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The Journal of Neuroscience, December 1, 2000, 20(23):8667-8676
A Conserved Clathrin Assembly Motif Essential for Synaptic
Vesicle Endocytosis
Jennifer R.
Morgan1, 3,
Kondury
Prasad2, 3,
Weihua
Hao2,
George J.
Augustine1, 3, and
Eileen M.
Lafer2, 3
1 Department of Neurobiology, Duke University Medical
Center, Durham, North Carolina 27710, 2 Department of
Biochemistry, University of Texas Health Science Center at San Antonio,
San Antonio, Texas, 78229, and 3 Marine Biological
Laboratory, Woods Hole, Massachusetts 02543
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ABSTRACT |
Although clathrin assembly by adaptor proteins (APs) plays a major
role in the recycling of synaptic vesicles, the molecular mechanism
that allows APs to assemble clathrin is poorly understood. Here we
demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal
domain of clathrin. Sequence analysis reveals a motif, containing the
sequence DLL, that exists in multiple copies in many clathrin APs.
Progressive deletion of these motifs caused a gradual reduction in the
ability of AP180 to assemble clathrin in vitro. Peptides
from AP180 or AP-2 containing this motif also competitively inhibited
clathrin assembly by either protein. Microinjection of these peptides
into squid giant presynaptic terminals reversibly blocked synaptic
transmission and inhibited synaptic vesicle endocytosis by preventing
coated pit formation at the plasma membrane. These results indicate
that the DLL motif confers clathrin assembly properties to AP180 and
AP-2 and, perhaps, to other APs. We propose that APs promote clathrin
assembly by cross-linking clathrin triskelia via multivalent
interactions between repeated DLL motifs in the APs and complementary
binding sites on the N-terminal domain of clathrin. These results
reveal the structural basis for clathrin assembly and provide novel
insights into the molecular mechanism of clathrin-mediated synaptic
vesicle endocytosis.
Key words:
AP180; AP-2; clathrin adaptors; coated vesicles; membrane
trafficking; presynaptic terminals; synaptic transmission
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INTRODUCTION |
Clathrin is important for membrane
trafficking in all eukaryotic cells (Goldstein et al., 1985 ; Pearse,
1988; Schmid, 1997). Clathrin assembles into cages that facilitate the
budding of membranes into smaller vesicles. Both clathrin assembly and
the connection of this cage to budding membranes are mediated by
clathrin adaptor/assembly proteins (APs; Keen et al., 1979;
Kirchhausen, 1999). These clathrin APs exist in various cellular
compartments and are separated into two structurally distinct classes:
tetrameric and monomeric APs. Tetrameric APs have two large subunits
( / / / and  ), a medium subunit (µ), and a small subunit
( ) (Keen, 1987; Ahle et al., 1988; Matsui and Kirchhausen, 1990).
AP-1 is involved in vesicle trafficking from the trans-Golgi
(Robinson, 1987; Ahle et al., 1988). AP-2 mediates protein sorting and
endocytosis from the plasma membrane of all cells, including neurons
(Robinson, 1987; Ahle et al., 1988; Gonzalez-Gaitan and Jackle, 1997;
Kirchhausen et al., 1997). AP-3 mediates synaptic vesicle formation
from endosomes and protein transport to vacuoles (Cowles et al., 1997;
Dell'Angelica et al., 1998; Faundez et al., 1998). No function has
been assigned to AP-4, although its cellular localization suggests a
role in trans-Golgi transport (Dell'Angelica et al., 1999).
Monomeric APs include AP180 and clathrin assembly lymphoid myeloid
leukemia protein (CALM; Ahle and Ungewickell, 1986; Kohtz and Puszkin, 1988; Murphy et al., 1991; Sousa et al., 1992; Zhou et al., 1992, 1993;
Tebar et al., 1999). AP180 is a synapse-specific protein that is
important for synaptic vesicle endocytosis (Zhang et al., 1998;
McMahon, 1999; Morgan et al., 1999; Nonet et al., 1999). CALM is a
ubiquitously expressed AP180 homolog that also may be involved in the
formation of clathrin-coated pits (Dreyling et al., 1996; Tebar et al.,
1999).
What remains to be determined is how these structurally diverse APs
promote clathrin assembly. Clathrin binding regions of the 2 subunit
of AP-2 and of the 3A subunit of AP-3 have been mapped to amino
acids 616-663 (Shih et al., 1995) and 630-638 (Dell'Angelica et al.,
1998), respectively. Alignment of these regions with other clathrin
binding proteins suggested that the sequence L(L,I)(D,E,N)(L,F)(D,E)
defines the core of a clathrin binding site, referred to as the
"clathrin box" (Dell'Angelica et al., 1998; ter Haar et al.,
2000). Here we extend this work with the discovery that the large
subunits of tetrameric APs and monomeric AP180 contain
multiple copies of a similar clathrin binding element. The
element is ~23 amino acids long and comprises both a central core
motif of DLL (for AP-1, AP-2, and AP180) or SLL (for AP-3 and AP-4) and
surrounding conserved residues. We provide several lines of evidence
that DLL motifs confer clathrin assembly properties on APs. The
clathrin assembly domain of AP180 containing 12 DLL motifs binds
directly to the N-terminal domain of the clathrin heavy chain.
Progressive deletion of the DLL motifs in AP180 quantitatively reduces
the clathrin assembly activity according to the number of remaining
motifs. Peptides containing the DLL motif competitively inhibit both
AP180- and AP-2-mediated clathrin assembly in vitro and
synaptic transmission and synaptic vesicle endocytosis in
vivo. Because DLL motifs are necessary for clathrin assembly and
are present in multiple copies in many APs, we propose that they allow
APs to assemble clathrin coats by cross-linking adjacent clathrin triskelia.
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MATERIALS AND METHODS |
Expression and purification of recombinant proteins.
