From the
Dynamin is a GTPase that appears to be required for endocytosis.
Even though this molecule is known to be in surface-coated pits, the
identity of the resident coat proteins that account for this
localization is not known. Here we show that dynamin is one of three
synaptic terminal proteins that bind with specificity to the appendage
domain of
Clathrin AP2 (adaptor) together with triskelion are the major
structural subunits of plasma membrane clathrin-coated pits
(1, 2) . AP2 is a hetero-oligomer composed of two
100-kDa adaptin molecules (
Rapid freeze, deep
etch images of the AP subunit show that it consists of a brick-shaped
core with two appendages that extend from either side
(5) . The
appendages correspond to the 300-amino acid COOH terminus of each
adaptin molecule. The core, by contrast, is constructed from the
remainder of the two adaptins (designated the trunk domain
(1) )
plus the other two protein components. The architecture of this
subunit, together with the sequence variability of the COOH terminus of
the adaptin molecule
(6) , suggests that the appendage domain
may interact with molecules that transiently associate with
clathrin-coated pits during endocytosis
(2) . So far there have
not been any molecules identified that bind to this domain. The trunk
domain, on the other hand, has been shown to interact with the
cytoplasmic tail of several receptors that cluster in coated pits
(7, 8, 9) . In addition, this domain is known to
bind tightly to synaptotagmin I
(4, 10) and to clathrin
(4, 11) .
A molecule that has emerged as an important
functional component of the clathrin-coated pit is dynamin
(12, 13) . The GTPase activity of this molecule appears
to be required for the conversion of a plasma membrane-coated pit into
a coated vesicle
(14, 15) . Furthermore, dynamin has
recently been found to bind tightly to several different signal
transducing molecules that contain SH3 domains
(16, 17, 18) . The protein responsible for
directing dynamin to coated pits is not known, but a likely candidate
is one of the adaptin molecules
(15) . We constructed fusion
proteins between glutathione S-transferase (GST)
To see if
We used immunoblotting to further characterize the various
proteins that bound to GST
The expression of
GTPase-deficient dynamin in fibroblasts has a dominant negative affect
on coated pit-mediated endocytosis
(14, 15) . Therefore,
we analyzed how nucleotides and ionic conditions affected the
association of dynamin with GST
Coated pit budding is arrested in the Drosophila shibire fly without there being any detectable change in the budding of
vesicles from the Golgi apparatus
(25, 26) . Therefore,
only surface-coated pits appear to require this protein to function.
Damke et al. (27) have shown that dynamin is located
in plasma membrane-coated pits but not coated pits in the Golgi
apparatus. The current results offer an explanation for this
selectivity. Dynamin bound directly to the
Several
laboratories have found that dynamin contains an SH3 binding domain
capable of interacting with phospholipase C
These results do not explain how GTPase-defective
dynamin prevents endocytosis, because GTP was not required for dynamin
binding to GST
In contrast to dynamin, the other
two proteins that were identified in the GST
The
third sequence from the 94-kDa protein matched a sequence in the
GenBankdata base with accession number U14913. This protein has no
known function.
We thank Dr. H.-C. Lin for assistance with the
expression and purification of rat brain dynamin 1a, Dr. Clive
Slaughter for doing the sequencing, and Dr. M. S. Robinson for
providing monoclonal antibodies against
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adaptin. Binding is sensitive to both salt and pH
levels but is not affected by nucleotides. Using recombinant dynamin
expressed in SF9 cells, we estimate that the binding affinity is
200 nM. Binding does not require GTP, and the GTPase
activity of dynamin is not stimulated by this interaction. These
results suggest that the COOH terminus of
-adaptin may be a domain
within AP2 that mediates the initial interactions between dynamin and
surface-coated pits. This may be an essential step in the regulation of
coated pit budding.
and
) plus one copy each of a 50-
and a 17-kDa protein
(1) . AP2 is only found in plasma
membrane-coated pits, but a similarly constructed subunit, called AP1,
serves the same purpose in coated pits of the Golgi apparatus
(1) . One of the chief differences between the two subunits is
that the
- and
-adaptins are replaced in the AP1 subunit with
- and
`-adaptins. A major function of both AP subunits is to
provide a linkage between the triskelion lattice and the membranes of
coated pits and vesicles
(3, 4) .
