5) (15). Despite these common
properties, the sequence homology in the LBM domain between endophilin
B1 and endophilin A1 is significantly less than that between the endophilins A. This should facilitate the identification of amino acid
residues that are of critical importance for the LPA-AT activity and
liposome tubulation of the endophilins, such as phenylalanine 10 in
endophilin A1 whose hydrophobic nature has been shown to be crucial for
liposome binding and tubulation (15).
However, in contrast to endophilin A1, which is essential for synaptic
vesicle endocytosis from the plasma membrane (7-12), and endophilin
A2, which has been implicated in the formation of tubular plasma
membrane invaginations at podosomes of non-neuronal cells (20),
endophilin B1 expressed in non-neuronal cells does not appear to be
involved in endocytosis at the plasma membrane (Fig. 4) but, rather, is
associated with intracellular membranes (Fig. 3), suggesting a role in
intracellular membrane dynamics. Consistent with this, the protein
interaction partners of endophilin B1 are not identical to those of
endophilin A1. On the one hand, endophilin B1, like endophilin A1 (2),
directly interacts via its SH3 domain with dynamin (Fig. 2), whose
various isoforms and splice variants have been implicated in membrane
tubulation and vesicle formation not only from the plasma membrane but
also from intracellular membranes (34, 35). On the other hand, however, in contrast to endophilin A1, endophilin B1 does not bind to
synaptojanin 1 and synapsin 1 (Fig. 2), which have been implicated
specifically in the membrane dynamics of synaptic vesicles (14,
33).
Another direct interaction partner of endophilin B1, huntingtin (Fig.
2), deserves special comment. Huntingtin is a protein of unknown
function that has been reported to be associated with vesicles in
neuronal cell bodies and dendrites (36, 37). At the trans-Golgi network
and endosomes, huntingtin has been detected on both clathrin-coated and
non-coated vesicles and buds (38). Endophilin A3 was previously
identified as an interaction partner of huntingtin, specifically the
Huntington's disease exon 1 protein containing the pathogenic
glutamine repeat (31). Because endophilin A3 was reported to promote
the formation of insoluble polyglutamine-containing aggregates in
vivo and, therefore, hypothesized to be involved in the
progressive pathology of Huntington's disease, it will be of interest
to determine whether the same is true for endophilin B1.
The conclusion that the endophilins have a broader role in membrane
dynamics than previously assumed is also supported not only by the
existence of a second endophilin B gene, endophilin B2 (21), but also
by the presence of several endophilin B1 transcripts resulting from
alternative use of polyadenylation signals and alternative splicing
(Fig. 1). These transcripts yield endophilin B1 variants with a
widespread tissue distribution, i.e. endophilin B1a, as well
as a tissue-specific distribution such as the brain-specific variants
endophilin B1b and B1c (Fig. 1). Although the widespread tissue
distribution of endophilin B1a is consistent with a role of endophilin
B1 in ubiquitous membrane dynamics, our data do not exclude an
involvement of the brain-specific endophilin B1 variants B1b and B1c in
cell type-specific membrane dynamics such as those at synapses, which
have been reported to show endophilin B1 immunoreactivity (15).
Whatever the precise function of the endophilins B will be, it is
likely to be an essential one, because the endophilins B, like the
endophilins A, are conserved from yeast to humans (6, 11, 12, 15, 21,
22).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter assays. Positive clones were isolated by
re-streaking and growth on cycloheximide-containing medium and verified
for the interaction with PACSIN 1 by mating with Y187 yeast cells
transformed with pAS2-1 PACSIN 1. Plasmid DNA of positive clones was
isolated and sequenced in both directions with universal and internal
primers using the ABI Prism Big Dye Terminator Cycle Sequencing Ready
Reaction kit, and products were resolved on an ABI Prism 377 automated
sequencer (PerkinElmer Life Sciences/Applied Biosystems). DNA and
protein sequence analyses were performed using the GCG software package
(University of Wisconsin, Madison, WI), and multiple gene databases
were searched using BLAST programs (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
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Fig. 1.
Genomic organization, splice variants, and
tissue distribution of endophilin B1. A, sequence
comparison of the exon 6- and 7-containing region of murine
(m) endophilin B1b (accession number AF272946) with human
(h) endophilin B1a (accession number AF263293),
Drosophila (d) endophilin B1 (accession number
AE003796), human endophilin B2 (SH3GLB2, accession number BC014635
(21)), C. elegans (c) endophilin B1 (F35A5-8,
accession number U46675), and the murine endophilins A1, A2, and A3
(accession numbers U58886, U58885, and U58887, respectively). Identical
amino acids are shown with a black background, and
conservative substitutions, as defined by the BoxShade program, are
gray. Gaps in the sequences, needed to optimize the
alignment, are represented by dots. B, Western
blot of adult murine total tissue protein (80 µg per lane) using an
antiserum against the recombinant C-terminal half of murine endophilin
B1b. Open arrowhead, endophilin B1b (42 kDa); filled
arrowhead, endophilin B1a (40 kDa); asterisk,
additional endophilin B1 splice variant (43 kDa) distinct from
endophilin B1b (see "Results" and Fig. 1D).
C, genomic structure of human endophilin B1, showing the
position and size of the exons (black bars and
boxes) as well as several splice variants. Endophilin B1a
lacks exons 6 and 7; endophilins B1b and B1c contain both these exons,
but endophilin B1b uses a splice site within exon 6, i.e.
contains a short version of exon 6 (6s), whereas endophilin B1c
contains the long version of exon 6 (6l). The table lists the
exon-intron boundary sequences and the size of the known exons and
introns. D, RT-PCR using primers in exons 5 and 10 and
poly(A)+ RNA isolated from various mouse tissues. Note the
widespread tissue distribution of endophilin B1a and the brain-specific
expression of endophilins B1b and B1c.