The N-terminal domain [TD; amino acids (aa) 1-494] of the rat
clathrin heavy chain (construct from T. Kirchhausen, Harvard
University, Cambridge, MA) was expressed recombinantly in
Escherichia coli BL21 cells and purified as described (ter
Haar et al., 1998). Constructs expressing full-length AP180 (mouse
AP180, aa 1-901) and deletion constructs C58 (aa 305-901), C42 (aa
469-901), C38 (aa 511-901), C27 (aa 623-901), C22 (aa 681-901), and
C16 (aa 739-901) were prepared as described previously (Hao et al.,
1999). C51 (aa 371-901) and C49 (aa 392-901) were made by PCR
with the following sense primers:
5'-AGCTTCGGTCGACTCTCTGGGGGTGCGACCGCC-3' (C51),
5'-AGCTTCGGTCGACTCGTTCCCTGTGAAGCACCG-3' (C49), and a common antisense primer, 5'-GCTGAAATGCGGCCGCTCTTACAAGAAATCCTT-3'. A
SalI restriction site was designed in the sense primers, and
a NotI site was designed in the antisense primer. In all of
the reactions, plasmid pGEX3X-F1-20 (AS15-) was used as the template
(Ye and Lafer, 1995a). The PCR products were subcloned into the
pGEX4T-1 expression vector (Pharmacia, Piscataway, NJ) at the
SalI/NotI site. The recombinant proteins were
expressed in BL21 cells and purified under the same conditions as
described previously for glutathione S-transferase
(GST)-AP180 (Ye and Lafer, 1995a). GST was removed by cleavage with
either Factor Xa or thrombin for the experiments displayed in Figure
3.
Surface plasmon resonance. Experiments were performed on a
BIAcore 2000 Surface Plasmon Resonance instrument, using CM5 research grade chips (BIAcore, Piscataway, NJ). The experiments were performed at 25°C with HEPES-buffered saline [HBS; containing (in
mM): 10 HEPES, 150 NaCl, and 3 EDTA plus 0.005% surfactant
P20, pH 7.0] as running buffer at a flow rate of 5 µl/min. The
surfaces of research grade CM5 chips were activated by a 6 min
injection of a solution containing 50%
N-ethyl-N'-(dimethylaminopropyl) carbodiimide and
50% N-hydroxy-succinimide. GST-C58 (3.6 kRU, where 1 kRU = 1000 response units = a change of ~1
ng/mm2 in surface protein concentration)
was immobilized on a chip at pH 4.7, and GST (3.4 kRU) was immobilized
at pH 5.0. After immobilization, each surface was blocked by a 6 min
injection of 1 M ethanolamine at pH 8.5. TD was
passed over the surfaces for 3 min at the indicated concentrations,
followed by a 4 min injection of HBS. Then the surfaces were
regenerated by a 3 min injection of 4 M
guanidine-HCl.
Sequence analysis. The self-homology plot of AP180 in Figure
2A was generated with PLALIGN (Huang and Miller,
1991). The multiple sequence alignment in Figure 2B
was generated with Megalign running under the Lasergene software
package (DNAStar, Madison, WI), using the Clustal method (Thompson et
al., 1994) and the PAM250 residue weight table. The alignments
displayed in Figure 2B included the following
sequences, with the indicated accession numbers: mouse AP180 (M83985),
rat 1 adaptin (M77245), rat 2 adaptin (M77246), mouse A
adaptin (X14971), mouse C adaptin (X14972), mouse adaptin
(X54424), human 3A adaptin (U91931), human 3B adaptin (U37673),
human 4 adaptin (AF092094), and human adaptin (U91930). Residues
were grouped according to a structural similarity consensus that
considers both the physical/chemical properties and the size/shape of
the residues. Residue groupings of the consensus are: D/E, H/K/R, N/Q,
W/Y, S/T, F/L/I/M, and A/G/V, P, C. Statistical analysis of the
AP180/AP-1/AP-2 alignment revealed that the scored motifs displayed an
average identity to the structural similarity consensus of 48% (range
30-70%). Statistical analysis of the AP-3/AP-4 alignment revealed
that the scored motifs displayed an average identity to the structural similarity consensus of 45% (range 35-57%).
Purification of coat proteins and clathrin assembly assays.
Clathrin, AP180, and AP-2 were purified from bovine brain as
described previously (Prasad and Lippoldt, 1988; Hao et al., 1999). The assembly of bovine brain clathrin into coats was measured by
ultracentrifugation in the presence of the indicated clathrin APs
(Morgan et al., 1999). Briefly, each AP was combined with
clathrin in 10 mM Tris-HCl, pH 8.5, and clathrin assembly
was initiated by adding 0.1 vol of 1 M MES-NaOH, pH 6.7. The final conditions in the assembly reaction were 0.5 µM
clathrin, 0.1 M MES-NaOH, 9 mM Tris-HCl, pH
6.7, and the indicated concentrations of APs in a volume of 200 µl.
The mixture was incubated on ice for 45 min and then was centrifuged at
400,000 × g at 4°C for 6 min. The upper 80% of the
supernatant was removed and analyzed by SDS-PAGE and Coomassie blue
staining. The percentage of clathrin that was assembled was determined
by the relative depletion of clathrin from the supernatants of the
centrifuged solutions before and after assembly. This was quantified by
densitometry of Coomassie blue-stained gels (Molecular Dynamics
Personal Densitometer SI, Sunnyvale, CA), using Image Quant software.
In the absence of APs, ~5% of the clathrin sedimented, and this
background clathrin assembly was subtracted from all data. Inhibitory
peptides were designed from mouse AP180 and rat AP-2 (2; see Table
1 for amino acid sequences). The effects of these peptides were examined under the following conditions: 0.5 µM clathrin, 1.0-1.2
µM AP180 or 1.5-2.0 µM
AP-2, 0.1 M MES-NaOH, 9 mM
Tris-HCl, pH 6.7, and 0-1000 µM peptide. The
amount of clathrin that was assembled was determined as described
above. Clathrin assembled in the presence of AP180 or AP-2, but without
peptides, was in the range of 50-55%. Inhibition by the peptides was
calculated as the relative amount of assembly in the presence of the
peptides as compared with the assembly in the absence of the peptides. Peptides were synthesized at the Institutional Protein Core Facility of
the University of Texas Health Science Center (San Antonio, TX). The
formation of clathrin coats and the inhibition of their formation by
peptides was assessed by electron microscopy, as described previously
(Ye and Lafer, 1995b). Assembled clathrin coat structures were applied
to freshly glow-discharged Formvar carbon-coated grids and negatively
stained with 1% uranyl acetate. The specimens were viewed in a JEOL
1200 EX Transmission electron microscope (Peabody, MA) at a
magnification of 72,400×.