(
)
and three different domains within the
- and
-adaptins to see if any other of these proteins would interact
with dynamin as well as other cytosolic proteins in brain extracts. We
found that dynamin binds with specificity and high affinity to the
appendage domain of
-adaptin.
Materials
The following reagents were obtained
from Sigma: ampicillin (A-9518), benzamidine (B-6506), HEPES (H-3375),
EDTA (ED2SS), glutathione (G-4251), glutathione-agarose (G-4510),
magnesium chloride ), potassium chloride (P-3911), pepstatin A
(P-4265), sodium chloride (S-9625), Tween 20 (P-1379), Triton X-100
(X-100), trichloroacetic acid (T-6399), monoclonal anti--adaptin
(clone 100/3, A-4200), and monoclonal anti-assembly protein AP180
(clone AP180-I, A-4825). Phenylmethanesulfonyl fluoride was purchased
from Boehringer Mannheim. 1,4-Dithio-DL-threitol was from
Fluka Chemical Corp. (Ronkonkomam, NY). Leupeptin was obtained from
Peptide Institute Inc. (Osaka, Japan).
Isopropyl-
-D-thiogalactopyranoside was purchased from
Stratagene (La Jolla, CA). Horseradish peroxidase-conjugated goat IgG
fractions against mouse IgG (
(50) or goat IgG fractions against
rabbit IgG (55676) were from Organon Teknika Corp. (Durham, NC). The
ECL detection kit was purchased from Amersham Corp. Prokaryotic
expression vector pGEX2T was obtained from Pharmacia Biotech Inc. The
TA cloning kit was from Invitrogen Corp. (San Diego, CA). Reagents used
for bacterial culture were purchased from Difco. All restriction
enzymes were obtained from Promega Corp. (Madison, WI). The reagents
and apparatus for acrylamide gel electrophoresis were purchased from
Bio-Rad. The Immobilon-P blotting membrane was obtained from Millipore
Corp. (Bedford, MA). The agarose and the equipment for agarose gel
electrophoresis were from Life Technologies, Inc. Monoclonal antibodies
that recognize
-adaptin (AC1M11) and full-length mouse cDNAs of
- and
-adaptin were kindly provided by Dr.
Margaret S. Robinson from the University of Cambridge, UK. Monoclonal
IgG against clathrin (OZ-71) and polyclonal antisera against dynamin
were prepared as previously described
(19, 32) .
Buffers
The cytosol buffer contained 20
mM HEPES, pH 7.2, 100 mM KCl, 2 mM
MgCl, and 1 mM dithiothreitol. The protease
inhibitor mixture was 10 µM leupeptin, 1 µg/ml
pepstatin A, 0.5 mM benzamidine, and 1 mM
phenylmethanesulfonyl fluoride in cytosol buffer. Tris-buffered saline
included 20 mM Tris and 137 mM NaCl, pH 7.6. The
antibody buffer contained Tris-buffered saline plus 1% dry milk and
0.1% Tween 20.