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Fig. 2.
Protein interactions of endophilin B1.
A, a mouse brain detergent extract was incubated with
immobilized GST fusion proteins of mouse endophilins B1b, A1, and A3,
as well as GST as control. Bound proteins were analyzed by
immunoblotting. BE, an aliquot of the brain extract used as
starting material. Longer exposure of the synaptojanin and synapsin
immunoblots (comparable to the amphiphysin immunoblots) did not yield
detectable signals for endophilin B1 (data not shown). B,
proteins in a rat brain detergent extract were separated by SDS-PAGE
and blotted on PVDF membrane. Stripe aliquots of the blot were
incubated (i) either with GST (control) or GST fusion proteins
containing the SH3 domain of endophilin B1 (SH3-B1) or
endophilins A1-3 (SH3-A1/2/3), followed by detection of
bound GST fusion protein by immunoblotting using anti-GST antibody
(Overlay); (ii) or with antibodies against the indicated
proteins (Immunoblot). Open squares,
synaptojanin; filled squares, amphiphysin 1; filled
circles, dynamin; open circles, amphiphysin 2.
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Fig. 3.
Subcellular localization of endophilin
B1b. A, subcellular fractions from NIH 3T3 cells
transiently transfected with HA-tagged endophilin B1b were obtained by
differential centrifugation and analyzed by Western blotting using an
antibody against the HA tag of endophilin B1 (upper panel).
A post-nuclear supernatant of mouse brain was subjected to
discontinuous sucrose gradient centrifugation, and the fractions were
analyzed by Western blotting using antibodies against endophilin B1
(lower panel). The molarity of the various sucrose phases
and interfaces is indicated. P, pellet. Open
arrowhead, endophilin B1b (42 kDa); filled arrowhead,
endophilin B1a (40 kDa); diamond, additional endophilin B1
splice variant, presumably endophilin B1c. B, NIH 3T3 cells
transiently transfected with HA-tagged endophilin B1b were analyzed by
double immunofluorescence using an antibody against the HA tag
(green, left panel) and antibody recognizing
endogenous dynamin (red, middle panel).
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Fig. 4.
Transferrin uptake of endophilin
SH3-domain-transfected cells. NIH 3T3 cells were transiently
transfected with the SH3-domain of mouse endophilin A1, A2, and B1,
each fused to DsRed, or with DsRed alone as control. FITC-transferrin
uptake of DsRed-expressing cells was quantitated by FACS and is
expressed as the percentage of that observed for control cells. The
values are the mean of three independent experiments; bars, ± S.D.
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Fig. 5.
Lipid binding and LPA-AT activity of
endophilin B1. A, binding of endophilin B1 to
palmitoyl-CoA agarose. Detergent lysates of NIH 3T3 cells transfected
with HA-tagged endophilin B1b were incubated in duplicate with
palmitoyl-CoA agarose, and bound proteins were eluted in the absence
( ) and presence (+) of free palmitoyl-CoA. Aliquots of the lysate
(10%), the unbound material (10%), the protein bound to palmitoyl-CoA
agarose (50%), and the eluates (50%) were analyzed by Western
blotting using an antibody against the HA tag of endophilin B1.
B and C, LPA-AT activity of endophilin B1. LPA-AT
assays were performed in duplicate using either 500 nM
recombinant His-tagged murine endophilin A1, GST-murine endophilin B1b
fusion protein and GST in the presence of 5 µM
[14C]arachidonoyl-CoA and 10 µM LPA
(B), or 500 nM recombinant murine endophilin B1b
proteolytically cleaved off GST and His-tagged murine endophilin A1 in
the presence of 10 µM [14C]oleoyl-CoA and
the absence (
) and presence (+) of 10 µM LPA as
indicated (C). An unidentified radioactive compound only
present in preparations containing GST is indicated by the
asterisk in B.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
---|
We thank Renate Knaup for technical assistance with the FACS analysis.
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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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF263364, AF263293, and AF272946.
§ Recipient of a fellowship from the Fonds der Chemischen Industrie. Present address: Dept. of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06510.
Present address: Montreal Neurological Institute, McGill
University, 3801 University St., Montreal, Quebec H3A2B4, Canada.
Supported by grants from the Deutsche Forschungsgemeinschaft
(SPP GTPases, Hu 275/5-1) and the Fonds der Chemischen Industrie.
§§ Supported by grants from the Köln Fortune program of the Medical Faculty of the University of Cologne and the Center for Molecular Medicine Cologne (ZMMK; TP78). To whom correspondence should be addressed. Tel.: 49-221-478-6944; Fax: 49-221-478-6977; E-mail: markus.plomann@uni-koeln.de.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M208568200
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ABBREVIATIONS |
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The abbreviations used are: LPA, lysophosphatidic acid; EST, expressed sequence tag; SH3, src homology 3; UTR, untranslated region; AT, acyl transferase; GST, glutathione S-transferase; HA, hemagglutinin; LBM, lipid binding and modifying; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; CMV, cytomegalovirus; PVDF, polyvinylidene difluoride; RT, reverse transcription; PACSIN, PKC and CK2 substrate in neurons; PKC, protein kinase C; CKZ, casein kinase Z.
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