Physiological methods. Electrical measurements were made on
giant synapses in isolated stellate ganglia of the squid, Loligo pealei (Morgan et al., 1999). Ganglia were superfused with
oxygenated physiological saline at 10-15°C containing (in
mM): 466 NaCl, 54 MgCl2, 11 CaCl2, 10 KCl, 3 NaHCO3,
and 10 HEPES, pH 7.2. Electrodes filled with 3 M
KCl were inserted into the presynaptic axon and the postsynaptic axon
for the purposes of stimulating synaptic transmission and recording
transmitter release, respectively. Synaptic transmission was evoked by
injecting a current pulse (0.7-1.9 µA) every 30 sec (0.03 Hz) into
the presynaptic axon to elicit single action potentials. Transmitter
release was measured by recording the initial rate of rise of
postsynaptic potentials. Data were acquired via an Axoclamp-2A
amplifier and analyzed with Axobasic programs written by Dr. Felix E. Schweizer (UCLA, Los Angeles, CA).
For microinjection the peptides were dissolved in physiological squid
saline containing (in mM): 250 K-isothionate, 100 KCl, 100 taurine, and 50 HEPES, pH 7.4. These solutions were delivered from a
microinjection pipette by delivering pulses of positive pressure
(10-80 msec; 10-100 psi; N2 gas) from a
Picospritzer injector (General Valve, NJ). Peptides were coinjected
with FITC-dextran (3 kDa molecular weight; 100 µM;
Molecular Probes, Eugene, OR) to monitor the intraterminal peptide
concentration. Fluorescence was imaged with a Zeiss Axioskop (10×,
0.25 numerical aperture objective; Oberkochen, Germany) and detected
with a Cohu SIT camera attached to an image processor (Image-1,
Universal Imaging, West Chester, PA).
To correlate the actions of AP2 peptides and mutants on clathrin
assembly and synaptic transmission (see Fig. 7D), we
chose a standard concentration at which to compare both the
inhibition of clathrin assembly in vitro and the inhibition
of synaptic transmission in vivo by each peptide. For
clathrin assembly by AP-2, the standard concentration chosen was 0.3 mM, the IC50 of AP2 pep for
AP-2-mediated assembly (see Table 1). For clathrin assembly by AP180,
the standard concentration chosen was 0.4 mM, the
IC50 of AP2 peptide on AP180-mediated clathrin
assembly. The standard concentration for examining the effect of AP2
peptide and mutants on synaptic transmission was 2.3 mM, the IC50 for inhibition
of synaptic transmission by AP2 pep (see Fig. 7C). The mean
inhibitory effects of each peptide in the in vitro and
in vivo assays were measured at the standard concentrations
and plotted as shown in Figure 7D.
Electron microscopy. Terminals were fixed with 2.5%
glutaraldehyde and processed for electron microscopy as described in
Sanchez et al. (1990). An 80-nm-thick section was taken every 50 µm
through the entire length of the synaptic terminal. Each section was
magnified 12,000× and examined with a JEM-1200 ExII (JEOL) electron
microscope. All sections from peptide-injected and control terminals
were analyzed with Image-1 software (Universal Imaging).
Distances between the plasma membrane and centers of individual
synaptic vesicles or coated vesicles were measured as described previously (Hess et al., 1993). Because active zones typically are
spaced ~1 µm apart, we measured vesicles within 500 nm of the
active zone to ensure that each vesicle was counted in association with
only one active zone. The plasma membrane perimeter of each terminal
was measured from low-magnification (500×) micrographs (Burns et al.,
1998). Such measurements underestimate plasma membrane area because of
small invaginations (Morgan et al., 1999). To determine the magnitude
of this effect, we measured the perimeter of small regions of the
plasma membrane in high-magnification images (12,000×) and used it to
correct perimeter measurements made at low magnification. Plasma
membrane perimeter was multiplied by the thickness of the section (80 nm) to calculate the surface area of plasma membrane in each section.
The surface area of synaptic and coated vesicles was calculated as
4 r2, where r is
the measured radius of these vesicles. Surface area per vesicle was
multiplied by the mean number of vesicles per active zone, yielding the
amount of vesicle area per active zone, and multiplied by the number of
active zones per section to yield the mean surface area in synaptic and
coated vesicles in each section. Vesicular structures that were
100-200 nm in diameter were counted as vesicular endosomes (Burns et
al., 1998). Because their diameters were larger than the thickness of
the section, their surface area was calculated as 2r ,
where is the thickness of the section (80 nm).
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RESULTS |
AP180 binds to the N-terminal domain of clathrin heavy chain
Recent work has demonstrated that both an AP-2 2 peptide and an
AP-3 3A peptide interact directly with the N-terminal domain (TD) of
clathrin heavy chain (ter Haar et al., 2000). However, the
identification of a binding site for AP180 on clathrin has been
problematic. Previous studies revealed that "clipped cages" from
which the TD was removed by proteolysis lost the ability to bind to
AP180, suggesting that AP180 may bind to the TD. However, this may not
be the case because the proteolytically released TD did not bind
measurably to AP180 (Murphy and Keen, 1992). We used recombinant TD and
sensitive surface plasmon resonance (SPR) detection to evaluate the
binding interaction between AP180 and the TD of clathrin. The clathrin
assembly domain of AP180 (C58, aa 305-901), fused to GST, was
immobilized on an SPR chip. As a control, GST was immobilized on a
second surface. Purified recombinant TD was passed over the two
surfaces, and the refractive index at the surfaces was monitored
continuously. The binding of TD to GST-C58 is expected to change the
surface refractive index in proportion to the mass of TD that is bound.
When TD was passed over the GST-C58 surface, a time-dependent increase
of the response was observed, indicating that TD was binding to GST-C58
(Fig. 1A). When the TD
solution was replaced by protein-free buffer, a slow dissociation of
the bound TD was observed. This is in contrast to the small SPR signal
observed while TD passed over the GST surface, presumably attributable
to changes in solution flow (Fig. 1A). Furthermore,
SPR analysis indicated that TD specifically bound to C58 in a
dose-dependent manner (Fig. 1B). Similar results were
obtained when full-length bovine AP180 was used instead of the C58
domain (data not shown). TD did not interact with control surfaces
prepared with BSA and IgG, and BSA and IgG also did not interact with
the specific GST-C58 surface. These results indicate that AP180, like
AP-2 (Murphy and Keen, 1992; ter Haar et al., 2000) and AP-3
(Dell'Angelica et al., 1998; ter Haar et al., 2000), specifically
binds to TD.