Preparation of Fusion Proteins
The construction of
GST fusion proteins containing different adaptin domains was performed
according to standard methods. The appropriate regions of cDNA for each
fusion protein were amplified from plasmids containing a full-length
cDNA insert of - or
-adaptin by using polymerase
chain reaction. The sequences of the first seven and the last seven
codons of each segment were synthesized as the primers. The names and
inserted regions of the three fusion proteins used in this experiment
are: GST
T, amino acids 1-620 of
-adaptin;
GST
A, amino acids 701-938 of
-adaptin; and
GST
A, amino acids 704-822 of
-adaptin. Amplified
fragments were subcloned into the pCR II cloning vector, and the
EcoRI fragments were subsequently transferred into the pGEX2T
expression vector. The plasmids containing the cDNAs encoding for all
fusion proteins were then transformed into either BL21 or HB101 Escherichia coli strains. The fusion proteins were
induced and attached to the affinity matrix as described below. A 10-ml
aliquot of the overnight culture was inoculated into 1 liter of fresh
LB medium containing 80 µg/ml ampicillin and grown at 37 °C
with vigorous shaking. When the density of the culture reached about
A
0.6, the flasks were transferred to room
temperature, and the production of fusion proteins was induced by
adding isopropyl-
-D-thiogalactopyranoside to a final
concentration of 50 µM for another 4 h with shaking. The
bacteria were harvested by centrifuging at 4000
g for
10 min at 4 °C, and the resulting pellet was resuspended in 40 ml
of ice-cold cytosol buffer. The suspension was then sonicated for four
15-s pulses with a half-inch probe and with the power setting at 3
(Ultrasonic Processor model W-380, Heat System-Ultrasonics Inc.). The
debris was removed by spinning the sonicated lysate at 12,000
g for 20 min at 4 °C. The supernatant was carefully
transferred to a new tube and supplemented with Triton X-100 to a final
concentration of 1%. The resulting supernatant was incubated with 1.5
ml of preswollen glutathione-agarose beads for at least 2 h at 4 °C
with tumbling. The beads were then washed three times by repeated
centrifugation and resuspension with 40 ml of cytosol buffer containing
0.1% Triton X-100. After the third wash, the beads with attached fusion
proteins were stored in the form of 50% slurry at 4 °C. The fusion
proteins remained stable without any degradation for at least 1 month.
Preparation of Cellular Extract
Bovine brains were
obtained from a local slaughterhouse. After removing meninges, whole
brain tissue was dissected into small pieces and snap-frozen in liquid
nitrogen. All the following procedures were performed at 4 °C.
Bovine brain membrane extract was prepared by homogenizing frozen brain
tissue in cytosol buffer containing the protease inhibitor mixture at a
ratio of 1 ml of buffer to 1 g (wet weight) of tissue. The mixture was
centrifuged for 1 h at 100,000 g at 4 °C, and the
pellet was resuspended in the same volume of fresh cytosol buffer.
Triton X-100 was added slowly with stirring to a final concentration of
1%, and then the mixture was incubated at 4 °C for 1 h. The mixture
was centrifuged at 100,000
g for 1 h at 4 °C, and
the supernatant fraction was collected (designated brain extract). The
protein concentration of this extract was approximately 15 mg/ml.
Identification of AP Binding Proteins
The membrane
extract was precleared by incubating each fraction of brain extract
with 75 µl of packed glutathione-agarose beads for 4 h at 4 °C.
The beads were removed by centrifugation, and 75 µl of beads
containing the indicated fusion proteins was added. The mixture was
then incubated overnight at 4 °C with tumbling. The
glutathione-agarose beads were recovered by spinning at 8500
g for 2 min at 4 °C. The beads were washed 6 times by
repeated resuspension and centrifugation with 1 ml of cytosol buffer
containing 0.1% Triton X-100. Bound proteins were eluted by incubating
beads in 650 µl of 50 mM glutathione in cytosol buffer for
10 min and spinning at 8500
g to remove the beads. The
supernatant fraction was precipitated with trichloroacetic acid, and
one-fifth of each sample was analyzed by electrophoresis on a 10% SDS
gel. After separation, proteins were visualized by Coomassie Blue R-250
staining.
Electrophoresis and Immunoblots
Protein samples
were separated by SDS-polyacrylamide gel electrophoresis and then
transferred to Immobilon-P polyvinylidene difluoride membranes. After
transferring, the blots were preblocked for 60 min with 5% dry milk
(Carnation) in Tris-buffered saline containing 0.5% Tween 20. Both
primary antibody and secondary antibody were diluted in antibody
buffer. Anti-dynamin rabbit polyclonal antiserum (E765) was diluted at
a ratio of 1/2,000. AC1M11, AP180-1, and 100/3 ascites fluids
were used at dilutions of 1/2,000, 1/15,000, and 1/200, respectively.
OZ71-purified IgG was used at a concentration of 20 µg/ml. The
preblocked membranes were rinsed twice with antibody buffer and
incubated with primary antibody solutions for 1-2 h at room
temperature. The appropriate second antibody coupled to horseradish
peroxidase was used at a dilution of 1/30,000 and detected by enhanced
chemiluminescence reagents. The relative amount of dynamin was
determined by densitometry with a personal densitometer (Molecular
Dynamics).
Peptide Microsequencing
Proteins eluted from the
GSTA fusion proteins were transferred onto polyvinylidene
difluoride membranes. The membranes were stained with Ponceau S. The
bands were then excised and digested in situ with Lyc-C
endoprotease. The resulting peptides were separated by reverse phase
high pressure liquid chromatography and sequenced using an automated
protein sequenator.
Other Methods
Protein concentration was determined
with Bio-Rad Bradford protein assay reagent using bovine serum albumin
as the standard.
-adaptin may be involved in targeting dynamin
to surface-coated pits, we constructed plasmids that express either the
-adaptin appendage (GST
A) and trunk (GST
T) domains or
the
-adaptin appendage (GST
A) domain fused to glutathione
S-transferase. Either GST alone or each of the fusion proteins
was then attached to glutathione beads. Brain is a rich source of
dynamin
(20) , so we incubated the beads overnight at 4 °C
in the presence or absence of 15 mg/ml Triton X-100 bovine brain
extract (Fig. 1 A). The beads were washed with 50
mM glutathione, and the eluted proteins were separated by gel
electrophoresis before staining with Coomassie Blue. Each bead
contained approximately the same amount of fusion protein
(Fig. 1 A, heavy band in each lane).
GST alone did not bind any proteins (Fig. 1 A, lanes
1 and 2). GST
A specifically bound four proteins with
molecular weights of 180,000, 120,000, 100,000, and 94,000
(Fig. 1 A, lane 4). GST
A, by contrast,
bound specifically a 180-kDa protein plus a cluster of proteins that
migrated at about 100 kDa (Fig. 1 A, lane 6).
GST
T did not bind any proteins in this molecular weight range.
Therefore, the plasma membrane- and Golgi-specific appendage domains
appeared to form a complex with different sets of proteins present in
the brain extract.
Figure 1:
Identification of proteins that bind
selectively to the appendage domain of - and
-adaptin. The
indicated GST fusion protein was attached to glutathione-agarose beads,
incubated in the presence (+) or absence (
) of a Triton
X-100 brain extract (15 mg/ml) and then eluted with 50 mM
glutathione. A, Coomassie Blue staining of the eluted
fractions from each of the indicated beads. B, sequence
analysis of four prominent bands that bound to the GST
A. We
compared the bovine sequences obtained with those sequences in data
base and found perfect matches between the 180-kDa band and mouse AP180
( top sequence, residues 135-149), the 120-kDa band and
chicken amphiphysin ( middle sequence, residues 31-47),
and the 100-kDa band and rat dynamin ( bottom sequence,
residues 90-107).
We used microsequencing to identify the four
proteins that were in the GSTA complex (Fig. 1 B).
Each of the bands was cut from the gel and sequenced by standard
methods. We obtained multiple sequences () from each
protein (legend to Fig. 1 B) and identified the 180-kDa
band as AP180
(21, 22) , the 120-kDa band as amphiphysin
(23) , and the 100-kDa band as dynamin
(20) . One of the
sequences obtained from the 94-kDa band matched a sequence in a yeast
protein of unknown function (GenBank
accession number
U14913).