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Figure 1.
Binding of clathrin TD to AP180. A,
Time course of binding of TD to AP180 was monitored by SPR. Traces
illustrate the SPR response, expressed in kilo response units
(kRU). During the time indicated by the
bar, TD was passed over surfaces to which either GST-C58
(solid line) or GST (dashed line) had
been coupled covalently. B, TD binds to GST-C58 in a
dose-dependent manner, whereas TD does not bind to GST at all of the
concentrations that were tested. Different concentrations of TD were
passed over the surfaces, and the responses were followed over time.
Maximum response values are plotted as a function of TD concentration.
The data points represent the mean response in two
experiments. Error bars indicate ± SEM. The data were fit by a
rectangular hyperbolic function.
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A repeated clathrin assembly motif in many APs
We next wanted to identify the site on AP180 to which clathrin
binds. The entire C-terminal domain of AP180 is required for efficient
assembly of clathrin (Ye and Lafer, 1995a), suggesting that clathrin
assembly activity is distributed throughout this domain. Further,
self-homology plots reveal that this domain of mammalian AP180 consists
of a large number of repeated sequence elements (Zhou et al., 1993)
(Fig. 2A). To consider
whether the repeating structures might represent clathrin binding
sites, we made an internal alignment of the primary amino acid sequence of AP180, guided by the use of a structural similarity consensus. This
alignment revealed that the clathrin assembly domain of AP180 contains
12 repeats of a degenerate sequence, ~23 amino acids in length, that
frequently contains a central DLL motif (Fig. 2B).
Very similar repeating motifs also are found in CALM, as well as in
squid, Drosophila, and Caenorhabditis elegans
AP180 (data not shown). The clathrin assembly (2) subunit of AP-2
possesses three similar repeats (Fig. 2B), two of
which map to the clathrin binding region previously identified between
amino acids 616-663 (Shih et al., 1995). Multiple repeats also were
found in the corresponding regions of the AP-1 1 subunit and in the
other large subunits of AP-1 () and AP-2 (A and C; Fig.
2B). The subunits of AP-2 have been reported to
bind clathrin, although it is not known whether they can assemble
clathrin (Prasad and Keen, 1991; Goodman and Keen, 1995). The large
subunits of AP-3 (; 3A, and 3B) and AP-4 (4) contain
similar repeats, typically centered on an SLL sequence (Fig.
2B). Previous mutagenesis of 3A revealed that the
sequence SLLDLDDFN, contained within one of these repeats (Fig. 2B), constitutes a clathrin binding site
(Dell'Angelica et al., 1998). Thus, our analysis identified a repeated
motif centered on a DLL/SLL that is common to many APs and might confer clathrin binding and assembly properties on these proteins.
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Figure 2.
Identification of a repetitive element in the APs.
A, A PLALIGN self-homology plot of AP180 reveals that
the structure of the clathrin assembly domain of AP180 (residues
305-901) is highly repetitive. The parallel
lines indicate regions of similarity. B,
Inspection of the LALIGN alignments revealed that the repetitive
element is an ~23 amino acid degenerate sequence, typically included
on a core DLL motif. The AP180 repeats were aligned with similar
repeats found in the large subunits of the AP-1 and AP-2 adaptors by
Megalign. Below, Alignment of the large subunits of the
AP-3 and AP-4 adaptors revealed a related repetitive element that
typically included a core SLL motif. The residues surrounded by
black in the alignments are identical to a structural
similarity consensus (see Materials and Methods).
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To determine whether the repeated motifs are important for clathrin
assembly, we first constructed AP180 mutants with
progressive deletions throughout the C-terminal clathrin assembly
domain (Fig. 3A). Progressive
deletion of the clathrin assembly domain of AP180 gradually decreased
the number of DLL motifs and caused a parallel loss in the clathrin
assembly activity of AP180 (Fig. 3B). For the nine
constructs that we studied, there was a linear relationship (r = 0.96) between the number of DLL motifs and the clathrin
assembly activity, indicated by the EC50 (Fig.
3C). We found that clathrin assembly is dependent on the
concentration of DLL motifs; when the EC50 for
each construct was normalized for the number of DLLs, by multiplying
the EC50 times the number of DLLs, the product was remarkably constant (Fig. 3D; mean = 9.1 ± 0.5 µM). This indicates that clathrin assembly
is directly proportional to the concentration of DLL motifs and thereby
implicates these repeated motifs in clathrin assembly.
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Figure 3.
Deletion of DLL motifs reduces the ability
of AP180 to assemble clathrin. A, Recombinant deletion
mutants of AP180 were constructed to vary the number of DLL motifs in
the clathrin assembly domain (C58). The DLL motifs
within full-length AP180 are denoted by asterisks.
B, Deletion of the DLL motifs gradually reduced the
ability of the AP180 mutants to assemble clathrin in
vitro. Colors correspond to the constructs
indicated in A for B-D; the data
points represent the mean of three to six independent
experiments; error bars indicate ± SEM. Smooth
lines indicate the Hill equation fit to the data.
C, The clathrin assembly activity of the AP180 mutants
was a linear function of the number of DLL motifs. The concentration of
assembly protein to give half-maximal assembly
(EC50) was determined from fitting the
data in B to the Hill equation. The error bars
indicate ± SEM. D, Clathrin assembly is
proportional to the concentration of DLL motifs, because the product of
the EC50 × the number of DLLs motifs was constant for
each construct. The EC50 values were determined from the
data in B.
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DLL motif peptide from AP180 inhibits clathrin assembly and
synaptic transmission
If the DLL motifs are important for clathrin binding and assembly,
then peptides bearing these motifs should act as competitive blockers
of AP-induced clathrin assembly. Clathrin assembly was studied as a
stringent measure of biological activity, because some proteins can
bind clathrin without assembling it (Ye and Lafer, 1995b; Goodman et
al., 1997). A DLL-containing peptide from AP180 (AP180 pep; aa
371-391) was synthesized and tested in vitro for its
ability to inhibit clathrin assembly by APs. Mammalian APs were used in
this analysis, because squid AP-2 has not been purified or cloned.
AP180 pep inhibited AP180-mediated clathrin assembly in a
dose-dependent manner (Fig.