A, GST
T, and GST
A
(Fig. 2). Anti-dynamin IgG reacted with the 100-kDa band in the
GST
A complex (Fig. 2, lane 2). This antibody did
not blot any of the proteins that bound to either GST
T or
GST
A (Fig. 2, lanes 3 and 4). When we
blotted with an anti-AP180 IgG, we found a strongly reactive band in
the GST
A complex (Fig. 2, lane 14) but very little
reactivity in the GST
A complex (Fig. 2, lane 16).
Because a protein of similar molecular weight was associated with
GST
A (Fig. 1 A, lane 8), we immunoblotted
each lane with anti-clathrin IgG. This antibody recognized the 180-kDa
band in the GST
A complex (Fig. 2, lane 20).
Anti-clathrin IgG did not bind to any of the proteins in the GST
A
complex (Fig. 2, lane 18).
Figure 2:
Immunoblot identification of proteins that
bind to appendage domain of adaptin fusion proteins. The indicated
fusion protein was incubated in the presence of brain membrane extract
and washed with glutathione, and the eluted proteins were separated by
gel electrophoresis. The indicated antibodies were then used to
immunoblot each of the fractions.
The presence of clathrin
in the GSTA complex suggested that the cluster of associated
proteins in the 100,000 molecular weight region (Fig. 1, lane
8) might correspond to the adaptins. Therefore, we blotted each of
the four fusion protein complexes with either anti-
-adaptin IgG or
anti-
-adaptin IgG. Surprisingly, both antibodies recognized the
bands associated with GST
A (Fig. 2, lanes 8 and
12). There were no immunoreactive bands in either the
GST
A or the GST
T lane (Fig. 2, lanes 6,
7, 10, and 11). The differential intensity
of the bands suggested that the GST
A complex bound more
-adaptin than
-adaptin. These results indicate that the
-appendage domain selectively interacts with APs but is not able
to discriminate between AP2 and AP1.
A. The addition of GTP, GTP
S,
AMP-PNP, ATP
S, or ATP to the brain extract had no effect on the
binding of either dynamin or the other proteins in the complex
(Fig. 3 A, upper and lower panels). We
also found that GTP did not stimulate the dissociation of dynamin that
had been prebound to GST
A (data not shown). On the other hand,
when the salt concentration of the extract was increased, there was a
reciprocal decline in the amount of dynamin that bound to the beads
(Fig. 3 B). Above 400 mM KCl, dynamin did not
bind at all (Fig. 3 B, lower panel). The binding
of the other three proteins, however, was relatively insensitive to
salt concentration (Fig. 3 B, upper panel). We
also found that dynamin binding was extremely sensitive to pH levels
(Fig. 3 C). Good binding was observed at neutral pH but
rapidly declined as the pH was lowered (Fig. 3 C,
lower panel). At pH 6.0, there was more than a 90% decrease in
the amount of bound dynamin. pH levels had little effect on the binding
of the other proteins in the complex (Fig. 3 C, upper
panel). Both pH and salt were also found to dissociate dynamin
that had been prebound to GST
A (data not shown).
Figure 3:
Effects of nucleotides ( A), ionic
strength ( B), and pH ( C) on the binding of dynamin to
the -adaptin appendage domain. GST
A was attached to
glutathione-agarose beads and incubated with brain membrane extract
that had the indicated nucleotide ( A), KCl concentration
( B), or pH ( C) as labeled at the top of each
lane in the upper panels. The bound proteins were
eluted with glutathione and analyzed by electrophoresis. The gels were
either stained by Coomassie Blue ( upper panel) or transferred
and immunoblotted with dynamin-specific antiserum ( lower
panel). The relative amount of bound dynamin was quantified by
densitometry ( lower panel).