4A). The relationship between peptide concentration and the degree of inhibition of clathrin
assembly could be described by the Hill equation (smooth lines in Fig. 4A); the concentration of peptide
required for half-maximal inhibition of assembly
(IC50), 0.5 mM in this
case, is reported in Table 1. This inhibitory activity depended on the
presence of the DLL motif, because scrambled AP180 peptides (Scram
AP180 pep 1 and 2) and a peptide in which the DLL was mutated to AAA (AP180 pep  DLL) were poor inhibitors of clathrin assembly (Fig. 4A). Compared with AP180 pep, these mutant peptides
were fourfold to 10-fold weaker inhibitors (see Table 1). AP180 pep
also inhibited assembly of clathrin by squid AP180, whereas the mutated
and scrambled peptides did not (data not shown). If the DLL motifs
exist within a clathrin binding site common to many clathrin APs, then
AP180 pep should cross-inhibit the assembly of clathrin that is
mediated by other APs. We found that AP180 pep indeed could inhibit
AP-2-mediated clathrin assembly in vitro, whereas AP180 pep
DLL was eight times weaker (Fig. 4B, Table 1).
These results indicate that the DLL motif defines a clathrin assembly
motif for AP180 and AP-2 and suggest that this conclusion may be
applicable to other APs.
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Figure 4.
A DLL-containing peptide from the clathrin
assembly domain of AP180 (AP180 pep) inhibits clathrin
assembly in vitro by both AP180 and AP-2.
A, AP180 pep produced a concentration-dependent
inhibition of clathrin assembly by AP180. The inhibition by AP180 pep
was reduced when the DLL was mutated to AAA
(DLL) or when the sequence of amino
acids within the peptide was scrambled (Scram1 and
Scram2). B, Concentration-dependent
inhibition of AP-2-mediated clathrin assembly by AP180 pep. The
inhibition by AP180 pep was reduced when the DLL was mutated to AAA
(DLL). In both panels the data
points represent the mean of three to four independent
experiments; error bars indicate ± SEM. Smooth
lines are fits of the Hill equation.
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Given its actions in vitro, AP180 pep should act as a broad
spectrum inhibitor of clathrin assembly in vivo, and this
was examined by injecting this peptide into squid giant presynaptic terminals. As a first test, synaptic transmission was measured by
monitoring the postsynaptic response to presynaptic action potentials
elicited at a low frequency (0.03 Hz). If AP180 pep inhibits clathrin
assembly, then synaptic transmission should be inhibited as synaptic
vesicles continue to fuse with the plasma membrane but are not
retrieved and restored to the pool of releasable vesicles (Morgan et
al., 1999). AP180 pep inhibited synaptic transmission without affecting
the presynaptic action potential (Fig.
5A). Because AP180 pep was
injected into only the presynaptic terminal, this peptide works by
inhibiting transmitter release rather than by affecting the
postsynaptic neuron. This inhibition was reversible and therefore was
not attributable to microinjection damage (Fig. 5B, Table
1). AP180 pep DLL, which had very little effect on clathrin assembly
in vitro, had no effect on synaptic transmission when it was
injected into the presynaptic terminal (Fig. 5C). Thus, the
effects of these peptides on synaptic transmission paralleled their
actions on clathrin assembly in vitro, suggesting that the inhibition of transmission is attributable to the blockade of clathrin
assembly.
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Figure 5.
AP180 pep inhibits neurotransmitter release.
A, Recordings of presynaptic and postsynaptic responses
before (Control) and after the injection of AP180
pep. AP180 pep transiently reduced transmitter release below the
threshold for eliciting a postsynaptic action potential. The vertical
scale bar applies to Vpost (above) and
Vpre (below) traces. B,
Time course of the inhibition of transmitter release by AP180 pep,
(injected during the time indicated by the bar), as
measured by the slope of the postsynaptic potential (PSP).
C, AP180 pep DLL had no effect on transmitter
release.
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DLL motif peptide from AP-2 also inhibits clathrin assembly and
synaptic transmission
As another test of the role of DLL motifs in clathrin assembly, we
examined a DLL-containing peptide from the 2 subunit of AP-2 (AP2
pep; aa 624-644). Like AP180 pep, in vitro AP2 pep
inhibited the assembly of clathrin by either AP-2 (Fig.
6A, Table 1) or AP180
(Fig. 6B, Table 1). Replacement of the DLL by AAA
(AP2 pep DLL) greatly reduced the inhibitory effect of this peptide on clathrin assembly (Fig. 6A,B). To examine the
structural basis for this effect, we examined clathrin cages made with
purified AP180 via electron microscopy. Clathrin cages were formed in
the presence of AP2 pep DLL (Fig. 6C), but not in the
presence of AP2 pep (Fig. 6D). AP2 pep also
reversibly inhibited synaptic transmission when it was injected into
the squid giant presynaptic terminal (Fig.
7A, Table 1). This inhibition
was dose-dependent, with the peptide capable of completely inhibiting
synaptic transmission at maximal concentrations (Fig. 7C).
Inhibition of synaptic transmission occurred at peptide concentrations
somewhat higher than those needed to block clathrin assembly in
vitro; this may reflect differences in the affinity of the
peptides for squid clathrin (vs mammalian clathrin) or in the amount of
APs and clathrin that are present in vivo (vs in
vitro). In contrast, AP2 pep DLL had no effect on synaptic
transmission at all of the concentrations that were tested (Fig.
7B,C). Thus, DLL motifs within AP-2 are also important for
clathrin assembly and synaptic transmission.
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Figure 6.
A DLL-containing AP-2 peptide (AP2
pep) inhibits clathrin assembly in vitro.
A, B, Clathrin assembly by both AP-2
(A) and AP180 (B) was
inhibited by AP2 pep in a concentration-dependent manner, whereas a
peptide in which the DLL was mutated to AAA
(DLL) or in which the D and L residues
were exchanged (D/L) had little effect. Mutation of the
downstream PVN to AAA in AP2 pep
(PVN) did not reduce the
efficacy of the peptide, whereas the mutation of the adjacent NLD to
AAA (NLD) had a moderate effect.
Data points represent the mean of three to four
independent experiments; error bars indicate ± SEM. Smooth
lines are fits of the Hill equation. C, D, The
ability of AP2 pep to inhibit clathrin assembly also was examined by
negative-staining electron microscopy of clathrin cages that were
assembled in the presence of AP2 pep DLL
(Control) or AP2 peptide (AP2
pep). The scale bar in D applies to both
images.
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Figure 7.
AP2 pep also inhibits transmitter release.
A, AP2 pep reversibly inhibited transmitter release when
it was injected (bar) into the squid giant presynaptic
terminal. B, The inhibitory effect on transmission was
lost when the DLL was mutated (AP2 pep DLL).
C, Inhibition of transmitter release by AP2 pep was
dependent on peptide concentration. Data points
represent the mean inhibition measured in two to seven experiments;
error bars represent ± SEM. Smooth lines indicate
rectangular hyperbola fits to the data. D, Correlation
between the abilities of several AP-2 peptides to inhibit synaptic
transmission in vivo and clathrin assembly in
vitro by AP-2.
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To define further the sequence requirements for clathrin assembly and
peptide action, we prepared additional peptides that included mutations
both within the DLL sequence and elsewhere in AP2 pep. When the DLL was
replaced with AAA (AP2 pep DLL) or when the D and L residues were
exchanged (AP2 pep D/L swap), the ability to inhibit clathrin assembly
was reduced six- to 125-fold (Fig. 6 A,B, Table 1). In
contrast, mutation of the adjacent NLD residues (AP2 pep NLD)
reduced the inhibitory activity of the peptide only twofold, and
mutation of the PVN sequence further downstream (AP2 pep PVN) had no
effect (Fig. 6 A,B, Table 1). Each of these mutated AP-2
peptides also was tested for its ability to inhibit synaptic
transmission. The actions of these peptides in vivo were
very similar to their actions in vitro; AP-2 peptides containing DLLs were effective in inhibiting synaptic transmission, whereas those with mutated DLLs had no effect (see Table 1). There was
a strong correlation (r = 0.98) between the inhibitory actions of these peptides on clathrin assembly and synaptic
transmission (Fig. 7D). Identical results were seen when the
inhibition of clathrin assembly by AP180 was compared with the
inhibition of transmission by AP2 pep and mutants (data not shown).
This indicates that the DLL is essential for the activity of AP2 pep
and that the NLD makes a smaller contribution to the activity both
in vitro and in vivo. All of these conclusions
support the hypothesis that the DLL motif is an important determinant
of clathrin binding and assembly by APs.
DLL motif peptide inhibits endocytosis at the clathrin
assembly step
The ability of the AP peptides to inhibit synaptic transmission is
consistent with these peptides blocking clathrin-mediated endocytosis
in the nerve terminal. We next examined this possibility more directly
by examining the ultrastructure of squid presynaptic terminals that
were injected with AP2 pep. If AP2 pep inhibits clathrin assembly
in vivo, then this peptide should deplete the nerve terminal
of both synaptic vesicles and coated vesicles. Further, the
accumulation of vesicular membrane should cause a proportionate
increase in the area of the presynaptic plasma membrane (Heuser and
Reese, 1973; Morgan et al., 1999). To test these predictions, we
injected AP2 pep until synaptic transmission was inhibited by >90%.
This was followed by fixation in glutaraldehyde and subsequent analysis
by electron microscopy. As a control, similar concentrations of an
inert peptide, AP2 pep DLL, were injected into other terminals and
had no effect on synaptic transmission, as described above.
In comparison to controls, terminals injected with AP2 pep were
depleted of synaptic vesicles and coated vesicles (Fig.
8). These structural changes were
analyzed in a number of ways. We quantified the spatial distribution of
synaptic vesicles, by measuring the distance between these vesicles and
the active zones at the plasma membrane (Hess et al., 1993), and found
that AP2 pep caused a depletion of synaptic vesicles at all distances
(Fig. 9A). When normalized to
the distribution of vesicles in the control terminals, the AP2 peptide
caused a 23% reduction in the docked synaptic vesicles within 50 nm of
the active zone and a 66% depletion of vesicles farther away (Fig.
9B). This yielded a 62% overall loss of synaptic vesicles
in the AP2 peptide-injected terminals (Fig. 9C;
p < 0.01, Student's t test). We also
measured the number of coated vesicles and found that these were
depleted by 95%, even more extensively than the synaptic vesicles
(Fig. 9D; p < 0.01, Student's t
test). The two terminals injected with AP2 pep also had a larger plasma
membrane area than the control terminals (Fig. 9E). This
increase was attributable to the trapping of synaptic vesicle membrane
in the plasma membrane, because the gain in plasma membrane area was
very similar to the reduction in synaptic vesicle membrane area (Fig.
9E). The area of membrane in larger spherical structures,
which have been termed vesicular endosomes (Burns et al., 1998),
remained unchanged (Fig. 9E). Unlike disruptions of AP180
function alone (Zhang et al., 1998; Morgan et al., 1999; Nonet et al.,
1999), the DLL-containing AP2 peptide did not alter synaptic vesicle
size when it was injected into the presynaptic terminal (Fig.
9F-H; p < 0.01, Student's t
test). Apparently, simultaneously inhibiting both AP-2 and AP180 does
not cause a change in vesicle size, consistent with previous notions
that AP180 has a unique role in determining vesicle size (Ye and Lafer, 1995b; Zhang et al., 1998; Morgan et al., 1999; Nonet et al., 1999). In
summary, these results are consistent with AP2 pep blocking synaptic
vesicle endocytosis at the clathrin assembly step and provide the final
evidence that DLLs are functional clathrin assembly motifs.
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Figure 8.
Presynaptic terminals injected with AP2 pep are
depleted of synaptic vesicles. Terminals injected with AP2 pep had
fewer synaptic vesicles than those injected with AP2 pep DLL
(Control). The postsynaptic spines are indicated
by S; the scale bar applies to both images.
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Figure 9.
The DLL-containing AP2 pep inhibits synaptic
vesicle endocytosis at the clathrin assembly step. A,
Spatial distribution of synaptic vesicles (SVs) in
terminals injected with AP2 pep or controls. Data represent the mean
values and SEM from 209 active zones (AZs) analyzed from
two AP2 peptide-injected terminals and 184 AZs from two control
terminals. B, Relative spatial distribution of SVs,
determined by dividing the mean values for the AP2 pep by the control
shown in A. The dashed line indicates a
1:1 ratio and represents no difference between terminals injected with
AP2 pep or control peptide. C, D, AP2 pep significantly
reduced the mean number of SVs (C) and coated
vesicles (CVs; D) found per AZ.