As a final
test of binding specificity, we measured the affinity of interaction
between dynamin and the GSTA. The fusion protein was attached to
beads and incubated in the presence of different concentrations of
brain extract (Fig. 4 A). As the amount of extract
increased, there was an increase in the amount of dynamin that bound.
Binding began to saturate at an extract concentration of 12-15
mg/ml. We estimate from this curve that half-maximal binding occurred
at 5 mg/ml extract. Because 0.4% of the brain protein is dynamin
(24) , this corresponds to a dynamin concentration of
2
10
M.
Figure 4:
High affinity interaction of dynamin with
the appendage domain. GST
A was immobilized on beads and
incubated in the presence of the indicated concentration of either
brain extract ( A) or recombinant rat dynamin I ( B).
Bound proteins were eluted from the beads, separated by gel
electrophoresis, and immunoblotted with anti-dynamin antiserum. The
dynamin band in each immunoblot was then
quantified.
Dynamin could be binding
directly to the appendage or be linked to the domain by one or more of
the other three proteins present in the GSTA complex. To
distinguish between these possibilities, we expressed rat brain dynamin
I fused to polyhistidine in SF9 cells and purified the protein. We then
incubated GST
A in the presence of various concentrations of the
fusion protein and assayed for binding. Fig. 4 B shows
that the binding of dynamin I was concentration-dependent and that
half-maximal binding occurred at a concentration of
2
10
M (Fig. 4 B).
Polyhistidine-tagged dynamin did not bind to GST
A (data not
shown). Therefore, the affinity of interaction between GST
A and
either the pure protein or the cytosolic dynamin is similar. This
suggests that neither a cofactor nor a covalent modification is
required for the direct binding of dynamin to the
-appendage of
AP2.
-appendage and did not
require the presence of the other proteins in the GST
A complex for
binding. Other parts of the
-adaptin molecule did not interact nor
did the
-adaptin appendage domain. Although other combinations of
coated pit proteins need to be tested to be sure, the high affinity and
specificity of binding strongly suggest that
-adaptin is
responsible for targeting dynamin to surface-coated pits.
, GRB-2, and
phosphatidylinositol 3`-kinase
(16, 17, 18) .
There is also evidence that amphiphysin, one of the other proteins in
the GST
A complex, is an SH3-containing protein that binds dynamin
(28) . The presence of dynamin in coated pits places it in a key
location to interact with the receptors that bind these SH3-containing,
signal-transducing proteins
(29) . In this location, dynamin
could function as an exchange factor that removes SH3-containing
molecules from the cytoplasmic tails of receptors being internalized by
coated pits. Therefore, dynamin may be essential for turning off signal
transduction as the receptor-hormone complex moves to the lysosome for
degradation.
A. The GTPase activity of the dynamin must be
required for other steps in the budding reaction. Most likely
-adaptin functions as a tethering device that holds dynamin in the
proper location to optimize its interaction with other molecules
required to complete the endocytosis cascade. We do not know the exact
orientation of the AP2 subunit in the coated pit
(30) , but the
appendage domain could be quite close to the membrane. This would place
dynamin in an optimal position to interact with membrane lipids and
proteins that might be passing by.
A complex appear to be
brain-specific. Because they are abundant in synapses, they must play a
specific role in the endocytosis of synaptic vesicle membrane. A
special feature of endocytosis at the synapse is the requirement for
extremely rapid membrane retrieval
(31) . Therefore, amphiphysin
and AP180 may be molecules that accelerate the budding of coated pits
at these sites.
Table: Sequence and identity of the four proteins
that bind to the -appendage domain of AP2
S, guanosine
5`-3- O-(thio)triphosphate; AMP-PNP, 5`-adenylyl
,
-imidodiphosphate; ATP
S, adenosine
5`- O-(thiotriphosphate).
-adaptin and the
full-length cDNA for
- and
-adaptin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.