E, The mean area of membrane within vesicular endosomes
(Endo), SVs, CVs, and the presynaptic plasma membrane
(PM) was measured for each section in control and
AP2 peptide-injected terminals. F-H, Distribution of SV
diameters was measured from 20 active zones each in control
(F) or AP2 peptide-injected terminals
(G). The smooth lines indicate
Gaussian functions fit to these distributions. H,
Superimposition of the two distributions (normalized to the peak)
reveals no change in mean SV diameter after injection of the AP2
peptide.
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DISCUSSION |
We have identified a motif, containing a DLL or SLL sequence, that
is found in multiple copies in many APs, including AP180, AP-1, AP-2,
AP-3, and AP-4 (see Fig. 2). The conservation of these motifs suggests
that they may have a general role in interactions with clathrin and
could confer clathrin assembly properties to both tetrameric and
monomeric APs. Our experiments provide several lines of evidence that
this is the case. First, the clathrin assembly domain of AP180 (C58),
which contains numerous DLL motifs, binds directly to the N-terminal
domain of clathrin (see Fig. 1). Second, progressive deletion of the
DLL motifs from AP180 gradually reduces the clathrin assembly activity
of this protein in vitro (see Fig. 3). Third, peptides
bearing DLL motifs from either AP180 (AP180 pep) or AP-2 (AP2 pep)
cross-inhibit the ability of either full-length protein to promote
clathrin assembly in vitro (see Figs. 4, 6, Table 1).
Fourth, AP180 pep and AP2 pep reversibly inhibit synaptic transmission
in vivo, presumably by blocking clathrin-mediated endocytosis (see Figs. 5, 7, Table 1). The effect of AP2 pep on
clathrin assembly and synaptic transmission is attributable to the
presence of the DLL motif, because mutating the DLL reduced these
effects (see Table 1). For a series of mutant AP2 peptides the
inhibition of synaptic transmission in vivo was correlated strongly with effects on clathrin assembly in vitro (see
Figs. 6, 7, Table 1), suggesting that the inhibition of synaptic
transmission is attributable to the inhibition of clathrin assembly
in vivo. Finally, the DLL-containing AP2 pep produced
structural changes consistent with the blockade of synaptic vesicle
endocytosis at the clathrin assembly step; this peptide decreased the
number of synaptic vesicles (see Figs. 8, 9) and expanded the
presynaptic terminal plasma membrane area (see Fig. 9).
The AP180 and AP2 peptides used in this study have a low affinity for
clathrin, reflected in the high concentrations required to inhibit both
clathrin assembly in vitro and synaptic transmission in vivo. Despite their low affinity, our mutagenesis
experiments indicate that these peptides bind to clathrin with high
specificity (see Figs. 4, 6, Table 1). Thus, peptide affinity and
specificity are independent. This conclusion is consistent with the
finding that a peptide from the AP-2 -subunit (GDLLNLDLGP), which is contained within our AP2 pep, specifically binds to the N-terminal domain of clathrin heavy chain (ter Haar et al., 2000) yet has a low
binding affinity (T. Kirchhausen, personal communication).
It is unlikely that AP180 pep or AP2 pep prevented endocytosis by
interfering with the functions of other AP binding partners, such as
dynamin or amphiphysin. Although inhibiting these proteins causes
accumulation of coated pits (Takei et al., 1996; Shupliakov et al.,
1997), we never detected such structures in nerve terminals that were
injected with these peptides. Instead, the structural changes produced
by these peptides are consistent with the block of an earlier step in
endocytosis, such as coated pit formation (Morgan et al., 1999). Thus,
we conclude that DLL motifs within AP180 and AP-2 function as clathrin
assembly motifs that are essential for the formation of clathrin coats
during synaptic vesicle endocytosis. Given that AP-3 and AP-4 also
contain similar sequences (see Fig. 2), it is likely that these APs
also promote clathrin assembly and contribute to vesicular trafficking
through other intracellular compartments.
The role of clathrin and its APs in synaptic vesicle recycling has been
debated ever since Heuser and Reese (1973) first postulated that
synaptic vesicles are recycled by a coated vesicle pathway. Recent
observations of fast rates of endocytosis in some nerve terminals and
secretory cells have led to suggestions of clathrin-independent endocytosis (Palfrey and Artalejo, 1998). Thus, the relative
contribution of clathrin to synaptic vesicle recycling is not entirely
clear. We found that presynaptic injection of a DLL-containing peptide, which prevents clathrin assembly by both AP180 and AP-2, caused evoked
neurotransmitter release to cease and dramatically reduced the number
of synaptic vesicles and coated vesicles. Thus, our results indicate
that clathrin assembly is an important step in the recycling mechanism
even under low physiological rates (0.03 Hz) of neuronal activity, and
complement several other studies in emphasizing the predominance of
clathrin-mediated endocytosis in nerve terminals (Gonzalez-Gaitan and
Jackle, 1997; Zhang et al., 1998; Morgan et al., 1999; Ringstad et al.,
1999). Whereas previous studies examined single components of clathrin
assembly or later steps in clathrin-mediated endocytosis
(Gonzalez-Gaitan and Jackle, 1997; Zhang et al., 1998; Morgan et al.,
1999; Nonet et al., 1999; Ringstad et al., 1999), our experiments are
the first to block in general all clathrin assembly in vivo
by the clathrin APs.
The DLL motif that has been identified in this study bears a strong
relationship to the recently described clathrin box
sequence (consensus: LLpL-) that mediates
the direct binding of AP-1, AP-2, AP-3, and -arrestin to clathrin TD
(Dell'Angelica et al., 1998; ter Haar et al., 2000). For example the
DLL motif of AP2 pep is within a sequence (DLLNLD) that
would be defined as a clathrin box. However, the DLL motif we have
identified is somewhat more degenerate; in the case of AP180 pep the
DLL motif is within a sequence (DLLGED) that is
lacking the third L residue and intervening polar residue expected of a
clathrin box. Despite this degeneracy, AP180 pep clearly interacts with
clathrin, because it inhibits both AP180- and AP-2-mediated clathrin
assembly in vitro (see Fig. 4) and synaptic transmission
in vivo (see Fig. 5). In particular, the polar group that
follows the di-leucine is not well conserved between the DLL motif and
the clathrin box. However, available structural information indicates
that this polar residue projects outward and lacks any specific
interactions with clathrin (ter Haar et al., 2000). The DLL motif
identified in this paper also bears some resemblance to a DXF motif
hypothesized to mediate the binding of proteins to AP-2 (Owen et al.,
1999). However, it is clear that the DLL motifs are not involved in
AP-2 binding, because deletion of 10 of these motifs has no effect on
the ability of AP180 to bind to AP-2 (Hao et al., 1999).
Our discovery that APs have multiple copies of the DLL motif is
interesting in light of the structure of clathrin TD. TD has a
seven-blade -propeller structure with seven grooves between these
blades (ter Haar et al., 1998). Clathrin box-containing peptides from
AP-1, AP-2, AP-3, and -arrestin-2 bind to a groove between blades 1 and 2 of the TD, with the well conserved hydrophobic residues fitting
into two hydrophobic pockets (ter Haar et al., 2000). Although there
are no apparent repetitive features between the various grooves of TD,
the hydrophobic nature of the grooves is well conserved (ter Haar et
al., 1998). It is possible that the di-leucine pairs common to the DLL
motif and clathrin box are involved in similar hydrophobic interactions
with the grooves of TD, whereas the degeneracy of the DLL motifs may
reflect differing groove specificities. Multivalent
peptide-in-groove interactions have been observed in other
-propellers. For example, each groove of the five-blade
tachylectin-2 -propeller is an N-acetylglucosamine binding pocket (Beisel et al., 1999). Thus it is possible that APs have
multiple clathrin binding motifs to allow for multiple interactions
with the grooves between the blades of clathrin TDs.
The finding that individual APs contain multiple copies of the DLL
motif also suggests that APs may promote clathrin assembly by
cross-linking clathrin triskelia via multivalent interactions between
repeated motifs in the APs and complementary binding sites in the
clathrin TD (Fig. 10). In an assembled
clathrin lattice three TDs from three different clathrin triskelia come
together under each vertex (Smith et al., 1998; Musacchio et al.,
1999). This places anywhere from 3-21
possible AP binding sites in close proximity, depending on whether only
the groove between blades 1 and 2 contains an AP binding site or
whether all seven grooves contain AP binding sites. AP180 and AP-2 form
a complex that promotes clathrin assembly more efficiently than either
protein alone (Hao et al., 1999), and each AP180/AP-2 complex contains
up to 19 DLL motifs (see Fig. 2A). This leads us to
hypothesize that the AP180/AP-2 complex mediates clathrin assembly by
cross-linking the TDs of three adjacent triskelia. These interactions
could facilitate the movement of each triskelion into the lattice as it
is assembling and assure an ordered size to the assembled structure on
the basis of the geometric relationships between the proteins.
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Figure 10.
Cross-linking model for clathrin assembly by APs.
Multiple clathrin binding elements (shown as spikes)
exist within a single clathrin AP (top). These clathrin
binding elements allow a single AP to have multivalent interactions
with TDs from several clathrin molecules, thereby assembling them into
a cage (middle). The inset
(bottom) shows our proposed arrangement of TDs from
three clathrin molecules at a vertex and illustrates the multivalent
cross-linking of these clathrin molecules by APs.
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This model is supported by several observations. First, we show that
the C-terminal domain of AP180, containing all twelve DLL motifs, binds
directly to TD (see Fig. 1). Second, progressive deletion of the DLL
motifs from AP180 gradually reduces the ability of AP180 to promote
clathrin assembly, as though multiple sites on AP180 are binding to TD
(see Fig. 3B). AP180 also contains more DLL motifs than are
found in the sum of the and subunits of AP-2 (see Fig. 2),
which can explain why AP180 has a higher clathrin assembly activity
in vitro than AP-2 (Lindner and Ungewickell, 1992). In our
cross-linking model a given AP molecule interacts with TDs from three
separate clathrin molecules (see Fig. 10). Because each clathrin
molecule has three terminal domains, this could explain the observed
stoichiometry of one AP180 per clathrin triskelion (Prasad and
Lippoldt, 1988). Our model also may explain why -arrestin does not
promote clathrin assembly (Goodman et al., 1997); -arrestin has only
a single clathrin binding site, and our model would predict that this
would prevent it from cross-linking adjacent triskelia (Goodman et al.,
1997; Krupnick et al., 1997). One challenge to our model comes from the
observation that clathrin was assembled weakly by an AP180 construct
(C16) apparently containing only a single DLL motif (see Fig. 3).
However, a DFL sequence at the C-terminal end of C16 (aa 899-901) may
provide an additional clathrin binding site. This is consistent with a
report that a similar motif within the final eight amino acids of yeast
AP180A binds to clathrin (Wendland et al., 1999). Alternatively, it is possible that C16 contains still other clathrin binding sites that
remain unidentified because of the degeneracy of the DLL motif. In
either case the behavior of C16 then would be compatible with our
model; although binding of an AP to three clathrin molecules may be
optimal for efficient cross-linking, even binding to two molecules
should be sufficient for some cross-linking to occur.
Until now, the basis for clathrin assembly by structurally diverse APs
has been unclear. Our study indicates that this property is conferred
by a conserved DLL motif that is found in multiple copies in all of
these proteins. Further, our results suggest that this motif is part of
a clathrin binding site that serves to assemble clathrin by acting as a
multivalent cross-linker. These results provide novel insights into the
molecular mechanism of clathrin assembly, a process that is essential
for vesicle budding from donor membranes in presynaptic terminals and
many other cellular compartments.
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FOOTNOTES |
Received April 15, 2000; revised Aug. 8, 2000; accepted Aug. 16, 2000.
This work was supported by National Institutes of Health Grant NS29051,
a grant from the Muscular Dystrophy Association, and a Marine
Biological Laboratory fellowship (to E.M.L.); by National Institutes of
Health Grant NS21624 and a Human Frontier Science program grant (to
G.J.A.); and by Ruth K. Broad Biomedical Research Foundation and
National Research Service Award fellowships (to J.R.M.). We thank M. Hale and S. Miller for performing the electron microscopy and S. Jin
for performing the biosensor analysis. We also thank L. Bonewald and S. Mouton of the Institutional Protein Core Facility at University of
Texas Health Science Center at San Antonio for peptide synthesis and T. Kirchhausen and E. ter Haar for the clathrin terminal domain construct
and thoughtful discussions.
Correspondence should be addressed to Dr. George J. Augustine,
Department of Neurobiology; Box 3209, Duke University Medical Center;
Durham, NC 27710. E-mail: georgea{at}neuro.duke.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
